U.S. patent application number 13/457775 was filed with the patent office on 2012-11-01 for mirna396 and growth regulating factors for cyst nematode tolerance in plants.
This patent application is currently assigned to IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC.. Invention is credited to Thomas J. Baum, Tarek Abdel Fattah Hewezi.
Application Number | 20120278929 13/457775 |
Document ID | / |
Family ID | 47069060 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120278929 |
Kind Code |
A1 |
Baum; Thomas J. ; et
al. |
November 1, 2012 |
miRNA396 AND GROWTH REGULATING FACTORS FOR CYST NEMATODE TOLERANCE
IN PLANTS
Abstract
The present invention presents methods to alter the genetic
composition of crop plants susceptible to nematode infection to
improve tolerance to the same. Methods and compositions for
modulating key pathways involved in the syncytial event of nematode
infection and for preventing the cascade of differential gene
expression caused by the same as disclosed. Applicants have found
that the microRNA miR396 acts as a master switch of syncytial gene
expression changes in plants after infection, and further that
miR396 and certain growth regulating transcription factors (GRF)
are connected through feedback interaction in syncytium initiation
and maintenance.
Inventors: |
Baum; Thomas J.; (Ames,
IA) ; Hewezi; Tarek Abdel Fattah; (Ames, IA) |
Assignee: |
IOWA STATE UNIVERSITY RESEARCH
FOUNDATION, INC.
Ames
IA
|
Family ID: |
47069060 |
Appl. No.: |
13/457775 |
Filed: |
April 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61480093 |
Apr 28, 2011 |
|
|
|
Current U.S.
Class: |
800/265 ;
435/6.12; 800/279; 800/298 |
Current CPC
Class: |
C12N 15/8285 20130101;
Y02A 40/164 20180101; Y02A 40/146 20180101; C12N 15/8218
20130101 |
Class at
Publication: |
800/265 ;
435/6.12; 800/279; 800/298 |
International
Class: |
A01H 1/02 20060101
A01H001/02; C12N 15/82 20060101 C12N015/82; A01H 5/00 20060101
A01H005/00; A01H 5/10 20060101 A01H005/10; C12Q 1/68 20060101
C12Q001/68; A01H 1/00 20060101 A01H001/00 |
Goverment Interests
GRANT REFERENCE
[0002] This invention was made with government support under
Contract No. 2008-35302-18824 awarded by USDA. The government has
certain rights in the invention.
Claims
1. A method for improving nematode tolerance in plants comprising:
a) introducing into a plant cell a polynucleotide sequence encoding
a plant miRNA396 and/or GRF protein with a miRNA396 binding site
operably linked to a promoter functional in said plant cell; b)
regenerating a genetically modified plant from the plant cell;
whereby a genetically modified plant demonstrates improved
tolerance to nematode infection compared to a plant without such
modification.
2. The method of claim 1, wherein said plant miRNA396 is selected
from the group consisting of: miRNA396a, miRNA396b, miRNA396c, and
miRNA396e.
3. The method of claim 1, wherein said plant GRF protein is
selected from the group consisting of: GRF 1, GRF2, GRF3, GRF 4,
GRF 7, GRF8, GRF9, GRF13, GRF15, and GRF16.
4. The method of claim 1, wherein the plant cell is from a plant
selected from the group consisting of: Potato, tomato, Phaseolus
spp., sugarbeet, peas, Vicia faba, sugar cane, eggplant, peppers,
tobacco, wheat, rice, sorghum, barley, oat, lawn grass, rye,
soybean, canola, Brassica, sunflower, maize, sorghum, alfalfa,
cotton, millet, peanut and cacao.
5. The method of claim 1 wherein said miRNA396 is from
Arabidopsis.
6. The method of claim 1 wherein said GRF is from Arabidopsis.
7. The method of claim 1 wherein said miRNA396 is from Glycine
max.
8. The method of claim 1 wherein said GRF is from Glycine max.
9. A method of improving nematode tolerance in a plant comprising:
modulating the miRNA396-GRF interaction in said plant.
10. The method of claim 6 further comprising introducing a plant
miRNA396 nucleic acid into the plant.
11. The method of claim 6 further comprising increasing the
expression of one or more plant GRF nucleic acids in the plant.
12. A method for making a genetically modified plant with improved
nematode tolerance comprising: a) transforming a cell with an
expression cassette comprising an isolated nucleic acid which
encodes a miRNA396 operably linked to a promoter sequence
functional in a plant cell b) regenerating a genetically modified
plant from the cell; and c) selecting for a genetically modified
plant with improved nematode tolerance when compared to a
non-modified plant.
13. A plant made by the method of claim 9.
14. A method for making a genetically modified plant with improved
nematode tolerance comprising: a) transforming a cell with an
expression cassette comprising an isolated nucleic acid which
encodes a GRF protein with a miRNA396 binding site operably linked
to a promoter sequence functional in a plant cell b) regenerating a
genetically modified plant from the cell; and c) selecting for a
genetically modified plant with improved nematode tolerance when
compared to a non-modified plant.
15. A plant made by the method of claim 14.
16. A genetically modified plant, with improved tolerance to
nematode infection compared to a plant without such modification,
said plant comprising: a heterologous nucleotide sequence encoding
a plant miRNA396 and/or a GRF protein with a miRNA396 binding site,
operably linked to a promoter functional in a said plant.
17. Seed of the plant of claim 16.
18. A method for making a genetically modified plant with improved
nematode tolerance comprising: a) transforming a cell with an
expression cassette comprising an isolated nucleic acid which
encodes a GRF protein with a miRNA396 binding site (SEQ ID NO:28)
or an miRNA396 (SEQ ID NO:27) operably linked to a promoter
sequence functional in a plant cell, and regenerating a genetically
modified plant from the plant cell; whereby a genetically modified
plant demonstrates improved tolerance to nematode infection
compared to a plant without such modification.
19. A method for conferring or improving nematode resistance of a
plant, said method comprising: stably introducing into the genome
of a plant, at least one nucleotide construct comprising an
miRNA396 and/or GRF nucleic acid molecule operably linked to a
promoter that drives expression in a plant cell, wherein said
nucleic acid molecule encodes a product that modulates the
miRNA396-GRF interaction upon nematode infection.
20. A method of breeding for improved nematode tolerance in plants
comprising: measuring the amount or activity of an miRNA396 or GRF
protein or nucleic acid present in one or more plant cells, and
selecting a plant with higher levels of activity or amounts of
miRNA396 or GRF for breeding.
21. A method of identifying a marker for improved nematode
tolerance in plants comprising: screening an endogenous miRNA396 or
GRF nucleotide sequence for a polymorphism that is associated with
higher levels of expression or activity and selecting plants for
breeding based upon the presence of absence of said polymorphism.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.119
to provisional application Ser. No. 61/480,093 filed Apr. 28, 2011,
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to the field of plant
molecular biology.
BACKGROUND OF THE INVENTION
[0004] Nematodes are a very large group of invertebrate animals
generally referred to as roundworms, threadworms, eelworms, or
nemas. Some nematodes are plant parasites and can feed on stems,
buds, leaves, and in particular on roots. Cyst nematodes
(principally Heterodera and Globodera spp.) are key pests of major
crops. Cyst nematodes are known to infect tobacco, cereals, sugar
beets, potato, rice, corn, soybeans and many other crops.
Heterodera schachtii principally attacks sugar beets, and
Heterodera avenae has cereals as hosts. Heterodera zeae feeds on
corn, and Globodera rostochiensis and G. pallida feed on potatoes.
The soybean cyst nematode (Heterodera glycines) infests every
soybean-producing state in the U.S., with total soybean yield loss
estimates approaching $1 billion per year.
[0005] Plant-parasitic nematodes change shape as they go through
their life cycle. In its juvenile form, the animals penetrate plant
roots. The number of juveniles entering the plant root soon after
plant emergence can have a dramatic effect on plant growth and
development. Plant damage occurs from juvenile feeding which
removes cell materials and disrupts the vascular tissue by inducing
the formation of novel plant cell types that are associated in a
unique feeding organ, the syncytium. Due to the sedentary nature of
their parasitism, cyst nematodes need to obtain all their
nourishment from one location, in fact, through the contact with
the initial feeding cell.
[0006] Cyst nematodes infect as second-stage juveniles (J2), which
initiate the induction/formation of the syncytium. During this
phase, J2s begin feeding on the growing syncytium and then develop
into third-stage (J3) and fourth-stage juveniles (J4) followed by
the adult stage. Syncytium formation encompasses reprogramming of
differentiated plant root cells, and these redifferentiations are
accompanied and mediated by massive gene expression changes, which
have been documented in diverse research approaches using soybean
and the soybean cyst nematode Heterodera glycines (Alkharouf et
al., 2006; Ithal et al., 2007; Klink et al., 2009) and probably
most extensively in Arabidopsis infected by the sugar beet cyst
nematode H. schachtii (Szakasits et al., 2009). Regulatory networks
governing gene expression patterns in nematode-infected roots and
particularly in the developing syncytium are very poorly
understood.
[0007] Existing methods for treating or preventing nematode disease
include the use of chemicals, pesticides, and fumigants. The use of
pre-plant soil fumigants is highly effective in controlling cyst
nematodes and other plant-parasitic nematodes. However, the
majority of the fumigant-type nematicides is no longer available
and is also costly and difficult to apply properly under the
prevailing conditions.
[0008] Crop rotation has also been used to control nematode
disease. Rotating non-host plants can be effective in controlling
nematode disease. Unfortunately, these non-host crops are often
less valuable. Cover crops grown between the main crops is another
alternative management strategy. Ryegrain, barley, oats,
sudangrass, tall fescue, and annual ryegrass have been shown to be
non- or poor hosts for some nematodes. Using cover crops, however,
can be costly because the cover crops occupy space that could be
used to grow more valuable crops.
[0009] Biological control organisms have also been used to try to
control nematode disease in crops. Commercially available
preparations of biological control organisms are limited in their
use to regions that can support the growth of the control organism.
Moreover, the outcome of using one organism to control another is
unpredictable and subject to a variety of factors such as weather
and climate.
[0010] As can be seen, a continuing need exists for the development
of methods and strategies to control and inhibit plant nematode
invasion.
[0011] It is an object of the present invention to develop plants,
seeds, varieties and lines that have improved tolerance to nematode
infection and resultant effects on plants.
[0012] It is another object of the invention to provide methods for
controlling nematode infection that are environmentally friendly
and do not rely on chemicals, biological control organisms, or crop
rotation.
[0013] It is yet another object of the invention to provide novel
plant genetic engineering strategies to ascertain more about the
mechanism and plant response to nematode infection, to develop
resistant varieties and to modulate expression of key components of
regulatory pathways that inhibit nematode infection and its affects
in the plant.
SUMMARY OF THE INVENTION
[0014] The present invention includes methods to alter the genetic
composition of crop plants, particularly those that are susceptible
to nematode infection, thereby improving tolerance to nematode
infection and reducing the effects thereof in plants. This
invention provides methods and compositions for modulating key
pathways involved in the syncytial event of nematode infection and
for preventing the cascade of differential gene expression caused
by the same. Applicants have found that the microRNA miR396 acts as
a master switch of syncytial gene expression changes in plants
after infection, and further that miR396 and growth regulating
transcription factors (GRF) with miRNA396 binding sites are
connected through a negative feedback loop to establish an
irreversible plant gene regulatory switch from syncytium initiation
and maintenance.
[0015] This invention in one embodiment relates to modulation of
expression of miRNA396 and GRFs with miRNA396 binding sites to
engineer improved tolerance to cyst nematode infection in plants as
well as the hinder the development and maintenance of the
syncytium, essential for plant pathogen survival.
[0016] According to the invention, miR396 and GRF1/GRF3 are
connected through a negative feedback loop from a low miR396 high
GRF1/3 state during syncytium initiation, to high miR396 low GFR1/3
during maintenance. Modulated expression of this interaction alters
the outcome of the plant pathogen interaction and alters plant
susceptibility. In particular, overexpression of miRNA396 reduces
plant susceptibility to nematode infection by more than half. Other
methods of interfering with this miRNA396 and GRF interaction would
also be included within the scope of this invention, whether by
increasing activity of the same, through such mechanisms as
overexpression, inhibition of activity, such as through inhibition
of translation or transcription, or introduction of heterologous
interfering or competing proteins.
[0017] Thus the invention contemplates the regulation of miRNA396
and the pathway of regulatory transcription factors associated with
the same to engineer tolerance to nematode infection in plants,
preferably by modulation of miRNA sequences or activity in
plants.
[0018] As used herein the term "miRNA396" or "miR396" shall be
interpreted to include genes such as miR396a (Arabidopsis ATG10606,
Glycine max MI0001785, MIMAT0001687); miR396b (Arabidopsis
AT5G35407, Glycine max MI0001786, MIMAT0001688); miR396c (Glycine
max MI0010572, MIMAT0010079); and miR396e (Glycine max MI0016586,
MIMAT0018345) which regulate expression of growth regulating
transcription factor genes that have an miR396-binding site such as
GRF 1 through 4 and 7 through 9 in Arabidopsis, See Jones-Rhoades
and Bartel, 2004, "Computational identification of plant microRNAs
and their targets, including a stress-induced miRNA" Mol. Cell. 14,
787-799. Soybean GRFs include GRF8, 9, 12, 13, 15, 16, and 19,
Mi396 is a highly conserved micro RNA as many are, and has been
found in many other nematode susceptible plants including Citrus
unshiu, Glycine max (soybean), Lactuca sativa (lettuce), Lotus
japonicus, Medicago truncatula, Nicotiana benthaminiana (tobacco),
Oryza sativa (rice), and Populus euphratica. See, Zhang et al.,
"Conservation and Divergence of Plant MicroRNA Genes" The Plant
Journal (2006) 46 243-259. Additionally, other miRNA396 homologs
may be identified thought databases such as Genbank, and the
mircoRNA database, at world wide web mirbase.org.
[0019] Similarly, other growth regulatory transcription factor
genes are known and easily identifiable by one of skill in the art
through similar databases. Kim, J. H., Choi, D., Kende, H. (2003)
"The AtGRF Family of Putative Transcription Factors is Involved in
Leaf and Cotyledon Growth in Arabidopsis" The Plant Journal 36.
These include, for example Arabidopsis, At2g22840 AtGRF1
transcription activator (GRF1), At2g36400 AtGRF3 transcription
activator (GRF3), At3g52910 AtGRF4 expressed protein,
growth-regulating factor, At3g13960 AtGRF5 transcription activator
(GRF5), At2g06200 AtGRF6 expressed protein, At5g53660 AtGRF7
hypothetical protein At4g24150 AtGRF8 hypothetical protein. From
soybean these include but are not limited to: GmGRF8
(Glyma10g07790); GRF9 (XM.sub.--003537618); GmGRF12
(Glyma13g16920); GmGRF13 (Glyma13g21630); GmGRF15
(XM.sub.--003547454); GmGRF16 (Glyma16g00970) and GmGRF19
(XM.sub.--003553541). All GFR transcription factors useful for the
invention, will have an miRNA396 sequence (CAAGUUCUUUCGNACACCUU)
(SEQ ID NO:27) binding site AAGGUGUNCGAAAGAACUUGC (SEQ ID NO:28) in
common. Thus, although the invention is exemplified herein with
specific Arabidopsis and soybean genes, the invention is not so
limited and has applicability to any plant susceptible to nematode
or other plant pathogen infection by interaction with miRNA396 and
corresponding GRF transcription factors.
[0020] The invention provides methods for improving plant tolerance
to cyst nematode infection by modulating miRNA 396 interacting
pathway, such as, for example, increasing/modulating the activity
of at least one miRNA396. In other embodiments, other steps along
the signaling pathway could be modulated, such as the miRNA396
binding sites including GRF1, GRF 3 and other GRFs.
[0021] According to the invention, the methods for modulation
include modification of a plant cell by introducing at least one
polynucleotide sequence comprising a plant miRNA396 or plant GRF
nucleic acid sequence, or subsequence thereof, into said plant
cell, such that the polynucleotide sequence is operably linked to a
promoter functional in said plant cell. In another embodiment, the
method of modulating the production of miRNA396 or a GRF protein by
increasing/modulating includes a miRNA396 or GRF gene which
comprises, e.g., at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 99%, at least about 99.5% or more sequence
identity to miR396a (Arabidopsis ATG10606 (SEQ ID NO:1), Glycine
max MI0001785 (SEQ ID NO:12), or MIMAT0001687 (SEQ ID NO:13);
miR396b (Arabidopsis AT5G35407 (SEQ ID NO:2), Glycine max MI0001786
(SEQ ID NO:14), MIMAT0001688) (SEQ ID NO:15); or miR396c (Glycine
max MI0010572 (SEQ ID NO:116), MIMAT0010079 (SEQ ID NO:17)); or
miR396e (Glycine max MI0016586 (SEQ ID NO:18), MIMAT0018345 (SEQ ID
NO:10)) or to corresponding GRFs including GRF1 (At2g22840) (SEQ ID
NO:3), GRF2 (At4g37740)) (SEQ ID NO:4), GRF3 (At2g36400)) (SEQ ID
NO:5), GRF4 (At3g52910)) (SEQ ID NO:6), GRF7 (At5g53660) (SEQ ID
NO:9), GRF8 (At4g24150) (SEQ ID NO:10), GRF9 (At2g45480) (SEQ ID
NO:11), GmGRF8 (Glyma10g07790) (SEQ ID NO:20); GRF9
(XM.sub.--003537618) (SEQ ID NO:21); GmGRF12 (Glyma13g16920) (SEQ
ID NO:22); GmGRF13 (Glyma13g21630) (SEQ ID NO:23); GmGRF15
(XM.sub.--003547454) (SEQ ID NO:24); GmGRF16 (Glyma16g00970) (SEQ
ID NO:25) and GmGRF19 (XM.sub.--003553541)) (SEQ ID NO:26).
[0022] Many plant miRNA396s and GRFs are known to those of skill in
the art such as those from rice, Arabidopsis and soybean and are
readily available through sources such as GENBANK and the like.
[0023] In another embodiment, the invention relates to methods for
improving plant tolerance to cyst nematode infection by providing
an isolated or recombinant modified plant cell comprising at least
one modification that increases, decreases or otherwise modulates
miRNA396 or GRF activity. In certain embodiments, a plant cell
resulting from the methods of the invention is from a dicot or
monocot. In another aspect, the plant cell is in a plant comprising
a sterility phenotype, e.g., a male sterility phenotype.
[0024] The methods of the invention are practiced with an isolated
or recombinant polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide, or a complement thereof,
comprising, e.g., at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 99%, about 99.5% or more sequence identity to
an miRNA396 or GRF transcription factor or a subsequence thereof,
or a conservative variation thereof; (b) a polynucleotide, or a
complement thereof, encoding a polypeptide sequence of a (c) a
polynucleotide, or a complement thereof, that hybridizes under
stringent conditions over substantially the entire length of a
polynucleotide subsequence comprising at least 100 contiguous
nucleotides of SEQ a, or that hybridizes to a polynucleotide
sequence of (a) or (b); and, (d) a polynucleotide that is at least
about 85% identical to a polynucleotide sequence of (a), (b) or
(c).
[0025] Such polynucleotides for practice of the methods of the
invention can comprise or be contained within an expression
cassette or a vector (e.g., a viral vector). The vector or
expression cassette can comprise a promoter (e.g., a constitutive,
tissue-specific, or inducible promoter) operably linked to the
polynucleotide. In a preferred embodiment, the promoter is a root
specific promoter.
[0026] Detection of expression products is performed either
qualitatively (by detecting presence or absence of one or more
product of interest) or quantitatively (by monitoring the level of
expression of one or more product of interest). Aspects of the
invention optionally include monitoring an expression level of a
nucleic acid, polypeptide or chemical as noted herein for detection
of the same in a plant or in a population of plants.
[0027] In yet another embodiment, the present invention is directed
to a transgenic plant or plant cells with improved performance
under nematode infecting conditions, containing the nucleic acids
described herein. Preferred plants containing the polynucleotides
of the present invention include but are not limited to soybean,
sunflower, maize, sorghum, canola, wheat, alfalfa, cotton, oat,
rice, barley, tomato, cacao and millet. In another embodiment, the
transgenic plant is a soybean plant or plant cells. Plants produced
according to the invention can have at least one of the following
phenotypes in nematode infecting conditions as compared to a
non-modified control plant, including but not limited to: increased
root mass, increased plant survival, increased root length,
increased leaf size, increased ear size, increased seed size,
absence of syncytia, smaller or decreased syncytia, or increased
plant size when compared to a non-modified plant under conditions
of nematode infection.
[0028] In yet another embodiment, levels of miRNA396 or GRF
proteins or mutant polynucleotide or polypeptide (where
appropriate) sequences may be used as markers or selection traits
to identify and select nematode tolerant plants even in the absence
of transformation for breeding of tolerant lines, plants seeds,
varieties and the like. Marker assisted selection protocols are
thus included herein.
DETAILED DESCRIPTION OF THE FIGURES
[0029] FIG. 1: Characterization of transgenic plants overexpressing
miR396 or the target genes GRF1 and GRF3. (A) Overexpression of
miR396 reduces GRF gene expression. The mRNA expression level of
GRF1-9 was measured by quantitative real-time RT-PCR in the root
tissues of 10 d-old wild-type (Col-0) and transgenic plants
overexpressing miR396b (line 16-4). The expression levels were
normalized using Actin8 as an internal control. The relative
fold-change values represent changes of mRNA levels in the
transgenic plants relative to the wild-type control. Data are
averages of three biologically independent experiments.+-.SE. (B)
and (C) Transgenic plants overexpressing miR396a (line 22-5) (B) or
miR396b (line 15-1) (C) develop shorter roots than the wild-type
(Col-0). Homozygous T3 plants were planted on modified Knop's
medium along with the wild type (Col-0), and root lengths were
measured 10 days after planting. Root length values are averages of
at least 50 plants. Differences between miR396 overexpression lines
and the wild type were statistically significant as determined by
unadjusted paired t tests (P<0.01). (D) Schematic representation
of wild-type and miR396-resistant versions of GRF1 and GRF3
transcripts. Nucleotide pairing of miR396 with the corresponding
wild-type binding sites of GRF1 (wtGRF1) and GRF3 (wtGRF3) show 19
nucleotide matches, whereas in the miR396-resistant version of GRF1
(rGRF1) and GRF3 (rGRF3) the miR396 binding site contains 10
mismatches. Conserved nucleotides between wild-type and modified
miR396 binding sites are in bold. (E) and (F): Transgenic plants
overexpressing wtGRF1 or wtGRF3 (E) and rGRF1 or rGRF3 (F) develop
shorter roots than the wild type (Col-0). Homozygous T3 plants were
planted on modified Knop's medium along with the wild type, and
root lengths were measured as indicated above. Differences between
overexpression lines and the wild type were statistically
significant as determined by unadjusted paired t tests (P<0.01).
(G) Overexpression of GRF1 or GRF3 negatively regulates GRF gene
expression. The mRNA expression levels of GRF1 through 9 were
quantified in the root tissues of the transgenic plants
overexpressing the wild-type forms of GRF1 and GRF3
(.sup.35S:wtGRF1 and .sup.35S:wtGRF3) or the miR396-resistant forms
(.sup.35S:rGRF1 and .sup.35S:rGRF3) using qPCR. The expression
levels were normalized using Actin8 as an internal control. The
relative fold-change values represent changes of GRF expression
levels in the transgenic plants relative to the wild-type control.
Data are averages of three biologically independent
experiments.+-.SE. Note that the expression levels of GRF1 and GRF3
in the .sup.35S:rGRF1 and .sup.35S:rGRF3 plants include the
endogenous transcripts. (H) Overexpression of GRF1 or GRF3
negatively regulates miR396 expression. The levels of pre-miR396a,
pre-miR396b and mature miR396 were quantified in root tissues of
the transgenic plants described in (G) using qPCR. The expression
levels were normalized using U6 snRNA as an internal control. The
relative fold-change values represent changes of miRNA abundance in
the transgenic plant relative to the wild-type control. Data are
averages of three biologically independent experiments.+-.SE. The
expression levels of the transgenes are provided in Figure S3.
[0030] FIG. 2: Promoter activity of miR396a, miR396b and the target
genes GRF1 and GRF3 during Heterodera schachtii infection. Time
course experiments comparing the expression of miR396a:GUS (A-D),
miR396b:GUS (E-H), GRF1:GUS (1-L), and GRF3: GUS (M-P) transgenic
plants at the second-stage (J2), early and late third-stage (J3),
and fourth-stage juvenile (J4) time points. N indicates nematode
and S indicates syncytium. See also Figure S2.
[0031] FIG. 3: Post-transcriptional regulation of GRF1 and GRF3 by
miR396 in response to H. schachtii infection. The expression level
of pre-miR396a, pre-miR396b, mature miR396, GRF1 and GRF3 was
measured by qPCR in wild-type (Col-0) root tissues. Infected and
noninfected tissues were collected at 1, 3, 8, and 14 days after
inoculation (dpi). Down regulation of miR396 at 1 and 3 dpi was
associated with up regulation of both GRF1 and GRF3. In contrast,
up regulation of miR396 at 8 and 14 dpi activated the cleavage of
GRF1 and GRF3 resulting in low transcript accumulation of GRF1 and
GRF3. U6 snRNA was used as an internal control to normalize the
expression levels of miR396, whereas Actin8 was used to normalize
the expression levels of GRF1 and 3. The relative fold-change
values represent changes of the expression levels in infected
tissues relative to noninfected controls. Data are averages of
three biologically independent experiments.+-.SE.
[0032] FIG. 4: Nematode susceptibility assays of miR396
overexpression lines and GRF mutants (A) and (B) Nematode
susceptibility assays of miR396 overexpression lines. Transgenic
plants overexpressing miR396a (A) or miR396b (B) exhibited reduced
susceptibility to H. schachtii. Homozygous T3 lines overexpressing
miR396a (lines 22-5, 13-10, and 10-12) or miR396b (lines 16-4, 15-1
and 8-16) were planted on modified Knop's medium, and 10-d-old
seedlings were inoculated with .about.200 surface-sterilized J2 H.
schachtii nematodes. Three weeks after inoculation, the number of
J4 female nematodes per root system was determined. Data are
presented as the mean.+-.SE. Mean values significantly different
from the wild type (Col-0) were determined by unadjusted paired t
tests (P<0.05) and indicated by an asterisk. Identical results
were obtained from at least two independent experiments. (C)
Nematode susceptibility is not significantly altered in grf1 or
grf3 single mutant. The mutant alleles of grf1 (Salk069339C and
Sa1k0785 47C) and gfr3 (salk116709 and sa1k026786) along with
wild-type Col-0 plants were planted on modified Knop's medium and
assayed for nematode susceptibility. No statistically significant
differences between these mutant lines and wild type were observed.
Data are presented as means.+-.SE. Similar results were obtained
from at least three independent experiments. (D) The grf1/grf2/grf3
triple mutant exhibited reduced susceptibility to H. schachtii.
Seeds of the grf1/grf2/grf3 triple mutant and wild type (WS) were
planted on modified Knop's medium and assayed for nematode
susceptibility. Data are presented as means.+-.SE and the
statistically significant difference between the grf1/grf2/grf3
mutant and the wild type (WS) is denoted by asterisk as determined
by unadjusted paired t tests (P<0.05). Identical results were
obtained from two independent experiments. (E-H). Transgenic plants
overexpressing wtGRF1 (E), rGRF1 (F), wtGRF3 (G) or rGRF3 (H)
revealed reduced susceptibility to H. schachtii. Four independent
homozygous T3 lines for each construct were assayed for nematode
susceptibility. All lines showed significantly reduced
susceptibility compared with wild-type plants. Data are presented
as the mean.+-.SE. Mean values significantly different from the
wild-type (Col-0) were determined by unadjusted paired t tests
(P<0.05) and indicated by an asterisk. Identical results were
obtained from at least two independent experiments.
[0033] FIG. 5: Overexpression of miR396, GRF1 or GRF3 negatively
impacts syncytium size and nematode development. (A) Transgenic
plants overexpressing miR396, rGRF1 or rGRF3 developed smaller
syncytia than the wild type. Homozygous T3 lines overexpressing
miR396b (line 16-4), rGRF1 (lines 12-3) or rGRF3 (line 12-5) as
well as wild-type (Col-0) were planted on modified Knop's medium,
and 10-d-old seedlings were inoculated with .about.200
surface-sterilized J2 H. schachtii nematodes. Two weeks
post-inoculation, at least 20 single-nematode syncytia were
randomly selected and measured. Data are presented as means.+-.SE.
The asterisk indicates a statistically significance difference from
wild-type plants at P<0.05. (B) and (C) Overexpression of
miR396, rGRF1 or rGRF3 negatively impacts nematode development.
Seeds of the above-indicated lines along with wild-type (Col-0)
were planted and inoculated as described in (A). After inoculation,
the number of parasitic J2/J3 (B) and J4 females (C) was counted in
the same plants. Data are presented as means.+-.SE. The asterisk
indicates a statistically significance difference from wild-type
plants at P<0.05.
[0034] FIG. 6: Functional classification of the differentially
expressed genes identified in 35S:rGRF1, 35S:rGRF1 and
grf1/grf1/grf3 mutants. (A) Venn diagram showing overlaps between
differentially expressed genes in 35S:rGRF1, 35S:rGRF3 and
grf1/grf2/grf3 mutants. The total number of differentially
expressed genes in each set is shown in parentheses. Genes are
listed in Table S1A-C. (B) and (C) Venn diagram comparing the
overlapping differentially expressed genes between 35S:rGRF1 and
grf1/grf2/grf3 (B) or 35S:rGRF1 and grf1/grf2/grf3 (C). Numbers in
the areas highlighted in red indicate differentially expressed
genes that exhibit opposite expression whereas overlapping areas
highlighted in blue indicate the number of the differentially
expressed genes that exhibited similar expression. (Genes are
listed in Table S1D and E). (D) and (E) Gene ontology
categorization of the molecular functions (D) or the biological
processes (E) of the candidate target genes of GRF1 or GRF3. (Genes
used for this categorization are listed in Table S1D and E). (F)
Venn diagram showing overlaps between differentially expressed
genes in the syncytium and those identified in 35S:rGRF1, 35S:rGRF1
and grf1/grf2/grf3 mutants. The total number of differentially
expressed genes in each set is shown in parentheses.
[0035] FIG. 7: Expression profiles of GRF gene family members in
Arabidopsis roots.
[0036] FIG. 8 (A-L): Spatial expression patterns of miR396a and
miR396b and the target genes GRF1 and GRF3.
[0037] FIG. 9 (A-F): Quantification of transgene expression levels
in the transgenic Arabidopsis lines described in this study using
qPCR.
[0038] FIG. 10 (A-C): Characterization of Arabidopsis grf1 and grf3
mutants.
[0039] FIG. 11 (A-D): GRF2 promoter activity during Heterodera
schachtii infection.
[0040] FIG. 12: Soybean miR396/target GRFs Expression Analyses with
qRT-PCR after SCN Infection.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of botany,
microbiology, tissue culture, molecular biology, chemistry,
biochemistry and recombinant DNA technology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Langenheim and Thimann, (1982) Botany: Plant
Biology and Its Relation to Human Affairs, John Wiley; Cell Culture
and Somatic Cell Genetics of Plants, vol. 1, Vasil, ed. (1984);
Stanier, et al., (1986) The Microbial World, 5.sup.th ed.,
Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant Pathology
Methods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: A
Laboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985);
Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid
Hybridization, Hames and Higgins, eds. (1984); and the series
Methods in Enzymology, Colowick and Kaplan, eds, Academic Press,
Inc., San Diego, Calif.
[0042] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. The terms defined below are more
fully defined by reference to the specification as a whole. In
describing the present invention, the following terms will be
employed, and are intended to be defined as indicated below.
[0043] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), Q-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., Diagnostic Molecular
Microbiology: Principles and Applications, Persing, et al., eds.,
American Society for Microbiology, Washington, D.C. (1993). The
product of amplification is termed an amplicon.
[0044] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refer to
those nucleic acids that encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine; one exception is
Micrococcus rubens, for which GTG is the methionine codon
(Ishizuka, et al., (1993) J. Gen. Microbiol. 139:425-32) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid, which encodes a
polypeptide of the present invention, is implicit in each described
polypeptide sequence and incorporated herein by reference.
[0045] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" when
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Thus, any number of amino acid
residues selected from the group of integers consisting of from 1
to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10
alterations can be made. Conservatively modified variants typically
provide similar biological activity as the unmodified polypeptide
sequence from which they are derived. For example, substrate
specificity, enzyme activity, or ligand/receptor binding is
generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably
60-90% of the native protein for its native substrate. Conservative
substitution tables providing functionally similar amino acids are
well known in the art.
[0046] The following six groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
[0047] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0048] See also, Creighton, Proteins, W.H. Freeman and Co.
(1984).
[0049] As used herein, "consisting essentially of" means the
inclusion of additional sequences to an object polynucleotide where
the additional sequences do not selectively hybridize, under
stringent hybridization conditions, to the same cDNA as the
polynucleotide and where the hybridization conditions include a
wash step in 0.1.times.SSC and 0.1% sodium dodecyl sulfate at
65.degree. C.
[0050] By "encoding" or "encoded," with respect to a specified
nucleic acid, is meant comprising the information for translation
into the specified protein. A nucleic acid encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid, or may lack such intervening
non-translated sequences (e.g., as in cDNA). The information by
which a protein is encoded is specified by the use of codons.
Typically, the amino acid sequence is encoded by the nucleic acid
using the "universal" genetic code. However, variants of the
universal code, such as is present in some plant, animal, and
fungal mitochondria, the bacterium Mycoplasma capricolumn (Yamao,
et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9), or the
ciliate Macronucleus, may be used when the nucleic acid is
expressed using these organisms.
[0051] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended
host where the nucleic acid is to be expressed. For example,
although nucleic acid sequences of the present invention may be
expressed in both monocotyledonous and dicotyledonous plant
species, sequences can be modified to account for the specific
codon preferences and GC content preferences of monocotyledonous
plants or dicotyledonous plants as these preferences have been
shown to differ (Murray, et al., (1989) Nucleic Acids Res.
17:477-98 and herein incorporated by reference). Thus, the maize
preferred codon for a particular amino acid might be derived from
known gene sequences from maize. Maize codon usage for 28 genes
from maize plants is listed in Table 4 of Murray, et al.,
supra.
[0052] As used herein, "control plant" is a plant without
recombinant DNA disclosed herein. A control plant is used to
measure and compare trait improvement in a transgenic plant with
such recombinant DNA. A suitable control plant may be a
non-transgenic plant of the parental line used to generate a
transgenic plant herein. Alternatively, a control plant may be a
transgenic plant that comprises an empty vector or marker gene, but
does not contain the recombinant DNA that produces the trait
improvement. A control plant may also be a negative segregant
progeny of hemizygous transgenic plant.
[0053] As used herein, "gene" refers to chromosomal DNA, plasmid
DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide,
polypeptide, protein, or RNA molecule, and regions flanking the
coding sequences involved in the regulation of expression.
[0054] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0055] By "host cell" is meant a cell, which comprises a
heterologous nucleic acid sequence of the invention, which contains
a vector and supports the replication and/or expression of the
expression vector. Host cells may be prokaryotic cells such as E.
coli, or eukaryotic cells such as yeast, insect, plant, amphibian,
or mammalian cells. Preferably, host cells are monocotyledonous or
dicotyledonous plant cells, including but not limited to maize,
sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola,
lawn grass, barley, millet, and tomato. A particularly preferred
monocotyledonous host cell is a soybean host cell.
[0056] The term "hybridization complex" includes reference to a
duplex nucleic acid structure formed by two single-stranded nucleic
acid sequences selectively hybridized with each other.
[0057] As used herein, "improved trait" refers to a trait with a
detectable improvement in a transgenic plant relative to a control
plant or a reference. In some cases, the trait improvement can be
measured quantitatively. For example, the trait improvement can
entail at least a 2% desirable difference in an observed trait, at
least a 5% desirable difference, at least about a 10% desirable
difference, at least about a 20% desirable difference, at least
about a 30% desirable difference, at least about a 50% desirable
difference, at least about a 70% desirable difference, or at least
about a 100% difference, or an even greater desirable difference.
In other cases, the trait improvement is only measured
qualitatively. It is known that there can be a natural variation in
a trait. Therefore, the trait improvement observed entails a change
of the normal distribution of the trait in the transgenic plant
compared with the trait distribution observed in a control plant or
a reference, which is evaluated by statistical methods provided
herein. Trait improvement includes, but not limited to, yield
increase, including increased yield under non-stress conditions and
increased yield under environmental stress conditions. Stress
conditions may include, for example, drought, shade, fungal
disease, viral disease, bacterial disease, insect infestation,
nematode infestation, cold temperature exposure, heat exposure,
osmotic stress, reduced nitrogen nutrient availability, reduced
phosphorus nutrient availability and high plant density.
[0058] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be incorporated into the genome of the cell (e.g.,
chromosome, plasmid, plastid or mitochondrial DNA), converted into
an autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0059] The terms "isolated" or "isolated nucleic acid" or "isolated
protein" refer to material, such as a nucleic acid or a protein,
which is substantially or essentially free from components which
normally accompany or interact with it as found in its naturally
occurring environment. The isolated material optionally comprises
material not found with the material in its natural environment.
Nucleic acids which are "isolated", as defined herein, are also
referred to as "heterologous" nucleic acids.
[0060] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0061] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules, which comprise and substantially represent
the entire transcribed fraction of a genome of a specified
organism. Construction of exemplary nucleic acid libraries, such as
genomic and cDNA libraries, is taught in standard molecular biology
references such as Berger and Kimmel, (1987) Guide To Molecular
Cloning Techniques, from the series Methods in Enzymology, vol.
152, Academic Press, Inc., San Diego, Calif.; Sambrook, et al.,
(1989) Molecular Cloning: A Laboratory Manual, 2.sub.nd ed., vols.
1-3; and Current Protocols in Molecular Biology, Ausubel, et al.,
eds, Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc. (1994
Supplement).
[0062] As used herein "operably linked" includes reference to a
functional linkage between a first sequence, such as a promoter,
and a second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA corresponding to the second
sequence. Generally, operably linked means that the nucleic acid
sequences being linked are contiguous and, where necessary to join
two protein coding regions, contiguous and in the same reading
frame.
[0063] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
plant cells and progeny of same. Plant cell, as used herein
includes, without limitation, cells in or from seeds, suspension
cultures, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen, and microspores.
The class of plants which can be used in the methods of the
invention is generally as broad as the class of higher plants
amenable to transformation techniques, including both
monocotyledonous and dicotyledonous plants including species from
the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus,
Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,
Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus,
Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,
Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium,
Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis,
Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio,
Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus,
Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum. A
particularly preferred plant is Zea mays.
[0064] As used herein, "yield" may include reference to bushels per
acre of a grain crop at harvest, as adjusted for grain moisture
(15% typically for maize, for example), and/or the volume of
biomass generated (for forage crops such as alfalfa, and plant root
size for multiple crops). Grain moisture is measured in the grain
at harvest. The adjusted test weight of grain is determined to be
the weight in pounds per bushel, adjusted for grain moisture level
at harvest. Biomass is measured as the weight of harvestable plant
material generated.
[0065] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof
that have the essential nature of a natural ribonucleotide in that
they hybridize, under stringent hybridization conditions, to
substantially the same nucleotide sequence as naturally occurring
nucleotides and/or allow translation into the same amino acid(s) as
the naturally occurring nucleotide(s). A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term
is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated
bases, to name just two examples, are polynucleotides as the term
is used herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term
polynucleotide as it is employed herein embraces such chemically,
enzymatically or metabolically modified forms of polynucleotides,
as well as the chemical forms of DNA and RNA characteristic of
viruses and cells, including inter alia, simple and complex
cells.
[0066] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0067] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells. Exemplary plant promoters
include, but are not limited to, those that are obtained from
plants, plant viruses, and bacteria which comprise genes expressed
in plant cells such Agrobacterium or Rhizobium. Examples are
promoters that preferentially initiate transcription in certain
tissues, such as leaves, roots, seeds, fibres, xylem vessels,
tracheids, or sclerenchyma. Such promoters are referred to as
"tissue-preferred." A "cell type" specific promoter primarily
drives expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" or
"regulatable" promoter is a promoter which is under environmental
control. Examples of environmental conditions that may affect
transcription by inducible promoters include anaerobic conditions
or the presence of light. Another type of promoter is a
developmentally regulated promoter, for example, a promoter that
drives expression during pollen development. Tissue preferred, cell
type specific, developmentally regulated, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter, which is active under most
environmental conditions.
[0068] As used herein "recombinant" includes reference to a cell or
vector that has been modified by the introduction of a heterologous
nucleic acid, or that the cell is derived from a cell so modified.
Thus, for example, recombinant cells express genes that are not
found in identical form within the native (non-recombinant) form of
the cell or express native genes that are otherwise abnormally
expressed, under expressed or not expressed at all as a result of
deliberate human intervention; or may have reduced or eliminated
expression of a native gene. The term "recombinant" as used herein
does not encompass the alteration of the cell or vector by
naturally occurring events (e.g., spontaneous mutation, natural
transformation/transduction/transposition) such as those occurring
without deliberate human intervention.
[0069] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements, which permit
transcription of a particular nucleic acid in a target cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed, and a promoter.
[0070] The terms "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogs of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0071] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 40% sequence identity, preferably 60-90% sequence identity,
and most preferably 100% sequence identity (i.e., complementary)
with each other.
[0072] The terms "stringent conditions" or "stringent hybridization
conditions" include reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences can be identified which can be up to 100% complementary
to the probe (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Optimally, the probe is approximately 500 nucleotides in
length, but can vary greatly in length from less than 500
nucleotides to equal to the entire length of the target
sequence.
[0073] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide or Denhardt's. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to lx SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times.SSC at 60 to 65.degree. C. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, (1984) Anal. Biochem.,
138:267-84: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61
(% form)-500/L; where M is the molarity of monovalent cations, % GC
is the percentage of guanosine and cytosine nucleotides in the DNA,
% form is the percentage of formamide in the hybridization
solution, and L is the length of the hybrid in base pairs. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of a complementary target sequence hybridizes to a
perfectly matched probe. T.sub.m is reduced by about 1.degree. C.
for each 1% of mismatching; thus, T.sub.m, hybridization and/or
wash conditions can be adjusted to hybridize to sequences of the
desired identity. For example, if sequences with >90% identity
are sought, the T.sub.m can be decreased 10.degree. C. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
and its complement at a defined ionic strength and pH. However,
severely stringent conditions can utilize a hybridization and/or
wash at 1, 2, 3 or 4.degree. C. lower than the thermal melting
point (T.sub.m); moderately stringent conditions can utilize a
hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than
the thermal melting point (T.sub.m); low stringency conditions can
utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or
20.degree. C. lower than the thermal melting point (T.sub.m). Using
the equation, hybridization and wash compositions, and desired
T.sub.m, those of ordinary skill will understand that variations in
the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a T.sub.m of less than 45.degree. C. (aqueous solution) or
32.degree. C. (formamide solution) it is preferred to increase the
SSC concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," Elsevier, N.Y. (1993); and Current
Protocols in Molecular Biology, chapter 2, Ausubel, et al., eds,
Greene Publishing and Wiley-Interscience, New York (1995). Unless
otherwise stated, in the present application high stringency is
defined as hybridization in 4.times.SSC, 5.times.Denhardt's (5 g
Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml
of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na
phosphate at 65.degree. C., and a wash in 0.1.times.SSC, 0.1% SDS
at 65.degree. C.
[0074] As used herein, "trait" refers to a physiological,
morphological, biochemical, or physical characteristic of a plant
or particular plant material or cell. In some instances, this
characteristic is visible to the human eye, such as seed or plant
size, or can be measured by biochemical techniques, such as
detecting the protein, starch, or oil content of seed or leaves, or
by observation of a metabolic or physiological process, e.g., by
measuring uptake of carbon dioxide, or by the observation of the
expression level of a gene or genes, e.g., by employing Northern
analysis, RT-PCR, microarray gene expression assays, or reporter
gene expression systems, or by agricultural observations such as
stress tolerance, yield, or pathogen tolerance.
[0075] As used herein, "transgenic plant" includes reference to a
plant, which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0076] As used herein, "transgenic seed" refers to a plant seed
whose genome has been altered by the incorporation of recombinant
DNA, e.g., by transformation as described herein. The term
"transgenic plant" is used to refer to the plant produced from an
original transformation event, or progeny from later generations or
crosses of a plant to a transformed plant, so long as the progeny
contains the recombinant DNA in its genome.
[0077] As used herein, "vector" includes reference to a nucleic
acid used in transfection of a host cell and into which can be
inserted a polynucleotide. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0078] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides
or polypeptides: (a) "reference sequence," (b) "comparison window,"
(c) "sequence identity," (d) "percentage of sequence identity," and
(e) "substantial identity."
[0079] As used herein, "reference sequence" is a defined sequence
used as a basis for sequence comparison. A reference sequence may
be a subset or the entirety of a specified sequence; for example,
as a segment of a full-length cDNA or gene sequence, or the
complete cDNA or gene sequence.
[0080] As used herein, "comparison window" means includes reference
to a contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e.,
gaps) compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length, and optionally can be 30, 40, 50, 100 or
longer. Those of skill in the art understand that to avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence a gap penalty is typically introduced and
is subtracted from the number of matches.
[0081] Methods of alignment of nucleotide and amino acid sequences
for comparison are well known in the art. The local homology
algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math
2:482, may conduct optimal alignment of sequences for comparison;
by the homology alignment algorithm (GAP) of Needleman and Wunsch,
(1970) J. Mol. Biol. 48:443-53; by the search for similarity method
(Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad.
Sci. USA 85:2444; by computerized implementations of these
algorithms, including, but not limited to: CLUSTAL in the PC/Gene
program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT,
BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Version 8 (available from Genetics Computer Group
(GCG.RTM. programs (Accelrys, Inc., San Diego, Calif.).). The
CLUSTAL program is well described by Higgins and Sharp, (1988) Gene
73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et
al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992)
Computer Applications in the Biosciences 8:155-65, and Pearson, et
al., (1994) Meth. Mol. Biol. 24:307-31. The preferred program to
use for optimal global alignment of multiple sequences is PileUp
(Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60 which is
similar to the method described by Higgins and Sharp, (1989) CABIOS
5:151-53 and hereby incorporated by reference). The BLAST family of
programs which can be used for database similarity searches
includes: BLASTN for nucleotide query sequences against nucleotide
database sequences; BLASTX for nucleotide query sequences against
protein database sequences; BLASTP for protein query sequences
against protein database sequences; TBLASTN for protein query
sequences against nucleotide database sequences; and TBLASTX for
nucleotide query sequences against nucleotide database sequences.
See, Current Protocols in Molecular Biology, Chapter 19, Ausubel et
al., eds., Greene Publishing and Wiley-Interscience, New York
(1995).
[0082] GAP uses the algorithm of Needleman and Wunsch, supra, to
find the alignment of two complete sequences that maximizes the
number of matches and minimizes the number of gaps. GAP considers
all possible alignments and gap positions and creates the alignment
with the largest number of matched bases and the fewest gaps. It
allows for the provision of a gap creation penalty and a gap
extension penalty in units of matched bases. GAP must make a profit
of gap creation penalty number of matches for each gap it inserts.
If a gap extension penalty greater than zero is chosen, GAP must,
in addition, make a profit for each gap inserted of the length of
the gap times the gap extension penalty. Default gap creation
penalty values and gap extension penalty values in Version 10 of
the Wisconsin Genetics Software Package are 8 and 2, respectively.
The gap creation and gap extension penalties can be expressed as an
integer selected from the group of integers consisting of from 0 to
100. Thus, for example, the gap creation and gap extension
penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,
50 or greater.
[0083] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA
89:10915).
[0084] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using the BLAST 2.0
suite of programs using default parameters (Altschul, et al.,
(1997) Nucleic Acids Res. 25:3389-402).
[0085] As those of ordinary skill in the art will understand, BLAST
searches assume that proteins can be modeled as random sequences.
However, many real proteins comprise regions of nonrandom
sequences, which may be homopolymeric tracts, short-period repeats,
or regions enriched in one or more amino acids. Such low-complexity
regions may be aligned between unrelated proteins even though other
regions of the protein are entirely dissimilar. A number of
low-complexity filter programs can be employed to reduce such
low-complexity alignments. For example, the SEG (Wooten and
Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Clayerie and
States, (1993) Comput. Chem. 17:191-201) low-complexity filters can
be employed alone or in combination.
[0086] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences, which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences, which differ by such conservative substitutions, are
said to have "sequence similarity" or "similarity." Means for
making this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17,
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0087] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0088] The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has between
50-100% sequence identity, preferably at least 50% sequence
identity, preferably at least 60% sequence identity, preferably at
least 70%, more preferably at least 80%, more preferably at least
90%, and most preferably at least 95%, compared to a reference
sequence using one of the alignment programs described using
standard parameters. One of skill will recognize that these values
can be appropriately adjusted to determine corresponding identity
of proteins encoded by two nucleotide sequences by taking into
account codon degeneracy, amino acid similarity, reading frame
positioning and the like. Substantial identity of amino acid
sequences for these purposes normally means sequence identity of
between 55-100%, preferably at least 55%, preferably at least 60%,
more preferably at least 70%, 80%, 90%, and most preferably at
least 95%.
[0089] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. The degeneracy of the genetic code
allows for many amino acids substitutions that lead to variety in
the nucleotide sequence that code for the same amino acid, hence it
is possible that the DNA sequence could code for the same
polypeptide but not hybridize to each other under stringent
conditions. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is that the polypeptide, which the first
nucleic acid encodes, is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0090] The terms "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with between 55-100%
sequence identity to a reference sequence preferably at least 55%
sequence identity, preferably 60% preferably 70%, more preferably
80%, most preferably at least 90% or 95% sequence identity to the
reference sequence over a specified comparison window. Preferably,
optimal alignment is conducted using the homology alignment
algorithm of Needleman and Wunsch, supra. An indication that two
peptide sequences are substantially identical is that one peptide
is immunologically reactive with antibodies raised against the
second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. In addition, a peptide can be
substantially identical to a second peptide when they differ by a
non-conservative change if the epitope that the antibody recognizes
is substantially identical. Peptides which are "substantially
similar" share sequences as noted above, except that residue
positions which are not identical may differ by conservative amino
acid changes.
[0091] Many agronomic traits can affect "yield", including without
limitation, plant height, pod number, pod position on the plant,
number of internodes, incidence of pod shatter, grain size,
efficiency of nodulation and nitrogen fixation, efficiency of
nutrient assimilation, resistance to biotic and abiotic stress,
carbon assimilation, plant architecture, resistance to lodging,
percent seed germination, seedling vigor, and juvenile traits.
Other traits that can affect yield include, efficiency of
germination (including germination in stressed conditions), growth
rate (including growth rate in stressed conditions), ear number,
seed number per ear, seed size, composition of seed (starch, oil,
protein) and characteristics of seed fill. Also of interest is the
generation of transgenic plants that demonstrate desirable
phenotypic properties that may or may not confer an increase in
overall plant yield. Such properties include enhanced plant
morphology, plant physiology or improved components of the mature
seed harvested from the transgenic plant.
[0092] As used herein, "increased yield" of a transgenic plant of
the present invention may be evidenced and measured in a number of
ways, including test weight, seed number per plant, seed weight,
seed number per unit area (i.e., seeds, or weight of seeds, per
acre), bushels per acre, tons per acre, kilo per hectare. For
example, maize yield may be measured as production of shelled corn
kernels per unit of production area, e.g., in bushels per acre or
metric tons per hectare, often reported on a moisture adjusted
basis, e.g., at 15.5% moisture. Increased yield may result from
improved utilization of key biochemical compounds, such as
nitrogen, phosphorous and carbohydrate, or from improved tolerance
to environmental stresses, such as cold, heat, drought, salt, and
attack by pests or pathogens. Trait-improving recombinant DNA may
also be used to provide transgenic plants having improved growth
and development, and ultimately increased yield, as the result of
modified expression of plant growth regulators or modification of
cell cycle or photosynthesis pathways.
Nucleic Acids
[0093] The present invention provides, inter alia, for the use of
isolated nucleic acids of RNA, DNA, homologs, paralogs and
orthologs and/or chimeras thereof, comprising a plant miRNA396 and
plant GRF encoding polynucleotide. This includes naturally
occurring as well as synthetic variants and homologs of the
sequences.
[0094] Sequences homologous, i.e., that share significant sequence
identity or similarity, to those provided herein derived from
maize, Arabidopsis thaliana, rice or from other plants of choice,
are also an aspect of the invention. Homologous sequences can be
derived from any plant including monocots and dicots and in
particular agriculturally important plant species, including but
not limited to, crops such as soybean, wheat, corn (maize), potato,
cotton, rice, rape, oilseed rape (including canola), sunflower,
alfalfa, clover, sugarcane, and turf; or fruits and vegetables,
such as banana, blackberry, blueberry, strawberry, and raspberry,
cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant,
grapes, honeydew, lettuce, mango, melon, onion, papaya, peas,
peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco,
tomato, tomatillo, watermelon, rosaceous fruits (such as apple,
peach, pear, cherry and plum) and vegetable brassicas (such as
broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi).
Other crops, including fruits and vegetables, whose phenotype can
be changed and which comprise homologous sequences include barley;
rye; millet; sorghum; currant; avocado; citrus fruits such as
oranges, lemons, grapefruit and tangerines, artichoke, cherries;
nuts such as the walnut and peanut; endive; leek; roots such as
arrowroot, beet, cassava, turnip, radish, yam, and sweet potato;
and beans. The homologous sequences may also be derived from woody
species, such pine, poplar and eucalyptus, or mint or other
labiates. In addition, homologous sequences may be derived from
plants that are evolutionarily-related to crop plants, but which
may not have yet been used as crop plants. Examples include deadly
nightshade (Atropa belladona), related to tomato; jimson weed
(Datura strommium), related to peyote; and teosinte (Zea species),
related to corn (maize).
Orthologs and Paralogs
[0095] Homologous sequences as described above can comprise
orthologous or paralogous sequences. Several different methods are
known by those of skill in the art for identifying and defining
these functionally homologous sequences. Three general methods for
defining orthologs and paralogs are described; an ortholog, paralog
or homolog may be identified by one or more of the methods
described below.
[0096] Orthologs and paralogs are evolutionarily related genes that
have similar sequence and similar functions. Orthologs are
structurally related genes in different species that are derived by
a speciation event. Paralogs are structurally related genes within
a single species that are derived by a duplication event.
[0097] Within a single plant species, gene duplication may result
in two copies of a particular gene, giving rise to two or more
genes with similar sequence and often similar function known as
paralogs. A paralog is therefore a similar gene formed by
duplication within the same species. Paralogs typically cluster
together or in the same Glade (a group of similar genes) when a
gene family phylogeny is analyzed using programs such as CLUSTAL
(Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins
et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar
genes can also be identified with pair-wise BLAST analysis (Feng
and Doolittle (1987) J. Mol. Evol. 25: 351-360).
[0098] Speciation, the production of new species from a parental
species, can also give rise to two or more genes with similar
sequence and similar function. These genes, termed orthologs, often
have an identical function within their host plants and are often
interchangeable between species without losing function. Because
plants have common ancestors, many genes in any plant species will
have a corresponding orthologous gene in another plant species.
Once a phylogenic tree for a gene family of one species has been
constructed using a program such as CLUSTAL (Thompson et al. (1994)
Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) supra)
potential orthologous sequences can be placed into the phylogenetic
tree and their relationship to genes from the species of interest
can be determined. Orthologous sequences can also be identified by
a reciprocal BLAST strategy. Once an orthologous sequence has been
identified, the function of the ortholog can be deduced from the
identified function of the reference sequence.
[0099] Orthologous genes from different organisms have highly
conserved functions, and very often essentially identical functions
(Lee et al. (2002) Genome Res. 12: 493-502; Remm et al. (2001) J.
Mol. Biol. 314: 1041-1052). Paralogous genes, which have diverged
through gene duplication, may retain similar functions of the
encoded proteins. In such cases, paralogs can be used
interchangeably with respect to certain embodiments of the instant
invention (for example, transgenic expression of a coding
sequence).
Variant Nucleotide Sequences in the Non-Coding Regions
[0100] The plant miRNA396 or GRF1/3 nucleotide sequences maybe used
to generate variant nucleotide sequences having the nucleotide
sequence of the 5'-untranslated region, 3'-untranslated region, or
promoter region that is approximately 70%, 75%, 80%, 85%, 90% and
95% identical to the original nucleotide sequence. These variants
are then associated with natural variation in the germplasm for
component traits related to nematode infection. The associated
variants are used as marker haplotypes to select for the desirable
traits.
Variant Amino Acid Sequences of Polypeptides
[0101] Variant amino acid sequences of the plant GRF polypeptides
are generated. For one example, one amino acid is altered.
Specifically, the open reading frames are reviewed to determine the
appropriate amino acid alteration. The selection of the amino acid
to change is made by consulting the protein alignment (with the
other orthologs and other gene family members from various
species). An amino acid is selected that is deemed not to be under
high selection pressure (not highly conserved) and which is rather
easily substituted by an amino acid with similar chemical
characteristics (i.e., similar functional side-chain). Using a
protein alignment, an appropriate amino acid can be changed. Once
the targeted amino acid is identified, the procedure outlined
herein is followed. Variants having about 70%, 75%, 80%, 85%, 90%
and 95% nucleic acid sequence identity are generated using this
method. These variants are then associated with natural variation
in the germplasm for component traits related to plant pathogen
infection. The associated variants are used as marker haplotypes to
select for the desirable traits.
[0102] The present invention also includes polynucleotides
optimized for expression in different organisms. For example, for
expression of the polynucleotide in a maize plant, the sequence can
be altered to account for specific codon preferences and to alter
GC content as according to Murray, et al, supra. Maize codon usage
for 28 genes from maize plants is listed in Table 4 of Murray, et
al., supra.
[0103] The plant miRNA398 or GRF1/GRF3 nucleic acids which may be
used for the present invention comprise isolated plant
polynucleotides which are inclusive of:
(a) a polynucleotide encoding an plant GRF1, or GRF3 polypeptide or
a micro RNA 396 and conservatively modified and polymorphic
variants thereof; (b) a polynucleotide having at least 70% sequence
identity with polynucleotides of (a); (c) complementary sequences
of polynucleotides of (a) or (b).
Construction of Nucleic Acids
[0104] The isolated nucleic acids of the present invention can be
made using (a) standard recombinant methods, (b) synthetic
techniques, or combinations thereof. In some embodiments, the
polynucleotides of the present invention will be cloned, amplified,
or otherwise constructed from a fungus or bacteria.
[0105] The nucleic acids may conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites may be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences may
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention--excluding the polynucleotide sequence--is
optionally a vector, adapter, or linker for cloning and/or
expression of a polynucleotide of the present invention. Additional
sequences may be added to such cloning and/or expression sequences
to optimize their function in cloning and/or expression, to aid in
isolation of the polynucleotide, or to improve the introduction of
the polynucleotide into a cell. Typically, the length of a nucleic
acid of the present invention less the length of its polynucleotide
of the present invention is less than 20 kilobase pairs, often less
than 15 kb, and frequently less than 10 kb. Use of cloning vectors,
expression vectors, adapters, and linkers is well known in the art.
Exemplary nucleic acids include such vectors as: M13, lambda ZAP
Express, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV,
pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4,
pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/-, pSG5, pBK,
pCR-Script, pET, pSPUTK, p3'SS, pGEM, pSK+/-, pGEX, pSPORTI and II,
pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44,
pOG45, pFRT.beta.GAL, pNEO.beta.GAL, pRS403, pRS404, pRS405,
pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambda
MOSElox. Optional vectors for the present invention, include but
are not limited to, lambda ZAP II, and pGEX. For a description of
various nucleic acids see, e.g., Stratagene Cloning Systems,
Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life
Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
Synthetic Methods for Constructing Nucleic Acids
[0106] The isolated nucleic acids used in the methods of the
present invention can also be prepared by direct chemical synthesis
by methods such as the phosphotriester method of Narang, et al.,
(1979) Meth. Enzymol. 68:90-9; the phosphodiester method of Brown,
et al., (1979) Meth. Enzymol. 68:109-51; the diethylphosphoramidite
method of Beaucage, et al., (1981) Tetra. Letts. 22(20):1859-62;
the solid phase phosphoramidite triester method described by
Beaucage, et al., supra, e.g., using an automated synthesizer,
e.g., as described in Needham-VanDevanter, et al., (1984) Nucleic
Acids Res. 12:6159-68; and, the solid support method of U.S. Pat.
No. 4,458,066. Chemical synthesis generally produces a single
stranded oligonucleotide. This may be converted into double
stranded DNA by hybridization with a complementary sequence or by
polymerization with a DNA polymerase using the single strand as a
template. One of skill will recognize that while chemical synthesis
of DNA is limited to sequences of about 100 bases, longer sequences
may be obtained by the ligation of shorter sequences.
UTRs and Codon Preference
[0107] In general, translational efficiency has been found to be
regulated by specific sequence elements in the 5' non-coding or
untranslated region (5' UTR) of the RNA. Positive sequence motifs
include translational initiation consensus sequences (Kozak, (1987)
Nucleic Acids Res. 15:8125) and the 5<G> 7 methyl GpppG RNA
cap structure (Drummond, et al., (1985) Nucleic Acids Res.
13:7375). Negative elements include stable intramolecular 5' UTR
stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG
sequences or short open reading frames preceded by an appropriate
AUG in the 5' UTR (Kozak, supra, Rao, et al., (1988) Mol. and Cell.
Biol. 8:284). Accordingly, the present invention provides 5' and/or
3' UTR regions for modulation of translation of heterologous coding
sequences.
[0108] Further, the polypeptide-encoding segments of the
polynucleotides of the present invention can be modified to alter
codon usage. Altered codon usage can be employed to alter
translational efficiency and/or to optimize the coding sequence for
expression in a desired host or to optimize the codon usage in a
heterologous sequence for expression in maize. Codon usage in the
coding regions of the polynucleotides of the present invention can
be analyzed statistically using commercially available software
packages such as "Codon Preference" available from the University
of Wisconsin Genetics Computer Group. See, Devereaux, et al.,
(1984) Nucleic Acids Res. 12:387-395); or MacVector 4.1 (Eastman
Kodak Co., New Haven, Conn.). Thus, the present invention provides
a codon usage frequency characteristic of the coding region of at
least one of the polynucleotides of the present invention. The
number of polynucleotides (3 nucleotides per amino acid) that can
be used to determine a codon usage frequency can be any integer
from 3 to the number of polynucleotides of the present invention as
provided herein. Optionally, the polynucleotides will be
full-length sequences. An exemplary number of sequences for
statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
Sequence Shuffling
[0109] The present invention also includes the use of sequence
shuffling using polynucleotides disclosed for the methods of the
present invention, and compositions resulting therefrom. Sequence
shuffling is described in PCT publication No. 96/19256. See also,
Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-9; and
Zhao, et al., (1998) Nature Biotech 16:258-61. Generally, sequence
shuffling provides a means for generating libraries of
polynucleotides having a desired characteristic, which can be
selected or screened for. Libraries of recombinant polynucleotides
are generated from a population of related sequence
polynucleotides, which comprise sequence regions, which have
substantial sequence identity and can be homologously recombined in
vitro or in vivo. The population of sequence-recombined
polynucleotides comprises a subpopulation of polynucleotides which
possess desired or advantageous characteristics and which can be
selected by a suitable selection or screening method. The
characteristics can be any property or attribute capable of being
selected for or detected in a screening system, and may include
properties of: an encoded protein, a transcriptional element, a
sequence controlling transcription, RNA processing, RNA stability,
chromatin conformation, translation, or other expression property
of a gene or transgene, a replicative element, a protein-binding
element, or the like, such as any feature which confers a
selectable or detectable property. In some embodiments, the
selected characteristic will be an altered K.sub.m and/or K.sub.cat
over the wild-type protein as provided herein. In other
embodiments, a protein or polynucleotide generated from sequence
shuffling will have a ligand binding affinity greater than the
non-shuffled wild-type polynucleotide. In yet other embodiments, a
protein or polynucleotide generated from sequence shuffling will
have an altered pH optimum as compared to the non-shuffled
wild-type polynucleotide. The increase in such properties can be at
least 110%, 120%, 130%, 140% or greater than 150% of the wild-type
value.
Recombinant Expression Cassettes
[0110] The present invention provides the use of recombinant
expression/transcription cassettes comprising a polynucleotide for
a plant microRNA396, or a GRF useful for the methods of the present
invention. A nucleic acid sequence coding for the desired
polynucleotide, for example a cDNA or a genomic sequence encoding a
polypeptide long enough to code for an active GRF protein, or for a
desired mircor RNA can be used to construct a recombinant
expression cassette which can be introduced into the desired host
cell. A recombinant expression cassette will typically comprise a
polynucleotide of the present invention operably linked to
transcriptional initiation regulatory sequences which will direct
the transcription of the polynucleotide in the intended host cell,
such as tissues of a transformed plant.
[0111] For example, plant expression vectors may include (1) a
cloned plant gene under the transcriptional control of 5' and 3'
regulatory sequences and (2) a dominant selectable marker. Such
plant expression vectors may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible or constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0112] A plant promoter fragment can be employed which will direct
expression of a polynucleotide of the present invention in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the 1'-
or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the
Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S.
Pat. No. 5,633,439), the Nos promoter, the rubisco promoter, the
GRP1-8 promoter, the 35S promoter from cauliflower mosaic virus
(CaMV), as described in Odell, et al., (1985) Nature 313:810-2;
rice actin (McElroy, et al., (1990) Plant Cell 163-171); ubiquitin
(Christensen, et al., (1992) Plant Mol. Biol. 12:619-632 and
Christensen, et al., (1992) Plant Mol. Biol. 18:675-89); pEMU
(Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,
et al., (1984) EMBO J. 3:2723-30); and maize H3 histone (Lepetit,
et al., (1992) Mol. Gen. Genet. 231:276-85; and Atanassvoa, et al.,
(1992) Plant Journal 2(3):291-300); ALS promoter, as described in
PCT Application No. WO 96/30530; and other transcription initiation
regions from various plant genes known to those of skill. For the
present invention ubiquitin is the preferred promoter for
expression in monocot plants.
[0113] Alternatively, the plant promoter can direct expression in a
specific tissue or may be otherwise under more precise
environmental or developmental control. Such promoters are referred
to here as "inducible" promoters. Environmental conditions that may
affect transcription by inducible promoters include pathogen
attack, anaerobic conditions, or the presence of light. Examples of
inducible promoters are the Adh1 promoter, which is inducible by
hypoxia or cold stress, the Hsp70 promoter, which is inducible by
heat stress, and the PPDK promoter, which is inducible by
light.
[0114] Examples of promoters under developmental control include
promoters that initiate transcription only, or preferentially, in
certain tissues, such as leaves, roots, fruit, seeds, or flowers.
The operation of a promoter may also vary depending on its location
in the genome. Thus, an inducible promoter may become fully or
partially constitutive in certain locations.
[0115] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from a variety of plant genes, or from T-DNA. The 3' end
sequence to be added can be derived from, for example, the nopaline
synthase or octopine synthase genes, or alternatively from another
plant gene, or less preferably from any other eukaryotic gene.
Examples of such regulatory elements include, but are not limited
to, 3' termination and/or polyadenylation regions such as those of
the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan,
et al., (1983) Nucleic Acids Res. 12:369-85); the potato proteinase
inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic Acids Res.
14:5641-50; and An, et al., (1989) Plant Cell 1:115-22); and the
CaMV 19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).
[0116] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates in the
cytosol. Inclusion of a spliceable intron in the transcription unit
in both plant and animal expression constructs has been shown to
increase gene expression at both the mRNA and protein levels up to
1000-fold (Buchman and Berg, (1988) Mol. Cell. Biol. 8:4395-4405;
Callis, et al., (1987) Genes Dev. 1:1183-200). Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of maize introns
Adh1-S intron 1, 2 and 6, the Bronze-1 intron are known in the art.
See generally, The Maize Handbook, Chapter 116, Freeling and
Walbot, eds., Springer, N.Y. (1994).
[0117] Plant signal sequences, including, but not limited to,
signal-peptide encoding DNA/RNA sequences which target proteins to
the extracellular matrix of the plant cell (Dratewka-Kos, et al.,
(1989) J. Biol. Chem. 264:4896-900), such as the Nicotiana
plumbaginifolia extension gene (DeLoose, et al., (1991) Gene
99:95-100); signal peptides which target proteins to the vacuole,
such as the sweet potato sporamin gene (Matsuka, et al., (1991)
Proc. Natl. Acad. Sci. USA 88:834) and the barley lectin gene
(Wilkins, et al., (1990) Plant Cell, 2:301-13); signal peptides
which cause proteins to be secreted, such as that of PRIb (Lind, et
al., (1992) Plant Mol. Biol. 18:47-53) or the barley alpha amylase
(BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol. 12:119, and
hereby incorporated by reference), or signal peptides which target
proteins to the plastids such as that of rapeseed enoyl-Acp
reductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202)
are useful in the invention.
[0118] The vector comprising the sequences from a plant
nicroRNA396, GRF1 or GRF3 will typically comprise a marker gene,
which confers a selectable phenotype on plant cells. Usually, the
selectable marker gene will encode antibiotic resistance, with
suitable genes including genes coding for resistance to the
antibiotic spectinomycin (e.g., the aada gene), the streptomycin
phosphotransferase (SPT) gene coding for streptomycin resistance,
the neomycin phosphotransferase (NPTII) gene encoding kanamycin or
geneticin resistance, the hygromycin phosphotransferase (HPT) gene
coding for hygromycin resistance, genes coding for resistance to
herbicides which act to inhibit the action of acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the
acetolactate synthase (ALS) gene containing mutations leading to
such resistance in particular the S4 and/or Hra mutations), genes
coding for resistance to herbicides which act to inhibit action of
glutamine synthase, such as phosphinothricin or basta (e.g., the
bar gene), or other such genes known in the art. The bar gene
encodes resistance to the herbicide basta, and the ALS gene encodes
resistance to the herbicide chlorsulfuron.
[0119] Typical vectors useful for expression of genes in higher
plants are well known in the art and include vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers, et al. (1987), Meth. Enzymol. 153:253-77.
These vectors are plant integrating vectors in that on
transformation, the vectors integrate a portion of vector DNA into
the genome of the host plant. Exemplary A. tumefaciens vectors
useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,
(1987) Gene 61:1-11, and Berger, et al., (1989) Proc. Natl. Acad.
Sci. USA, 86:8402-6. Another useful vector herein is plasmid
pBI101.2 that is available from CLONTECH Laboratories, Inc. (Palo
Alto, Calif.).
Expression of Proteins in Host Cells
[0120] Using the methods of the present invention, one may express
an miRNA396 or GRF protein in a recombinantly engineered cell such
as bacteria, yeast, insect, mammalian, or preferably plant cells.
The cells produce the protein in a non-natural condition (e.g., in
quantity, composition, location, and/or time), because they have
been genetically altered through human intervention to do so.
[0121] It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding a protein of the present
invention. No attempt to describe in detail the various methods
known for the expression of proteins in prokaryotes or eukaryotes
will be made.
[0122] In brief summary, the expression of isolated nucleic acids
encoding a GRF 1 or GRF3 protein or microRNA will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or inducible), followed by
incorporation into an expression vector. The vectors can be
suitable for replication and integration in either prokaryotes or
eukaryotes. Typical expression vectors contain transcription and
translation terminators, initiation sequences, and promoters useful
for regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a cloned
gene, it is desirable to construct expression vectors which
contain, at the minimum, a strong promoter, such as ubiquitin, to
direct transcription, a ribosome binding site for translational
initiation, and a transcription/translation terminator.
Constitutive promoters are classified as providing for a range of
constitutive expression. Thus, some are weak constitutive
promoters, and others are strong constitutive promoters. Generally,
by "weak promoter" is intended a promoter that drives expression of
a coding sequence at a low level. By "low level" is intended at
levels of about 1/10,000 transcripts to about 1/100,000 transcripts
to about 1/500,000 transcripts. Conversely, a "strong promoter"
drives expression of a coding sequence at a "high level," or about
1/10 transcripts to about 1/100 transcripts to about 1/1,000
transcripts.
[0123] One of skill would recognize that modifications could be
made to a GRF protein or MicroRNA without diminishing its
biological activity. Some modifications may be made to facilitate
the cloning, expression, or incorporation of the targeting molecule
into a fusion protein. Such modifications are well known to those
of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
Expression in Prokaryotes
[0124] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the beta
lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp)
promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057)
and the lambda derived P L promoter and N-gene ribosome binding
site (Shimatake, et al., (1981) Nature 292:128). The inclusion of
selection markers in DNA vectors transfected in E. coli is also
useful. Examples of such markers include genes specifying
resistance to ampicillin, tetracycline, or chloramphenicol.
[0125] The vector is selected to allow introduction of the gene of
interest into the appropriate host cell. Bacterial vectors are
typically of plasmid or phage origin. Appropriate bacterial cells
are infected with phage vector particles or transfected with naked
phage vector DNA. If a plasmid vector is used, the bacterial cells
are transfected with the plasmid vector DNA. Expression systems for
expressing a protein of the present invention are available using
Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-35;
Mosbach, et al., (1983) Nature 302:543-5). The pGEX-4T-1 plasmid
vector from Pharmacia is the preferred E. coli expression vector
for the present invention.
Expression in Eukaryotes
[0126] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, the present invention
can be expressed in these eukaryotic systems. In some embodiments,
transformed/transfected plant cells, as discussed infra, are
employed as expression systems for production of the proteins of
the instant invention.
[0127] Synthesis of heterologous proteins in yeast is well known.
Sherman, et al., (1982) Methods in Yeast Genetics, Cold Spring
Harbor Laboratory is a well-recognized work describing the various
methods available to produce the protein in yeast. Two widely
utilized yeasts for production of eukaryotic proteins are
Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g., Invitrogen).
Suitable vectors usually have expression control sequences, such as
promoters, including 3-phosphoglycerate kinase or alcohol oxidase,
and an origin of replication, termination sequences and the like as
desired.
[0128] A plant protein, once expressed, can be isolated from yeast
by lysing the cells and applying standard protein isolation
techniques to the lysates or the pellets. The monitoring of the
purification process can be accomplished by using Western blot
techniques or radioimmunoassay of other standard immunoassay
techniques.
[0129] The sequences encoding plant GRF proteins or miRNA396 can
also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian, insect, or
plant origin. Mammalian cell systems often will be in the form of
monolayers of cells although mammalian cell suspensions may also be
used. A number of suitable host cell lines capable of expressing
intact proteins have been developed in the art, and include the
HEK293, BHK21, and CHO cell lines. Expression vectors for these
cells can include expression control sequences, such as an origin
of replication, a promoter (e.g., the CMV promoter, a HSV tk
promoter or pgk (phosphoglycerate kinase) promoter), an enhancer
(Queen, et al., (1986) Immunol. Rev. 89:49), and necessary
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site), and transcriptional terminator sequences. Other
animal cells useful for production of proteins of the present
invention are available, for instance, from the American Type
Culture Collection Catalogue of Cell Lines and Hybridomas (7.sup.th
ed., 1992).
[0130] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth, and Drosophila cell lines such as a
Schneider cell line (see, e.g., Schneider, (1987) J. Embryol. Exp.
Morphol. 27:353-65).
[0131] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague et al., J. Virol. 45:773-81 (1983)).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors (Saveria-Campo, "Bovine
Papilloma Virus DNA a Eukaryotic Cloning Vector," in DNA Cloning: A
Practical Approach, vol. II, Glover, ed., IRL Press, Arlington,
Va., pp. 213-38 (1985)).
[0132] In addition, the plant GRF or miRNA396 gene placed in the
appropriate plant expression vector can be used to transform plant
cells. The polypeptide can then be isolated from plant callus or
the transformed cells can be used to regenerate transgenic plants.
Such transgenic plants can be harvested, and the appropriate
tissues (seed or leaves, for example) can be subjected to large
scale protein extraction and purification techniques.
Plant Transformation Methods
[0133] Numerous methods for introducing foreign genes into plants
are known and can be used to insert a plant miRNA396 or GRF
encoding polynucleotide into a plant host, including biological and
physical plant transformation protocols. See, e.g., Miki et al.,
"Procedure for Introducing Foreign DNA into Plants," in Methods in
Plant Molecular Biology and Biotechnology, Glick and Thompson,
eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). The methods
chosen vary with the host plant, and include chemical transfection
methods such as calcium phosphate, microorganism-mediated gene
transfer such as Agrobacterium (Horsch et al., Science 227:1229-31
(1985)), electroporation, micro-injection, and biolistic
bombardment.
[0134] Expression cassettes and vectors and in vitro culture
methods for plant cell or tissue transformation and regeneration of
plants are known and available. See, e.g., Gruber et al., "Vectors
for Plant Transformation," in Methods in Plant Molecular Biology
and Biotechnology, supra, pp. 89-119.
[0135] The isolated plant polynucleotides or polypeptides may be
introduced into the plant by one or more techniques typically used
for direct delivery into cells. Such protocols may vary depending
on the type of organism, cell, plant or plant cell, i.e. monocot or
dicot, targeted for gene modification. Suitable methods of
transforming plant cells include microinjection (Crossway, et al.,
(1986) Biotechniques 4:320-334; and U.S. Pat. No. 6,300,543),
electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606, direct gene transfer (Paszkowski et al., (1984) EMBO
J. 3:2717-2722), and ballistic particle acceleration (see, for
example, Sanford, et al., U.S. Pat. No. 4,945,050; WOk 91/10725;
and McCabe, et al., (1988) Biotechnology 6:923-926). Also see,
Tomes, et al., "Direct DNA Transfer into Intact Plant Cells Via
Microprojectile Bombardment". pp. 197-213 in Plant Cell, Tissue and
Organ Culture, Fundamental Methods. eds. O. L. Gamborg & G. C.
Phillips. Springer-Verlag Berlin Heidelberg New York, 1995; U.S.
Pat. No. 5,736,369 (meristem); Weissinger, et al., (1988) Ann. Rev.
Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and
Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.
87:671-674 (soybean); Datta, et al., (1990) Biotechnology 8:736-740
(rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563
(maize); WO 91/10725 (maize); Klein, et al., (1988) Plant Physiol.
91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839;
and Gordon-Kamm, et al., (1990) Plant Cell 2:603-618 (maize);
Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London)
311:763-764; Bytebierm, et al., (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet, et al., (1985) In The
Experimental Manipulation of Ovule Tissues, ed. G. P. Chapman, et
al., pp. 197-209. Longman, N.Y. (pollen); Kaeppler, et al., (1990)
Plant Cell Reports 9:415-418; and Kaeppler, et al., (1992) Theor.
Appl. Genet. 84:560-566 (whisker-mediated transformation); U.S.
Pat. No. 5,693,512 (sonication); D'Halluin, et al., (1992) Plant
Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell
Reports 12:250-255; and Christou and Ford, (1995) Annals of Botany
75:407-413 (rice); Osjoda, et al., (1996) Nature Biotech.
14:745-750; Agrobacterium mediated maize transformation (U.S. Pat.
No. 5,981,840); silicon carbide whisker methods (Frame, et al.,
(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)
Physiologia Plantarum 93:19-24); sonication methods (Bao, et al.,
(1997) Ultrasound in Medicine & Biology 23:953-959; Finer and
Finer, (2000) Lett Appl Microbiol. 30:406-10; Amoah, et al., (2001)
J Exp Bot 52:1135-42); polyethylene glycol methods (Krens, et al.,
(1982) Nature 296:72-77); protoplasts of monocot and dicot cells
can be transformed using electroporation (Fromm, et al., (1985)
Proc. Natl. Acad. Sci. USA 82:5824-5828) and microinjection
(Crossway, et al., (1986) Mol. Gen. Genet. 202:179-185); all of
which are herein incorporated by reference.
Agrobacterium-Mediated Transformation
[0136] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria, which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of plants. See, e.g., Kado, (1991) Crit. Rev. Plant
Sci. 10:1. Descriptions of the Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are provided in
Gruber, et al., supra; Miki, et al., supra; and Moloney, et al.,
(1989) Plant Cell Reports 8:238.
[0137] Similarly, the gene can be inserted into the T-DNA region of
a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes,
respectively. Thus, expression cassettes can be constructed as
above, using these plasmids. Many control sequences are known which
when coupled to a heterologous coding sequence and transformed into
a host organism show fidelity in gene expression with respect to
tissue/organ specificity of the original coding sequence. See,
e.g., Benfey and Chua, (1989) Science 244:174-81. Particularly
suitable control sequences for use in these plasmids are promoters
for constitutive leaf-specific expression of the gene in the
various target plants. Other useful control sequences include a
promoter and terminator from the nopaline synthase gene (NOS). The
NOS promoter and terminator are present in the plasmid pARC2,
available from the American Type Culture Collection and designated
ATCC 67238. If such a system is used, the virulence (vir) gene from
either the Ti or Ri plasmid must also be present, either along with
the T-DNA portion, or via a binary system where the vir gene is
present on a separate vector. Such systems, vectors for use
therein, and methods of transforming plant cells are described in
U.S. Pat. No. 4,658,082; U.S. Pat. No. 913,914, filed Oct. 1, 1986,
as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993; and
Simpson, et al., (1986) Plant Mol. Biol. 6:403-15 (also referenced
in the '306 patent); all incorporated by reference in their
entirety.
[0138] Once constructed, these plasmids can be placed into A.
rhizogenes or A. tumefaciens and these vectors used to transform
cells of plant species, which are ordinarily susceptible to
Fusarium or Alternaria infection. Several other transgenic plants
are also contemplated by the present invention including but not
limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage,
banana, coffee, celery, tobacco, cowpea, cotton, melon and pepper.
The selection of either A. tumefaciens or A. rhizogenes will depend
on the plant being transformed thereby. In general A. tumefaciens
is the preferred organism for transformation. Most dicotyledonous
plants, some gymnosperms, and a few monocotyledonous plants (e.g.,
certain members of the Liliales and Arales) are susceptible to
infection with A. tumefaciens. A. rhizogenes also has a wide host
range, embracing most dicots and some gymnosperms, which includes
members of the Leguminosae, Compositae, and Chenopodiaceae. Monocot
plants can now be transformed with some success. European Patent
Application No. 604 662 A1 discloses a method for transforming
monocots using Agrobacterium. European Application No. 672 752 A1
discloses a method for transforming monocots with Agrobacterium
using the scutellum of immature embryos. Ishida, et al., discuss a
method for transforming maize by exposing immature embryos to A.
tumefaciens (Nature Biotechnology 14:745-50 (1996)).
[0139] Once transformed, these cells can be used to regenerate
transgenic plants. For example, whole plants can be infected with
these vectors by wounding the plant and then introducing the vector
into the wound site. Any part of the plant can be wounded,
including leaves, stems and roots. Alternatively, plant tissue, in
the form of an explant, such as cotyledonary tissue or leaf disks,
can be inoculated with these vectors, and cultured under
conditions, which promote plant regeneration. Roots or shoots
transformed by inoculation of plant tissue with A. rhizogenes or A.
tumefaciens, containing the gene coding for the fumonisin
degradation enzyme, can be used as a source of plant tissue to
regenerate fumonisin-resistant transgenic plants, either via
somatic embryogenesis or organogenesis. Examples of such methods
for regenerating plant tissue are disclosed in Shahin, (1985)
Theor. Appl. Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et
al., supra; and U.S. Pat. Nos. 913,913 and 913,914, both filed Oct.
1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16,
1993, the entire disclosures therein incorporated herein by
reference.
Direct Gene Transfer
[0140] Despite the fact that the host range for
Agrobacterium-mediated transformation is broad, some major cereal
crop species and gymnosperms have generally been recalcitrant to
this mode of gene transfer, even though some success has recently
been achieved in rice (Hiei, et al., (1994) The Plant Journal
6:271-82). Several methods of plant transformation, collectively
referred to as direct gene transfer, have been developed as an
alternative to Agrobacterium-mediated transformation.
[0141] A generally applicable method of plant transformation is
microprojectile-mediated transformation, where DNA is carried on
the surface of microprojectiles measuring about 1 to 4 .mu.m. The
expression vector is introduced into plant tissues with a biolistic
device that accelerates the microprojectiles to speeds of 300 to
600 m/s which is sufficient to penetrate the plant cell walls and
membranes (Sanford, et al., (1987) Part. Sci. Technol. 5:27;
Sanford, (1988) Trends Biotech 6:299; Sanford, (1990) Physiol.
Plant 79:206; and Klein, et al., (1992) Biotechnology 10:268).
[0142] Another method for physical delivery of DNA to plants is
sonication of target cells as described in Zang, et al., (1991)
BioTechnology 9:996. Alternatively, liposome or spheroplast fusions
have been used to introduce expression vectors into plants. See,
e.g., Deshayes, et al., (1985) EMBO J. 4:2731; and Christou, et
al., (1987) Proc. Natl. Acad. Sci. USA 84:3962. Direct uptake of
DNA into protoplasts using CaCl.sub.2 precipitation, polyvinyl
alcohol, or poly-L-ornithine has also been reported. See, e.g.,
Hain, et al., (1985) Mol. Gen. Genet. 199:161; and Draper, et al.,
(1982) Plant Cell Physiol. 23:451. Electroporation of protoplasts
and whole cells and tissues has also been described. See, e.g.,
Donn, et al., (1990) Abstracts of the VIIth Int'l. Congress on
Plant Cell and Tissue Culture IAPTC, A2-38, p. 53; D'Halluin, et
al., (1992) Plant Cell 4:1495-505; and Spencer, et al., (1994)
Plant Mol. Biol. 24:51-61.
[0143] Some embodiments may involve the improvement in nematode
tolerance by modulating the expression of a plant miRNA396,
GRF1/GRF3 in a way that decreases the activity/expression of the
protein or mircroRNA.
Reducing the Activity of a Plant GRF Polypeptide or MicroRNA
[0144] Methods are also provided to reduce or eliminate the
activity of a plant GRF Polypeptide or MicroRNA by transforming a
plant cell with an expression cassette that expresses a
polynucleotide that inhibits the expression of the plant
polypeptide or microRNA. The polynucleotide may inhibit the
expression of the plant a plant GRF Polypeptide or MicroRNA
directly, by preventing transcription or translation of the plant
messenger RNA, or indirectly, by encoding a polypeptide that
inhibits the transcription or translation of an plant a plant GRF
Polypeptide or MicroRNA gene encoding an plant a plant GRF
Polypeptide or MicroRNA. Methods for inhibiting or eliminating the
expression of a gene in a plant are well known in the art, and any
such method may be used in the present invention to inhibit the
expression of the plant a plant GRF Polypeptide or MicroRNA. Many
methods may be used to reduce or eliminate the activity of GRF
polypeptides. In addition, more than one method may be used to
reduce the activity of a plant GRF Polypeptide or MicroRNA.
1. Polynucleotide-Based Methods:
[0145] In some embodiments of the present invention, a plant is
transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of a plant
GRF Polypeptide or MicroRNA of the invention. For example, for the
purposes of the present invention, an expression cassette capable
of expressing a polynucleotide that inhibits the expression of at
least one a plant GRF Polypeptide or MicroRNA is an expression
cassette capable of producing an RNA molecule that inhibits the
transcription and/or translation of at least one plant a plant GRF
Polypeptide or MicroRNA of the invention.
[0146] Examples of polynucleotides that inhibit the expression of a
plant GRF Polypeptide or MicroRNA include sense
suppression/cosuppresion. In cosuppression, an expression cassette
is designed to express an RNA molecule corresponding to all or part
of a messenger RNA encoding a plant GRF Polypeptide or MicroRNA in
the "sense" orientation. Over expression of the RNA molecule can
result in reduced expression of the native gene. The polynucleotide
used for cosuppression may correspond to all or part of the
sequence encoding the a plant GRF Polypeptide or MicroRNA, all or
part of the 5' and/or 3' untranslated region of a plant GRF
Polypeptide or MicroRNA transcript, or all or part of both the
coding sequence and the untranslated regions of a transcript
encoding a plant GRF Polypeptide or MicroRNA. In some embodiments
where the polynucleotide comprises all or part of the coding region
for the plant a plant GRF Polypeptide or MicroRNA, the expression
cassette is designed to eliminate the start codon of the
polynucleotide so that no protein product will be translated.
[0147] In some embodiments of the invention, inhibition of the
expression of a plant GRF Polypeptide or MicroRNA may be obtained
by antisense suppression. For antisense suppression, the expression
cassette is designed to express an RNA molecule complementary to
all or part of a messenger RNA encoding the a plant GRF Polypeptide
or MicroRNA. Over expression of the antisense RNA molecule can
result in reduced expression of the native gene. The polynucleotide
for use in antisense suppression may correspond to all or part of
the complement of the sequence encoding the a plant GRF Polypeptide
or MicroRNA, all or part of the complement of the 5' and/or 3'
untranslated region of the plant a plant GRF Polypeptide or
MicroRNA transcript, or all or part of the complement of both the
coding sequence and the untranslated regions of a transcript
encoding the plant a plant GRF Polypeptide or MicroRNA. In
addition, the antisense polynucleotide may be fully complementary
(i.e., 100% identical to the complement of the target sequence) or
partially complementary (i.e., less than 100% identical to the
complement of the target sequence) to the target sequence.
[0148] In some embodiments of the invention, inhibition of the
expression of a plant GRF Polypeptide or MicroRNA may be obtained
by double-stranded RNA (dsRNA) interference. For dsRNA
interference, a sense RNA molecule like that described above for
cosuppression and an antisense RNA molecule that is fully or
partially complementary to the sense RNA molecule are expressed in
the same cell, resulting in inhibition of the expression of the
corresponding endogenous messenger RNA.
[0149] Expression of the sense and antisense molecules can be
accomplished by designing the expression cassette to comprise both
a sense sequence and an antisense sequence. Alternatively, separate
expression cassettes may be used for the sense and antisense
sequences. Multiple plant lines transformed with the dsRNA
interference expression cassette or expression cassettes are then
screened to identify plant lines that show the greatest inhibition
of plant a plant GRF Polypeptide or MicroRNA. Methods for using
dsRNA interference to inhibit the expression of endogenous plant
genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad.
Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol.
129:1732-1743, and WO 99/49029, WO 99/53050, WO 99/61631, and WO
00/49035; each of which is herein incorporated by reference.
[0150] In some embodiments of the invention, inhibition of the
expression of a plant GRF Polypeptide or MicroRNA may be obtained
by hairpin RNA (hpRNA) interference or intron-containing hairpin
RNA (ihpRNA) interference. These methods are highly efficient at
inhibiting the expression of endogenous genes. See, Waterhouse and
Helliwell, (2003) Nat. Rev. Genet. 4:29-38 and the references cited
therein.
[0151] For hpRNA interference, the expression cassette is designed
to express an RNA molecule that hybridizes with itself to form a
hairpin structure that comprises a single-stranded loop region and
a base-paired stem. The base-paired stem region comprises a sense
sequence corresponding to all or part of the endogenous messenger
RNA encoding the gene whose expression is to be inhibited, and an
antisense sequence that is fully or partially complementary to the
sense sequence. Alternatively, the base-paired stem region may
correspond to a portion of a promoter sequence controlling
expression of the gene to be inhibited. Thus, the base-paired stem
region of the molecule generally determines the specificity of the
RNA interference. hpRNA molecules are highly efficient at
inhibiting the expression of endogenous genes, and the RNA
interference they induce is inherited by subsequent generations of
plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl.
Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant
Physiol. 129:1723-1731; and Waterhouse and Helliwell, (2003) Nat.
Rev. Genet. 4:29-38. Methods for using hpRNA interference to
inhibit or silence the expression of genes are described, for
example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci.
USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.
129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.
4:29-38; Pandolfini et al., BMC Biotechnology 3:7, and U.S. Patent
Publication No. 2003/0175965; each of which is herein incorporated
by reference. A transient assay for the efficiency of hpRNA
constructs to silence gene expression in vivo has been described by
Panstruga, et al., (2003) Mol. Biol. Rep. 30:135-140, herein
incorporated by reference.
[0152] For ihpRNA, the interfering molecules have the same general
structure as for hpRNA, but the RNA molecule additionally comprises
an intron that is capable of being spliced in the cell in which the
ihpRNA is expressed. The use of an intron minimizes the size of the
loop in the hairpin RNA molecule following splicing, and this
increases the efficiency of interference. See, for example, Smith,
et al., (2000) Nature 407:319-320. In fact, Smith, et al., show
100% suppression of endogenous gene expression using
ihpRNA-mediated interference. Methods for using ihpRNA interference
to inhibit the expression of endogenous plant genes are described,
for example, in Smith, et al., (2000) Nature 407:319-320; Wesley,
et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)
Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003)
Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods
30:289-295, and U.S. Patent Publication No. 2003/0180945, each of
which is herein incorporated by reference.
[0153] The expression cassette for hpRNA interference may also be
designed such that the sense sequence and the antisense sequence do
not correspond to an endogenous RNA. In this embodiment, the sense
and antisense sequence flank a loop sequence that comprises a
nucleotide sequence corresponding to all or part of the endogenous
messenger RNA of the target gene. Thus, it is the loop region that
determines the specificity of the RNA interference. See, for
example, WO 02/00904; Mette, et al., (2000) EMBO J. 19:5194-5201;
Matzke, et al., (2001) Curr. Opin. Genet. Devel. 11:221-227;
Scheid, et al., (2002) Proc. Natl. Acad. Sci., USA 99:13659-13662;
Aufsaftz, et al., (2002) Proc. Nat'l. Acad. Sci. 99(4):16499-16506;
Sijen, et al., Curr. Biol. (2001) 11:436-440), herein incorporated
by reference.
[0154] Amplicon expression cassettes comprise a plant virus-derived
sequence that contains all or part of the target gene but generally
not all of the genes of the native virus. The viral sequences
present in the transcription product of the expression cassette
allow the transcription product to direct its own replication. The
transcripts produced by the amplicon may be either sense or
antisense relative to the target sequence. Methods of using
amplicons to inhibit the expression of endogenous plant genes are
described, for example, in Angell and Baulcombe, (1997) EMBO J.
16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362, and
U.S. Pat. No. 6,635,805, each of which is herein incorporated by
reference.
[0155] In some embodiments, the polynucleotide expressed by the
expression cassette of the invention is catalytic RNA or has
ribozyme activity specific for the messenger RNA of the plant
miRNA396 or GRF polypeptide. Thus, the polynucleotide causes the
degradation of the endogenous messenger RNA, resulting in reduced
expression of the plant GRF polypeptide or miRNA396. This method is
described, for example, in U.S. Pat. No. 4,987,071, herein
incorporated by reference.
[0156] In some embodiments of the invention, inhibition of the
expression of a plant GRF Polypeptide or MicroRNA activity may be
obtained by RNA interference by expression of a gene encoding a
micro RNA (miRNA). miRNAs are regulatory agents consisting of about
22 ribonucleotides. miRNA are highly efficient at inhibiting the
expression of endogenous genes. See, for example Javier, et al.,
(2003) Nature 425:257-263, herein incorporated by reference.
[0157] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous miRNA
gene. The miRNA gene encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to
another endogenous gene (target sequence). miRNA molecules are
highly efficient at inhibiting the expression of endogenous genes,
and the RNA interference they induce is inherited by subsequent
generations of plants.
2. Polypeptide-Based Inhibition of Gene Expression
[0158] In one embodiment, the polynucleotide encodes a zinc finger
protein that binds to a gene encoding a plant GRF Polypeptide or
MicroRNA, resulting in reduced expression of the gene. In
particular embodiments, the zinc finger protein binds to a
regulatory region a plant GRF Polypeptide or MicroRNA gene. In
other embodiments, the zinc finger protein binds to a messenger RNA
encoding a plant GRF Polypeptide or MicroRNA and prevents its
translation. Methods of selecting sites for targeting by zinc
finger proteins have been described, for example, in U.S. Pat. No.
6,453,242, and methods for using zinc finger proteins to inhibit
the expression of genes in plants are described, for example, in
U.S. Patent Publication No. 2003/0037355; each of which is herein
incorporated by reference.
3. Polypeptide-Based Inhibition of Protein Activity
[0159] In some embodiments of the invention, the polynucleotide
encodes an antibody that binds to at least one a plant GRF
Polypeptide or MicroRNA, and reduces the activity of the a plant
GRF Polypeptide or MicroRNA. In another embodiment, the binding of
the antibody results in increased turnover of the antibody-GRF
Polypeptide or MicroRNA complex by cellular quality control
mechanisms. The expression of antibodies in plant cells and the
inhibition of molecular pathways by expression and binding of
antibodies to proteins in plant cells are well known in the art.
See, for example, Conrad and Sonnewald, (2003) Nature Biotech.
21:35-36, incorporated herein by reference.
4. Gene Disruption
[0160] In some embodiments of the present invention, the activity
of a plant GRF Polypeptide or MicroRNA is reduced or eliminated by
disrupting the gene encoding a plant GRF Polypeptide or MicroRNA.
The gene encoding the plant a plant GRF Polypeptide or MicroRNA may
be disrupted by any method known in the art. For example, in one
embodiment, the gene is disrupted by transposon tagging. In another
embodiment, the gene is disrupted by mutagenizing plants using
random or targeted mutagenesis, and selecting for plants that have
increased nematode tolerance.
[0161] i. Transposon Tagging
[0162] In one embodiment of the invention, transposon tagging is
used to reduce or eliminate a plant GRF Polypeptide or MicroRNA
activity of one or more plant GRF Polypeptides or MicroRNA
polypeptides. Transposon tagging comprises inserting a transposon
within an endogenous plant a plant GRF Polypeptide or MicroRNA gene
to reduce or eliminate expression of the plant a plant GRF
Polypeptide or MicroRNA.
[0163] In this embodiment, the expression of one or more a plant
GRF Polypeptide or MicroRNA is reduced or eliminated by inserting a
transposon within a regulatory region or coding region of the gene
encoding a plant GRF Polypeptide or MicroRNA. A transposon that is
within an exon, intron, 5' or 3' untranslated sequence, a promoter,
or any other regulatory sequence of a plant GRF Polypeptide or
MicroRNA gene may be used to reduce or eliminate the expression
and/or activity of the encoded a plant GRF Polypeptide or
MicroRNA.
[0164] Methods for the transposon tagging of specific genes in
plants are well known in the art. See, for example, Maes, et al.,
(1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS
Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J.
22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot,
(2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000)
Nucleic Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics
153:1919-1928). In addition, the TUSC process for selecting Mu
insertions in selected genes has been described in Bensen, et al.,
(1995) Plant Cell 7:75-84; Mena, et al., (1996) Science
274:1537-1540; and U.S. Pat. No. 5,962,764; each of which is herein
incorporated by reference.
[0165] ii. Mutant Plants with Reduced
Transcription/Translation/Activity
[0166] Additional methods for decreasing or eliminating the
expression of endogenous genes in plants are also known in the art
and can be similarly applied to the instant invention. These
methods include other forms of mutagenesis, such as ethyl
methanesulfonate-induced mutagenesis, deletion mutagenesis, and
fast neutron deletion mutagenesis used in a reverse genetics sense
(with PCR) to identify plant lines in which the endogenous gene has
been deleted. For examples of these methods see, Ohshima, et al.,
(1998) Virology 243:472-481; Okubara, et al., (1994) Genetics
137:867-874; and Quesada, et al., (2000) Genetics 154:421-436; each
of which is herein incorporated by reference. In addition, a fast
and automatable method for screening for chemically induced
mutations, TILLING (Targeting Induced Local Lesions In Genomes),
using denaturing HPLC or selective endonuclease digestion of
selected PCR products is also applicable to the instant invention.
See, McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein
incorporated by reference.
[0167] Mutations that impact gene expression or that interfere with
the function of the encoded protein are well known in the art.
Insertional mutations in gene exons usually result in null-mutants.
Mutations in conserved residues are particularly effective in
inhibiting the activity of the encoded protein. Conserved residues
of plant GRF polypeptides and/or miRNA396 suitable for mutagenesis
with the goal to eliminate activity have been described. Such
mutants can be isolated according to well-known procedures, and
mutations in different loci can be stacked by genetic crossing.
See, for example, Gruis, et al., (2002) Plant Cell
14:2863-2882.
[0168] The methods of the invention provides for improved plant
tolerance to nematode infection. This performance may be
demonstrated in a number of ways including the following.
Improved or Modulated Root Development in Nematode Infected
Plants
[0169] Methods for improving tolerance to nematode infection and
root development in a plant are provided. By "modulating root
development" is intended any alteration in the development of the
plant root under nematode infection when compared to a control
plant. Such alterations in root development include, but are not
limited to, alterations in the growth rate of the primary root, the
fresh root weight, the extent of lateral and adventitious root
formation, the vasculature system, meristem development, or radial
expansion.
[0170] The methods comprise modulating the level and/or activity of
a miRNA396, GRF1 or GRF3 and their interaction in the plant. In one
method, a plant miRNA396 sequence expression construct is provided
to the plant. In other methods, root development is modulated by
increasing the level or activity of the GRF proteins that interact
with miRNA396 in the plant. A change in plant GRF activity can
result in at least one or more of the following alterations to root
development, including, but not limited to, alterations in root
biomass and length when the plant is grown under nematode
infection.
[0171] As used herein, "root growth" encompasses all aspects of
growth of the different parts that make up the root system at
different stages of its development in both monocotyledonous and
dicotyledonous plants. It is to be understood that enhanced root
growth can result from enhanced growth of one or more of its parts
including the primary root, lateral roots, adventitious roots,
etc.
[0172] Methods of measuring such developmental alterations in the
root system are known in the art. See, for example, U.S.
Application No. 2003/0074698 and Werner, et al., (2001) PNAS
18:10487-10492, both of which are herein incorporated by
reference.
As discussed above, one of skill will recognize the appropriate
promoter to use to modulate root development in the plant.
Exemplary promoters for this embodiment include constitutive
promoters and root-preferred promoters. Exemplary root-preferred
promoters have been disclosed elsewhere herein.
[0173] Stimulating root growth and increasing root mass in the
presence of nematode infection by increasing the activity and/or
level of miRNA396 or its targets such as the GRF proteins also
finds use in improving the standability of a plant. The term
"resistance to lodging" or "standability" refers to the ability of
a plant to fix itself to the soil. For plants with an erect or
semi-erect growth habit, this term also refers to the ability to
maintain an upright position under adverse (environmental)
conditions. This trait relates to the size, depth and morphology of
the root system. Furthermore, higher root biomass production has a
direct effect on the yield and an indirect effect of production of
compounds produced by root cells or transgenic root cells or cell
cultures of said transgenic root cells.
Modulating Shoot and Leaf Development in Nematode Infected
Plants
[0174] Methods are also provided for modulating shoot and leaf
development in a plant, particularly under nematode infection. By
"modulating shoot and/or leaf development" is intended any
alteration in the development of the plant shoot and/or leaf in
nematode infection. Such alterations in shoot and/or leaf
development include, but are not limited to, alterations in shoot
meristem development, in leaf number, leaf size, leaf and stem
vasculature, internode length, and leaf senescence. As used herein,
"leaf development" and "shoot development" encompasses all aspects
of growth of the different parts that make up the leaf system and
the shoot system, respectively, at different stages of their
development, both in monocotyledonous and dicotyledonous plants.
Methods for measuring such developmental alterations in the shoot
and leaf system are known in the art. See, for example, Werner, et
al., (2001) PNAS 98:10487-10492 and U.S. Application No.
2003/0074698, each of which is herein incorporated by
reference.
[0175] The method for modulating shoot and/or leaf development in a
plant in nematode infected conditions comprises increasing the
activity and/or level of plant mrRNA396 or its target GRF proteins.
In one embodiment, the plant nucleotide sequences can be provided
by introducing into the plant a polynucleotide comprising an plant
expression construct, expressing the same, and thereby modifying
shoot and/or leaf development in nematode infected plants. In other
embodiments, the plant expression nucleotide construct introduced
into the plant is stably incorporated into the genome of the
plant.
[0176] An increase in plant tolerance to nematode infection can
result in at least one or more of the following alterations in
shoot and/or leaf development under nematode infection when
compared to a nonmodified plant, including, but not limited to,
changes in leaf number, altered leaf surface, altered vasculature,
internodes and plant growth, and alterations in leaf senescence,
when compared to a control plant in the same conditions.
[0177] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate shoot and leaf development
of the plant. Exemplary promoters for this embodiment include
constitutive promoters, shoot-preferred promoters, shoot
meristem-preferred promoters, and leaf-preferred promoters.
Exemplary promoters have been disclosed elsewhere herein.
Method of Use for Plant miRNA, and/or GRF Polynucleotides in
Combination with Other Phenotype Changing Polynucleotides
[0178] The nucleotides, expression cassettes and methods disclosed
herein are useful in regulating expression of any heterologous
nucleotide sequence in a host plant in order to vary the phenotype
of a plant. Various other changes in phenotype are of interest
including modifying the fatty acid composition in a plant, altering
the amino acid content of a plant, altering a plant's stress
tolerance, and the like. These results can be achieved by providing
expression of heterologous products or increased expression of
endogenous products in plants. Alternatively, the results can be
achieved by providing for a reduction of expression of one or more
endogenous products, particularly enzymes or cofactors in the
plant. These changes result in a change in phenotype of the
transformed plant.
[0179] Genes of interest are reflective of the commercial markets
and interests of those involved in the development of the crop.
Crops and markets of interest change, and as developing nations
open up world markets, new crops and technologies will emerge also.
In addition, as our understanding of agronomic traits and
characteristics such as yield and heterosis increase, the choice of
genes for transformation will change accordingly. General
categories of genes of interest include, for example, those genes
involved in information, such as zinc fingers, those involved in
communication, such as kinases, and those involved in housekeeping,
such as heat shock proteins. More specific categories of
transgenes, for example, include genes encoding important traits
for agronomics, insect resistance, disease resistance, herbicide
resistance, sterility, grain characteristics, and commercial
products. Genes of interest include, generally, those involved in
oil, starch, carbohydrate, or nutrient metabolism as well as those
affecting kernel size, sucrose loading, and the like.
[0180] In certain embodiments the plant miRNA/GRF nucleic acid
sequences of can be used in combination ("stacked") with other
polynucleotide sequences of interest in order to create plants with
a desired phenotype. The combinations generated can include
multiple copies of any one or more of the polynucleotides of
interest. The polynucleotides of the present invention may be
stacked with any gene or combination of genes to produce plants
with a variety of desired trait combinations, including but not
limited to traits desirable for animal feed such as high oil genes
(e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,
hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and
5,703,049); barley high lysine (Williamson, et al., (1987) Eur. J.
Biochem. 165:99-106; and WO 98/20122); and high methionine proteins
(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et
al., (1988) Gene 71:359; and Musumura, et al., (1989) Plant Mol.
Biol. 12:123)); increased digestibility (e.g., modified storage
proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7,
2001); and thioredoxins (U.S. application Ser. No. 10/005,429,
filed Dec. 3, 2001)), the disclosures of which are herein
incorporated by reference. The polynucleotides of the present
invention can also be stacked with traits desirable for insect,
disease or herbicide resistance (e.g., Bacillus thuringiensis toxic
proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514;
5,723,756; 5,593,881; Geiser, et al., (1986) Gene 48:109); lectins
(Van Damme, et al., (1994) Plant Mol. Biol. 24:825); fumonisin
detoxification genes (U.S. Pat. No. 5,792,931); avirulence and
disease resistance genes (Jones, et al., (1994) Science 266:789;
Martin, et al., (1993) Science 262:1432; Mindrinos, et al., (1994)
Cell 78:1089); acetolactate synthase (ALS) mutants that lead to
herbicide resistance such as the S4 and/or Hra mutations;
inhibitors of glutamine synthase such as phosphinothricin or basta
(e.g., bar gene); and glyphosate resistance (EPSPS gene)); and
traits desirable for processing or process products such as high
oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty
acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516));
modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch
synthases (SS), starch branching enzymes (SBE) and starch
debranching enzymes (SDBE)); and polymers or bioplastics (e.g.,
U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate
synthase, and acetoacetyl-CoA reductase (Schubert, et al., (1988)
J. Bacteriol. 170:5837-5847) facilitate expression of
polyhydroxyalkanoates (PHAs)), the disclosures of which are herein
incorporated by reference.
[0181] One could also combine the polynucleotides of the present
invention with polynucleotides affecting agronomic traits such as
male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength,
flowering time, or transformation technology traits such as cell
cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364;
WO 99/25821), the disclosures of which are herein incorporated by
reference.
[0182] In one embodiment, sequences of interest improve plant
growth and/or crop yields. For example, sequences of interest
include agronomically important genes that result in improved
primary or lateral root systems. Such genes include, but are not
limited to, nutrient/water transporters and growth induces.
Examples of such genes, include but are not limited to, maize
plasma membrane H.sup.+-ATPase (MHA2) (Frias, et al., (1996) Plant
Cell 8:1533-44); AKT1, a component of the potassium uptake
apparatus in Arabidopsis, (Spalding, et al., (1999) J Gen Physiol
113:909-18); RML genes which activate cell division cycle in the
root apical cells (Cheng, et al., (1995) Plant Physiol 108:881);
maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol
Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J. Biol.
Chem. 27:16749-16752, Arredondo-Peter, et al., (1997) Plant
Physiol. 115:1259-1266; Arredondo-Peter, et al., (1997) Plant
Physiol 114:493-500 and references sited therein). The sequence of
interest may also be useful in expressing antisense nucleotide
sequences of genes that that negatively affects root
development.
[0183] Additional, agronomically important traits such as oil,
starch, and protein content can be genetically altered in addition
to using traditional breeding methods. Modifications include
increasing content of oleic acid, saturated and unsaturated oils,
increasing levels of lysine and sulfur, providing essential amino
acids, and also modification of starch. Hordothionin protein
modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801,
5,885,802, and 5,990,389, herein incorporated by reference. Another
example is lysine and/or sulfur rich seed protein encoded by the
soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the
chymotrypsin inhibitor from barley, described in Williamson, et
al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which
are herein incorporated by reference.
[0184] Derivatives of the coding sequences can be made by
site-directed mutagenesis to increase the level of preselected
amino acids in the encoded polypeptide. For example, the gene
encoding the barley high lysine polypeptide (BHL) is derived from
barley chymotrypsin inhibitor, U.S. application Ser. No.
08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of
which are herein incorporated by reference. Other proteins include
methionine-rich plant proteins such as from sunflower seed (Lilley,
et al., (1989) Proceedings of the World Congress on Vegetable
Protein Utilization in Human Foods and Animal Feedstuffs, ed.
Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.
497-502; herein incorporated by reference); corn (Pedersen, et al.,
(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene
71:359; both of which are herein incorporated by reference); and
rice (Musumura, et al., (1989) Plant Mol. Biol. 12:123, herein
incorporated by reference). Other agronomically important genes
encode latex, Floury 2, growth factors, seed storage factors, and
transcription factors.
[0185] Herbicide resistance traits may include genes coding for
resistance to herbicides that act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides (e.g., the acetolactate synthase (ALS) gene containing
mutations leading to such resistance, in particular the S4 and/or
Hra mutations), genes coding for resistance to herbicides that act
to inhibit action of glutamine synthase, such as phosphinothricin
or basta (e.g., the bar gene), or other such genes known in the
art. The bar gene encodes resistance to the herbicide basta, the
nptII gene encodes resistance to the antibiotics kanamycin and
geneticin, and the ALS-gene mutants encode resistance to the
herbicide chlorsulfuron.
[0186] Sterility genes can also be encoded in an expression
cassette and provide an alternative to physical detasseling.
Examples of genes used in such ways include male tissue-preferred
genes and genes with male sterility phenotypes such as QM,
described in U.S. Pat. No. 5,583,210. Other genes include kinases
and those encoding compounds toxic to either male or female
gametophytic development.
[0187] The quality of grain is reflected in traits such as levels
and types of oils, saturated and unsaturated, quality and quantity
of essential amino acids, and levels of cellulose. In corn,
modified hordothionin proteins are described in U.S. Pat. Nos.
5,703,049, 5,885,801, 5,885,802, and 5,990,389.
[0188] Commercial traits can also be encoded on a gene or genes
that could increase for example, starch for ethanol production, or
provide expression of proteins. Another important commercial use of
transformed plants is the production of polymers and bioplastics
such as described in U.S. Pat. No. 5,602,321. Genes such as
.beta.-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and
acetoacetyl-CoA reductase (see, Schubert, et al., (1988) J.
Bacteriol. 170:5837-5847) facilitate expression of
polyhyroxyalkanoates (PHAs).
[0189] Exogenous products include plant enzymes and products as
well as those from other sources including prokaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones, and
the like. The level of proteins, particularly modified proteins
having improved amino acid distribution to improve the nutrient
value of the plant, can be increased. This is achieved by the
expression of such proteins having enhanced amino acid content.
Production And Characterization Of Stably Transformed Plants
[0190] After effecting delivery of exogenous DNA to recipient
cells, the next steps generally concern identifying the transformed
cells for further culturing and plant regeneration. In order to
improve the ability to identify transformants, one may desire to
employ a selectable or screenable marker gene with a transformation
vector prepared in accordance with the invention. In this case, one
would then generally assay the potentially transformed cell
population by exposing the cells to a selective agent or agents, or
one would screen the cells for the desired marker gene trait.
Selection
[0191] It is believed that DNA is introduced into only a small
percentage of target cells in any one study. In order to provide an
efficient system for identification of those cells receiving DNA
and integrating it into their genomes one may employ a means for
selecting those cells that are stably transformed. One exemplary
embodiment of such a method is to introduce into the host cell, a
marker gene which confers resistance to some normally inhibitory
agent, such as an antibiotic or herbicide. Examples of antibiotics
which may be used include the aminoglycoside antibiotics neomycin,
kanamycin and paromomycin, or the antibiotic hygromycin. Resistance
to the aminoglycoside antibiotics is conferred by aminoglycoside
phosphotransferase enzymes such as neomycin phosphotransferase II
(NPT II) or NPT I, whereas resistance to hygromycin is conferred by
hygromycin phosphotransferase.
[0192] Potentially transformed cells then are exposed to the
selective agent. In the population of surviving cells will be those
cells where, generally, the resistance-conferring gene has been
integrated and expressed at sufficient levels to permit cell
survival. Cells may be tested further to confirm stable integration
of the exogenous DNA.
[0193] One herbicide which constitutes a desirable selection agent
is the broad spectrum herbicide bialaphos. Bialaphos is a
tripeptide antibiotic produced by Streptomyces hygroscopicus and is
composed of phosphinothricin (PPT), an analogue of L-glutamic acid,
and two L-alanine residues. Upon removal of the L-alanine residues
by intracellular peptidases, the PPT is released and is a potent
inhibitor of glutamine synthetase (GS), a pivotal enzyme involved
in ammonia assimilation and nitrogen metabolism (Ogawa et al.,
1973). Synthetic PPT, the active ingredient in the herbicide
Liberty.TM. also is effective as a selection agent Inhibition of GS
in plants by PPT causes the rapid accumulation of ammonia and death
of the plant cells.
[0194] The organism producing bialaphos and other species of the
genus Streptomyces also synthesizes an enzyme phosphinothricin
acetyl transferase (PAT) which is encoded by the bar gene in
Streptomyces hygroscopicus and the pat gene in Streptomyces
viridochromogenes. The use of the herbicide resistance gene
encoding phosphinothricin acetyl transferase (PAT) is referred to
in DE 3642 829 A, wherein the gene is isolated from Streptomyces
viridochromogenes.
[0195] Another example of a herbicide which is useful for selection
of transformed cell lines in the practice of the invention is the
broad spectrum herbicide glyphosate. Glyphosate inhibits the action
of the enzyme EPSPS which is active in the aromatic amino acid
biosynthetic pathway Inhibition of this enzyme leads to starvation
for the amino acids phenylalanine, tyrosine, and tryptophan and
secondary metabolites derived thereof. U.S. Pat. No. 4,535,060
describes the isolation of EPSPS mutations which confer glyphosate
resistance on polypeptides encoded by the Salmonella typhimurium
gene for EPSPS, aroA. The EPSPS gene was cloned from Zea mays and
mutations similar to those found in a glyphosate resistant aroA
gene were introduced in vitro. Mutant genes encoding glyphosate
resistant EPSPS enzymes are described in, for example,
International Patent WO 97/4103. The best characterized mutant
EPSPS gene conferring glyphosate resistance comprises amino acid
changes at residues 102 and 106, although it is anticipated that
other mutations will also be useful (PCT/WO97/4103).
[0196] To use a bar-bialaphos or the EPSPS-glyphosate selective
system, for example, transformed tissue can be cultured for 0-28
days on nonselective medium and subsequently transferred to medium
containing from 1-3 mg/l bialaphos or 1-3 mM glyphosate as
appropriate. While ranges of 1-3 mg/l bialaphos or 1-3 mM
glyphosate may be preferred, it is proposed that ranges of 0.1-50
mg/l bialaphos or 0.1-50 mM glyphosate will find utility.
[0197] Regeneration and Seed Production
[0198] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In an
exemplary embodiment, MS and N6 media may be modified by including
further substances such as growth regulators. One such growth
regulator is dicamba or 2,4-D. However, other growth regulators may
be employed, including NAA, NAA+2,4-D or picloram. Media
improvement in these and like ways has been found to facilitate the
growth of cells at specific developmental stages. Tissue may be
maintained on a basic media with growth regulators until sufficient
tissue is available to begin plant regeneration efforts, or
following repeated rounds of manual selection, until the morphology
of the tissue is suitable for regeneration, at least 2 wk, then
transferred to media conducive to maturation of embryoids. Cultures
are transferred every 2 wk on this medium. Shoot development will
signal the time to transfer to medium lacking growth
regulators.
[0199] The transformed cells, identified by selection or screening
and cultured in an appropriate medium that supports regeneration,
will then be allowed to mature into plants. Developing plantlets
are transferred to soiless plant growth mix, and hardened, e.g., in
an environmentally controlled chamber, for example, at about 85%
relative humidity, 600 ppm CO.sub.2, and 25-250 microeinsteins
m.sup.-2 s.sup.-1 of light. Plants may be matured in a growth
chamber or greenhouse. Plants can be regenerated from about 6 wk to
10 months after a transformant is identified, depending on the
initial tissue. During regeneration, cells are grown on solid media
in tissue culture vessels. Illustrative embodiments of such vessels
are petri dishes and Plant Cons. Regenerating plants can be grown
at about 19 to 28.degree. C. After the regenerating plants have
reached the stage of shoot and root development, they may be
transferred to a greenhouse for further growth and testing.
[0200] Seeds on transformed plants may occasionally require embryo
rescue due to cessation of seed development and premature
senescence of plants. To rescue developing embryos, they are
excised from surface-disinfected seeds 10-20 days post-pollination
and cultured. An embodiment of media used for culture at this stage
comprises MS salts, 2% sucrose, and 5.5 .mu.l agarose. In embryo
rescue, large embryos (defined as greater than 3 mm in length) are
germinated directly on an appropriate media. Embryos smaller than
that may be cultured for 1 wk on media containing the above
ingredients along with 10.sup.-5M abscisic acid and then
transferred to growth regulator-free medium for germination.
Characterization
[0201] To confirm the presence of the exogenous DNA or
"transgene(s)" in the regenerating plants, a variety of assays may
be performed. Such assays include, for example, "molecular
biological" assays, such as Southern and Northern blotting and
PCR.TM.; "biochemical" assays, such as detecting the presence of a
protein product, e.g., by immunological means (ELISAs and Western
blots) or by enzymatic function; plant part assays, such as leaf or
root assays; and also, by analyzing the phenotype of the whole
regenerated plant.
DNA Integration, RNA Expression and Inheritance
[0202] Genomic DNA may be isolated from cell lines or any plant
parts to determine the presence of the exogenous gene through the
use of techniques well known to those skilled in the art. Note,
that intact sequences will not always be present, presumably due to
rearrangement or deletion of sequences in the cell. The presence of
DNA elements introduced through the methods of this invention may
be determined, for example, by polymerase chain reaction (PCR.TM.).
Using this technique, discrete fragments of DNA are amplified and
detected by gel electrophoresis. This type of analysis permits one
to determine whether a gene is present in a stable transformant,
but does not prove integration of the introduced gene into the host
cell genome. It is typically the case, however, that DNA has been
integrated into the genome of all transformants that demonstrate
the presence of the gene through PCR.TM. analysis. In addition, it
is not typically possible using PCR.TM. techniques to determine
whether transformants have exogenous genes introduced into
different sites in the genome, i.e., whether transformants are of
independent origin. It is contemplated that using PCR.TM.
techniques it would be possible to clone fragments of the host
genomic DNA adjacent to an introduced gene.
[0203] Positive proof of DNA integration into the host genome and
the independent identities of transformants may be determined using
the technique of Southern hybridization. Using this technique
specific DNA sequences that were introduced into the host genome
and flanking host DNA sequences can be identified. Hence the
Southern hybridization pattern of a given transformant serves as an
identifying characteristic of that transformant. In addition it is
possible through Southern hybridization to demonstrate the presence
of introduced genes in high molecular weight DNA, i.e., confirm
that the introduced gene has been integrated into the host cell
genome. The technique of Southern hybridization provides
information that is obtained using PCR.TM., e.g., the presence of a
gene, but also demonstrates integration into the genome and
characterizes each individual transformant.
[0204] It is contemplated that using the techniques of dot or slot
blot hybridization which are modifications of Southern
hybridization techniques one could obtain the same information that
is derived from PCR.TM., e.g., the presence of a gene.
[0205] Both PCR.TM. and Southern hybridization techniques can be
used to demonstrate transmission of a transgene to progeny. In most
instances the characteristic Southern hybridization pattern for a
given transformant will segregate in progeny as one or more
Mendelian genes (Spencer et al., 1992) indicating stable
inheritance of the transgene.
[0206] Whereas DNA analysis techniques may be conducted using DNA
isolated from any part of a plant, RNA will only be expressed in
particular cells or tissue types and hence it will be necessary to
prepare RNA for analysis from these tissues. PCR.TM. techniques
also may be used for detection and quantitation of RNA produced
from introduced genes. In this application of PCR.TM. it is first
necessary to reverse transcribe RNA into DNA, using enzymes such as
reverse transcriptase, and then through the use of conventional
PCR.TM. techniques amplify the DNA. In most instances PCR.TM.
techniques, while useful, will not demonstrate integrity of the RNA
product. Further information about the nature of the RNA product
may be obtained by Northern blotting. This technique will
demonstrate the presence of an RNA species and give information
about the integrity of that RNA. The presence or absence of an RNA
species also can be determined using dot or slot blot Northern
hybridizations. These techniques are modifications of Northern
blotting and will only demonstrate the presence or absence of an
RNA species.
Gene Expression
[0207] While Southern blotting and PCR.TM. may be used to detect
the gene(s) in question, they do not provide information as to
whether the corresponding protein is being expressed. Expression
may be evaluated by specifically identifying the protein products
of the introduced genes or evaluating the phenotypic changes
brought about by their expression.
[0208] Assays for the production and identification of specific
proteins may make use of physical-chemical, structural, functional,
or other properties of the proteins. Unique physical-chemical or
structural properties allow the proteins to be separated and
identified by electrophoretic procedures, such as native or
denaturing gel electrophoresis or isoelectric focusing, or by
chromatographic techniques such as ion exchange or gel exclusion
chromatography. The unique structures of individual proteins offer
opportunities for use of specific antibodies to detect their
presence in formats such as an ELISA assay. Combinations of
approaches may be employed with even greater specificity such as
western blotting in which antibodies are used to locate individual
gene products that have been separated by electrophoretic
techniques. Additional techniques may be employed to absolutely
confirm the identity of the product of interest such as evaluation
by amino acid sequencing following purification. Although these are
among the most commonly employed, other procedures may be
additionally used.
[0209] Very frequently the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
These assays also may take many forms including but not limited to
analyzing changes in the chemical composition, morphology, or
physiological properties of the plant. Chemical composition may be
altered by expression of genes encoding enzymes or storage proteins
which change amino acid composition and may be detected by amino
acid analysis, or by enzymes which change starch quantity which may
be analyzed by near infrared reflectance spectrometry.
Morphological changes may include greater stature or thicker
stalks. Most often changes in response of plants or plant parts to
imposed treatments are evaluated under carefully controlled
conditions termed bioassays.
Breeding Plants
[0210] In addition to direct transformation of a particular plant
genotype with a construct prepared according to the current
invention, transgenic plants may be made by crossing a plant having
a selected DNA of the invention to a second plant lacking the
construct. For example, a selected polypeptide coding sequence can
be introduced into a particular plant variety by crossing, without
the need for ever directly transforming a plant of that given
variety. Therefore, the current invention not only encompasses a
plant directly transformed or regenerated from cells which have
been transformed in accordance with the current invention, but also
the progeny of such plants. As used herein the term "progeny"
denotes the offspring of any generation of a parent plant prepared
in accordance with the instant invention, wherein the progeny
comprises a selected DNA construct prepared in accordance with the
invention. "Crossing" a plant to provide a plant line having one or
more added transgenes relative to a starting plant line, as
disclosed herein, is defined as the techniques that result in a
transgene of the invention being introduced into a plant line by
crossing a starting line with a donor plant line that comprises a
transgene of the invention. To achieve this one could, for example,
perform the following steps:
[0211] (a) plant seeds of the first (starting line) and second
(donor plant line that comprises a transgene of the invention)
parent plants;
[0212] (b) grow the seeds of the first and second parent plants
into plants that bear flowers;
[0213] (c) pollinate a flower from the first parent plant with
pollen from the second parent plant; and
[0214] (d) harvest seeds produced on the parent plant bearing the
fertilized flower.
Backcrossing is herein defined as the process including the steps
of:
[0215] (a) crossing a plant of a first genotype containing a
desired gene, DNA sequence or element to a plant of a second
genotype lacking the desired gene, DNA sequence or element;
[0216] (b) selecting one or more progeny plant containing the
desired gene, DNA sequence or element;
[0217] (c) crossing the progeny plant to a plant of the second
genotype; and
[0218] (d) repeating steps (b) and (c) for the purpose of
transferring a desired DNA sequence from a plant of a first
genotype to a plant of a second genotype.
[0219] Introgression of a DNA element into a plant genotype is
defined as the result of the process of backcross conversion. A
plant genotype into which a DNA sequence has been introgressed may
be referred to as a backcross converted genotype, line, inbred, or
hybrid. Similarly a plant genotype lacking the desired DNA sequence
may be referred to as an unconverted genotype, line, inbred, or
hybrid.
[0220] It is understood that modifications which do not
substantially affect the activity the various embodiments of this
invention are also provided within the definition of the invention
provided herein. Accordingly, the following examples are intended
to illustrate but not limit the present invention.
EXAMPLES
Example 1
[0221] Pathogens alter their hosts' biology to ensure successful
infection. Such modifications range from moderate to extensive, and
in the case of plant pathogens, few infections result in more
dramatic changes than those of sedentary endoparasitic nematodes,
which include the cyst nematodes (Heterodera spp.). Maybe rivaled
in complexity only by plant interactions with Agrobacterium and
Rhizobia, cyst nematodes are obligate parasitic roundworms that
induce the formation of novel plant cell types that are associated
in a unique feeding organ, the syncytium.
[0222] Cyst nematodes infect as second-stage juveniles (J2), which
initiate the induction/formation of the syncytium. During this
phase, J2s begin feeding on the growing syncytium and then develop
into third-stage (J3) and fourth-stage juveniles (J4) followed by
the adult stage. Syncytium development can be separated into an
induction/formation phase followed by a maintenance phase.
Induction/formation involves effector-mediated communication
between the nematode and plant cells leading to cytoplasmic and
nuclear changes followed by successive cell-to-cell fusions of the
cells surrounding an initial feeding cell (IFC). Through continuous
cell fusions, syncytium formation and enlargement continues. During
the maintenance phase no additional cells are incorporated and
syncytial cells have undergone their developmental changes and now
are fully engaged in maintaining syncytium function.
[0223] Due to their sedentary nature of parasitism, cyst nematodes
need to obtain all their nourishment from one location, in fact,
through the contact with the IFC. The severity of this constraint
becomes obvious when considering that the worm-shaped infective J2
nematode has a body length of approximately 500 um and then grows
to a large lemon-shaped sphere that produces several hundred eggs,
each containing a fully infective nematode. The sheer logistics of
nutrient availability and flux appear unrivalled for an individual
plant pathogen. This association is also impressive with regard to
the complete dependence of nematode survival on the well-being and
survival of the IFC and the syncytium. In other words, a single
hypersensitive response or an interruption of the newly induced
developmental programs of syncytium formation would eliminate
nematode parasitism. But despite a plant's well developed ability
to detect and defend against invaders, co-evolution of nematode and
plant has resulted in an uncannily robust and successful
pathosystem in which nematode contact with the IFC does not trigger
effective defenses. Instead, syncytial cells are dedicated to
nematode nourishment, and their plant defenses have been suppressed
by the nematode.
[0224] Syncytium formation encompasses reprogramming of
differentiated root cells, and these redifferentiations are
accompanied and mediated by massive gene expression changes, which
have been documented in diverse research approaches using soybean
and the soybean cyst nematode Heterodera glycines (Alkharouf et
al., 2006; Ithal et al., 2007; Klink et al., 2009) and probably
most extensively in Arabidopsis infected by the sugar beet cyst
nematode H. schachtii (Szakasits et al., 2009). These gene
expression changes clearly require powerful mechanisms of concerted
regulation, and the existence of major regulatory choke points,
i.e., master switches, can be hypothesized, although none have been
documented to date. Regulatory networks governing gene expression
patterns in nematode-infected roots and particularly in the
developing syncytium are very poorly understood.
[0225] miRNAs initially have been shown to be involved in the
regulation of a variety of plant developmental processes including
phase transition, hormone synthesis and signaling, pattern
formation, and morphogenesis (Chen, 2009). Recent studies indicate
that miRNAs and small endogenous RNAs also are involved in biotic
stress responses in plants (Navarro et al. 2006; Li et al., 2010;
He et al., 2008; Lu et al., 2007; Fahlgren et al. 2007; Hewezi et
al., 2008a; Pandey et al., 2008; Katiyar-Agarwal et al., 2006 and
2007). Also, consistent with a role of small RNAs in the regulation
of plant immune responses, Arabidopsis mutants deficient in siRNA
or miRNA biogenesis affected plant susceptibility to bacteria
(Navarro et al., 2008) and the sugar beet cyst nematode H.
schachtii (Hewezi et al., 2008a). Collectively, these emerging data
indicate that small RNA-mediated gene regulation is a fundamental
mechanism in plant-pathogen interactions.
[0226] Despite these advances, little is known about the molecular
mechanisms controlling cell differentiation and development in the
nematode-induced syncytium. The miR396 family, miR396a and miR396b,
governs the expression of seven growth regulating transcription
factor genes (GRFs) (Jones-Rhoades and Bartel, 2004). The GRF gene
family in Arabidopsis is known to act in a functionally redundant
fashion to positively control cell proliferation and size in leaves
(Kim et al., 2003; Kim and Kende, 2004; Horiguchi et al., 2005; Kim
and Lee, 2006). Consistent with the fact that miR396 acts as a
negative regulator of GRF gene expression, overexpression of miR396
negatively impacted cell proliferation in leaves and meristem size
(Liu et al., 2009; Rodriguez et al., 2010). However, the roles of
the miR396/GRF regulatory module in controlling developmental
events during plant-pathogen interactions or in root developmental
processes are completely unknown. In this study we demonstrate that
miR396 is differentially expressed in the syncytium, that the
miR396-GRF regulatory unit is subject to extensive feedback
regulation, and that this microRNA functions as a true master
switch in syncytium formation.
Results
[0227] In Arabidopsis, miR396 is encoded by two genes, miR396a
(AT2G10606) (SEQ ID NO:1) and miR396b (AT5G35407) (SEQ ID NO:2) and
regulates the expression of seven of the nine Arabidopsis growth
regulating transcription factor genes (GRF1 through 4 and 7 through
9), which share the miR396-binding site (Jones-Rhoades and Bartel,
2004). To determine which GRF genes could be targeted by miR396 in
roots, we measured the mRNA steady-state levels in root tissues of
10-d old seedlings of all 9 GRF genes by quantitative real-time
RT-PCR (qPCR). GRF1 and GRF3 showed by far the highest root
expression levels (FIG. 7). This observation implies that if
miRNA396 is active in post-transcriptional gene regulation in
Arabidopsis roots in general and during nematode infection in
particular, GRF1 and GRF3 are its most likely targets, which is
consistent with our previous findings that GRF1 and GRF3 are the
genes most responsive to H. schachtii infection among the GRF
family members (Hewezi et al., 2008a).
miR396a and miR396b have Similar Spatial Expression Patterns and
Overlap With GRF1 and GRF3 Expression in Roots
[0228] To examine tissue-specific expression patterns of the two
miR396 genes, we generated transgenic plants expressing constructs
containing the regions upstream of miRNA396 precursor sequences
fused to the .beta.-glucuronidase (GUS) reporter gene (miR396a:GUS
and miR396b:GUS). GUS staining of at least four independent lines
for each construct revealed that the miR396a and miR396b promoters
have very similar spatial expression patterns, both in leaf and
root tissues (FIG. 8). Despite the fact that miR396a and miR396b
have similar spatial expression patterns, GUS staining of miR396b:
GUS lines was in general much stronger than that of miR396a:GUS
lines. This was confirmed by real-time RT-PCR (qPCR) analysis of
miR396 precursors (pre-miR396) in roots of two-week-old Colombia-0
(Col-0) plants. We found an mRNA abundance of pre-miR396b about
70-fold higher than that of pre-miR396a. To determine whether the
spatial expression of miR396 coincides with that of the GRF1 and
GRF3 target genes, we generated and examined at least four
transgenic lines each expressing the reporter gene fusion
constructs GRF1: GUS or GRF3: GUS. Promoter activity of GRF1: GUS
and GRF3: GUS (FIG. 8H) revealed that expression locations of both
miR396a and miR396b spatially overlap with the expression of the
target genes GRF1 and GRF3, supporting a post-transcriptional
regulation of GRF1 and GRF3 by miR396 also in roots.
miRNA396 and GRF Transcription Factors Represent a Complex
Regulatory Unit Governed by Multiple Mechanisms Including Feedback
Regulation.
[0229] To gain insight into the effect of miR396 on GRF expression
in roots, we expressed the primary miRNA sequences of both miR396a
and miR396b in Arabidopsis under the control of the 35S promoter.
Independent homozygous T3 lines expressing between 2- and 5-fold
higher miRNA levels relative to the wild type were identified
(FIGS. 9A and B). While we used these lines in phenotypical
assessments (see below), we also determined whether miR396
overexpression resulted in the expected decreased mRNA abundance of
GRF target genes by using qPCR to quantify the mRNA levels of the
GRF gene family in the roots of transgenic miR396b-overexpression
plants (line 16-4). All GRF gene mRNA abundances were reduced as a
consequence of this manipulation (FIG. 1A). Interestingly, mRNA
levels of GRF5 and GRF6 also were down regulated in the miR396
overexpression plants (1.62 and 1.68 fold, respectively), even
though these genes are not directly targeted by miRNA396. These
data show that miRNA396 induction results in the expected mRNA
reduction of its target genes in roots but also that the GRF gene
family is subject to additional concerted regulatory mechanisms
that are sensitive to gene family member expression levels. Similar
results in support of the latter conclusion were also obtained by
Rodriguez et al. (2010) in shoots.
[0230] Having identified that miR396 is highly expressed in roots
it was of interest to determine the influence of miRNA396
overexpression on root development. Interestingly, we found that
overexpression of miR396 resulted in root length reductions of 12%
to 49% (FIGS. 1B and C). These data suggest that GRF transcription
factors are positive regulators of Arabidopsis root development.
Given the fact that GRF1 and GRF3 are the most abundant gene family
members in roots, their roles appear most prominent in this
developmental pathway.
[0231] In order to further explore GRF1 and GRF3 functions in
roots, we overexpressed the coding sequences of these two genes
under the control of the 35S promoter in two forms. First, we
generated plants expressing the wild-type variants (35S:wtGRF1 and
35S:wtGRF3) cleavable by miRNA396 and second, we generated plants
harboring miR396-resistant non-cleavable variants (35S:rGRF1 and
35S:rGRF3). While we expected that these lines would produce
phenotypes opposite to those found in miR396 overexpression lines,
unexpectedly, the transgenic lines overexpressing either the
wild-type or the resistant versions of GRF1 and GRF3 both showed
phenotypes similar to miR396 overexpression plants of shorter roots
(FIGS. 1E and F). In other words, overexpression of GRF 1 or GRF3
had similar effects on root morphology as the overexpression of
miRNA396a or miR396b, which was counter intuitive. However, this
observation was explained when we discovered that the majority of
GRF genes are down regulated at the mRNA level in these GRF1 and
GRF3 overexpression lines, particularly when overexpressing the
variants resistant to miRNA396 (FIG. 1G). In other words, a general
down regulation of GRF family members is a common feature of the
rGRF1 and rGRF3 overexpression lines on one hand and the miRNA396a
and miR396b overexpression lines on the other, which explains the
common phenotypes. These findings illustrate again that expression
levels of the GRF family members are intricately connected and
that, so far unknown, mechanisms govern a mutual influence among
gene family members.
[0232] Our findings that GRF 1 and GRF3 overexpression resulted in
a down regulation of other GRFs in theory could be reconciled by
the hypothesis that GRF1 and GRF3 expression levels provide a
positive feedback regulation stimulus for miRNA396 expression.
I.e., elevated GRF1 and GRF3 gene expression would result in a
miRNA396 induction, which would re-equilibrate the regulatory
equilibrium disturbed by GRF overexpression. Similar examples of
feedback regulation of miRNAs through the expression levels of
their target genes recently have been identified (Gutierrez et al.,
2009; Wu et al., 2009; Marin et al., 2010). We, therefore, assessed
the abundances of pre-miRNA396a, pre-miRNA396b and mature miRNA396
in the 35S:wtGRF1, 35S:wtGRF3, 35S: rGRF1 and 35S:rGRF3 transgenic
lines. While a miRNA396 increase in these overexpression lines
would have explained the observed decreased root length as well as
the decreased GRF mRNA levels, we unexpectedly measured significant
decreases in abundance of miRNA396 in both of GRF1 and GRF3
overexpression lines (FIG. 1H). This observation adds additional
complexity to the regulatory mechanisms not only of the GRF gene
family but also the miRNA396-GRF regulatory unit. Clearly, the GRF
expression changes constituted a negative feedback on the
expression of miRNA396. The mutual influence of GRF family members
on each other coupled with a GRF feedback on miRNA396 expression
reveal a complex regulatory module for these regulatory genes.
[0233] As a final step towards understanding the regulatory
mechanisms of the miRNA396-GRF system, further insight could be
expected from GRF mutants. We identified two independent T-DNA
insertional alleles each for GRF1 (Salk.sub.--069339C and
Salk.sub.--0785 47C) and GRF3 (Salk.sub.--116709 and
Salk.sub.--026786) (FIGS. 10A and B) and obtained the
grf1/grf2/grf3 triple knockout mutant of Kim et al. (2003). Also
here we observed counter-intuitive effects of GRF expression on
miRNA396 abundance. While a simple model would imply that knocking
out a miRNA target gene would result in a down-regulation of the
miRNA, we observed in all mutants significant increases in miRNA
abundance (FIG. 10C), which is consistent with our results obtained
with the rGRF1 and rGRF3 overexpression plants showing significant
decrease in miR396 expression. In summary, the miRNA396-GRF gene
family system constitutes a non-trivial and complex,
multi-dimensional regulatory network.
miR396a/b and GRF1 and GRF3 are Expressed in Syncytia of Heterodera
schachtii.
[0234] We previously observed marked RNA abundance changes for
miR396a/b as well as GRF1 and GRF3 following root infections by H.
schachtii (Hewezi et al. 2008a) and, thus, it was of highest
interest to identify the location of these altered expressions. We
explored this question by analyzing the promoter activities of
miR396a, miR396b and of the target genes GRF1 and GRF3 at different
time points after H. schachtii infection using our transgenic
Arabidopsis GUS lines. Most remarkably, the activities of the
promoters of both miRNA396a and miR396b were strongly
down-regulated in developing syncytia at early time points of H.
schachtii infection (i.e., the parasitic J2 and early J3 stages)
(FIGS. 2 A-B and E-F). At the same time, the GRF1 and GRF3
promoters became very active at the same locations (FIGS. 2 I-J and
M-N). In other words, these observations of transcriptional
miRNA396 down-regulation with simultaneous target gene
up-regulation should result in a very pronounced peak of GRF 1 and
GRF3 mRNA abundance in the syncytium at the time of syncytium
induction and formation.
[0235] Maybe more interestingly, after this initial early phase,
the promoters of both miRNA396a and miR396b became very active in
the syncytia of late J3 and J4 nematodes (FIGS. 2 C-D and G-H),
thus delineating the two distinct phases of syncytium
induction/formation versus syncytium maintenance. At the same time,
GRF1 and GRF3 promoters remained highly active in late J3 syncytia,
with only GRF3 becoming less active at the J4 stage (FIGS. 2 K-L
and O-P). In other words, following the initial phase of syncytium
induction/formation, GRF1 and GRF3 mRNA abundance should markedly
decrease in syncytia during the maintenance phase as a function of
miRNA396-mediated post-transcriptional transcript degradation.
[0236] Because the GRF gene family in Arabidopsis is known to act
in a functionally redundant manner (Kim et al., 2003) and because
GRF2 shares highest sequence similarity with GRF1, we also tested
whether the GRF2 promoter is active in the syncytium. Transgenic
plants expressing the reporter gene fusion GRF2:rGRF2-GUS
(Rodriguez et al., 2010) were inoculated with H. schachtii. No GUS
activity was detected in early syncytia during the J2 and early J3
infective stages (FIG. 11), while at late J3 and J4 stages very
weak syncytial GUS activity was observed (FIG. 11). These
observations indicate that GRF2 does not work in concert with GRF1
and GRF3 during the early induction/formation period of the
syncytium. Given the overall low expression of other GRF genes in
roots, similar conclusions can be drawn for the remaining GRF
genes.
GRF1 and GRF3 are Post-Transcriptionally Regulated by miR396 During
Nematode Infection
[0237] Our promoter analyses clearly show a co-expression of
miRNA396 with its GRF1 and GRF3 target genes in the syncytium,
which indicates a posttranscriptional GRF expression regulation
following nematode infection. To investigate any such
posttranscriptional regulation of GRF1 and GRF3 by miR396, we
quantified the abundances of miR396 precursors (pre-miR396a and
pre-miR396b) and mature micro RNAs (miR396) along with GRF1 and
GRF3 mRNA steady state levels in response to H. schachtii infection
over time using qPCR. Ten-day-old wild-type Arabidopsis seedlings
were inoculated with H. schachtii, and root tissues were collected
from inoculated and non-inoculated control plants at 1, 3, 8, and
14 days post inoculation (dpi) for RNA isolation and cDNA
synthesis. Data from three independent experiments revealed that
the accumulation of pre-miR396a, pre-miR396b and mature miR396 was
down regulated in H. schachtii-inoculated roots at 1 and 3 dpi time
points when compared with non-inoculated roots (FIG. 3), confirming
the down regulation of the miR396a/b promoters in the developing
syncytium (FIG. 2). Consistent with a posttranscriptional
regulation of GRF1 and GRF3, this down regulation was accompanied
by elevated mRNA abundance for both GRF1 and GRF3 (FIG. 3), most
probably as a result of decreased cleavage of GRF1 and GRF3 mRNA by
miR396. In contrast, at 8 and 14 dpi, pre-miRNAs and mature miR396
were elevated more than 2-fold in inoculated roots (FIG. 3). Again
consistent with a posttranscriptional regulation of GRF1 and GRF3,
this miR396 increase was correlated with low transcript abundance
of GRF1 and GRF3 (FIG. 3). In other words, despite the
nematode-induced increased GRF promoter activities in syncytia
(FIG. 2), GRF1 and GRF3 steady-state mRNA levels decrease in the
syncytia of late J3 (8 dpi) and J4 (14 dpi) nematodes.
Overexpression of miR396 and Altered GRF Expression Modulate
Nematode Susceptibility
[0238] Our finding that miR396 and GRF1 and GRF3 are differentially
expressed in syncytia strongly suggests that miR396-mediated
regulation of GRFs is of importance in the plant-nematode
interaction, and the timing of these expression changes implies a
possible function in the early events of syncytium
induction/formation and even a delineation of the transition from a
period of syncytium initiation/formation to the period of syncytium
maintenance. To test this hypothesis, we determined the effect of
miR396 overexpression on nematode susceptibility using our
homozygous T3 lines overexpressing miR396a or miR396b. Ten-day-old
plants were inoculated with H. schachtii J2, and the number of
adult females was counted 3 weeks after inoculation for both the
transgenic lines and the wild-type control and used to quantify
plant susceptibility. A remarkable effect of miR396 overexpression
on nematode susceptibility was observed. All transgenic lines
overexpressing miR396a (FIG. 4A) or miR396b (FIG. 4B) were
dramatically less susceptible than the wild-type control, as shown
by the statistically significant reduction in number of females per
root system.
[0239] It appeared most logic that this reduction of susceptibility
in miRNA396 overexpression lines is mediated through a resultant
down-regulation of GRFs, particularly GRF1 and GRF3. Therefore, we
hypothesized that mutants of GRF1 and GRF3 will phenocopy the
decreased nematode susceptibility of miRNA396 overexpression lines.
The single knockdown mutants of GRF1 and GRF3 exhibited small or no
effects on nematode susceptibility (FIG. 4C), confirming the
previously reported results of Kim et al. (2003) that GRF gene
family members are functionally redundant. However, the
grf1/grf2/grf3 triple knockout mutant (Kim et al., 2003) showed a
statistically significant decrease in susceptibility to H.
schachtii relative to the wild-type control (FIG. 4D), supporting
our hypothesis that the low susceptibility phenotypes of miR396a/b
overexpression lines are mediated by a post-transcriptional
down-regulation of GRF1 and GRF3 in the syncytium.
[0240] In order to take this analysis one step further, we also
assessed the susceptibility of the Arabidopsis lines
over-expressing the wild type or the resistant versions of GRF1 and
GRF3. As we have shown above, these lines unexpectedly phenocopied
the miRNA396 overexpression lines by showing reduced root length
and down regulation of other GRFs. Therefore, it was interesting to
determine if also nematode susceptibility would follow the same
direction. We therefore tested 35S:wtGRF1, 35S:rGRF1, 35S:wtGRF3,
and 35S:rGRF3 homozygous T3 lines in nematode susceptibility
assays. All tested lines exhibited significantly reduced
susceptibility relative to wild-type plants (FIG. 4E-H). These
results again firmly connect GRF transcription factors,
particularly GRF1 and GRF3, to determining the outcome of the cyst
nematode--Arabidopsis interaction.
miR396 and Its Target Genes GRF1 and GRF3 Control Syncytium Size
and Nematode Development.
[0241] In addition to merely determining the number of females that
mature on the different Arabidopsis genotypes, it is of particular
interest to elucidate when and how altered susceptibility
phenotypes are established. For this purpose, we measured syncytium
sizes and quantified different nematode developmental stages at
different assessment times. Two weeks post-inoculation, we measured
the size of fully formed syncytia in transgenic plants
overexpressing miR396b or the resistant versions of GRF1 or GRF3 as
well as in wild-type Arabidopsis. Interestingly, the syncytia
formed in the transgenic lines were significantly smaller than
those in the wild-type control (FIG. 5A). The average reduction in
syncytium size was up to 33% in miR396-overexpression plants and
19% and 14% in the transgenic plants expressing rGRF1 and rGRF3,
respectively. These results indicate that the mode of action
responsible for the reduced susceptibility in the transgenic lines
overexpressing miR396 or the target genes GRF1 and GRF3 is
manifested during the formation phase of the syncytium, i.e., at
early stages of parasitism.
[0242] To investigate whether the activity of miR396 and its target
genes GRF1 and GRF3 are associated with arrested nematode
development at a specific stage of parasitism, we counted the
number of parasitic J2/J3 at 7 dpi in the transgenic lines
overexpressing miR396 or the target genes rGRF1 and rGRF3. The
number of developing (i.e., already swollen) J2 and J3 was
significantly reduced in these transgenic plants relative to the
wild-type control (FIG. 5B), and the reduction ranged between 42%
for miR396 overexpressing plants and 20% and 39% for the transgenic
plants expressing rGRF1 and rGRF3, respectively. These reductions
in nematode numbers were also evident when the number of J4 was
counted at 21 dpi in the same plants (FIG. 5C). In fact, the
percentages of nematode reduction were not significantly changed
from the 7 dpi assessment. These data indicate that the reduced
susceptibility of these transgenic lines is associated with early
arrested nematode development during the J2/J3 stages, which again
points to a mode of action during the early stages of parasitism
when the syncytium is being formed.
Identification of Potential Targets of GRF1 and GRF3 Using
Microarray Analysis
[0243] Because both GRF1 and GRF3 function as transcription
factors, identifying their direct or indirect target genes will
elucidate the pathways in which these transcription factors
function. To this end, we used Arabidopsis Affymetrix ATH1
GeneChips to compare the mRNA profiles of root tissues of the
grf1/grf2/grf3 triple mutant and transgenic plants expressing rGRF1
or rGRF3 with those of the corresponding wild-type (Col-0 or Ws).
We identified 3,944, 2,293 and 2,410 genes as differentially
expressed in the grf1/grf2/grf3 triple mutant, rGRF1 and rGRF3
plants, respectively, at a false discovery rate (FDR) of <5% and
a P value of <0.05 (Table S1A-C). In order to mine these
expression data for the most likely GRF-dependent target gene
candidates, we hypothesized that bona fide target genes of GRF1 and
GRF3 likely would exhibit opposite expression patterns in the
grf1/grf2/grf3 triple mutant and rGRF1 or rGRF3 over-expression
plants. We first compared the differentially expressed genes in
grf1/grf2/grf3 triple mutant (3,944 genes) with those identified as
differentially expressed in rGRF1 (2,293 genes) (FIG. 6A). We
identified 1,135 overlapping genes of which 1,098 had opposite
expression patterns in both lines (FIG. 6B). Of these 1,098 genes,
507 genes were found to be up regulated in rGRF land down regulated
in grf1/grf2/grf3 triple mutant, and 591 genes were up regulated in
the grf1/grf2/grf3 mutant and down regulated in rGRF1 (FIG. 6B and
Table S1D). Similarly, we compared the differentially expressed
genes of the grf1/grf2/grf3 triple mutant (3,944 genes) with those
identified as differentially expressed in rGRF3 (2,410 genes) (FIG.
6A). We identified 796 overlapping genes of which 600 have opposite
expression patterns in rGRF3 and grf1/grf2/grf3 triple mutant, and
of these, 299 genes were found to be up regulated in rGRF3 and down
regulated in grf1/grf2/grf3 triple mutant, and 301 genes were up
regulated in the grf1/grf2/grf3 triple mutant and down regulated in
rGRF3 (FIG. 6C). We considered these 1,098 and 600 genes as
candidate targets of GRF1 and GRF3, respectively.
[0244] GRFs in Arabidopsis function redundantly in controlling
various aspects of plant development (Kim et al., 2003; Kim and
Kende, 2004; Horiguchi et al., 2005; Kim and lee, 2006). To address
the potential redundant function of GRF1 and GRF3 in regulating
gene expression, we compared the 1,098 candidate target genes of
GRF1 with the 600 candidate target genes of GRF3 to identify genes
that are common to both. Interestingly, we discovered 264 genes as
overlapping targets between GRF1 and GRF3 reducing the total number
of targets to 1,434 unique putative target genes of GRF1 and GRF3.
Interestingly, the 264 overlapping target genes all showed the same
trend of expression in the rGRF1 and rGRF3 overexpression lines, in
which 124 genes were up regulated and 140 genes were down regulated
in both lines, indicating that GRF1 and GRF3 activate and inhibit
gene expression in a similar manner.
[0245] In addition to apparently targeting identical genes, careful
examination of the putative function/annotation of the GRF1 and
GRF3 target genes revealed that both transcription factors regulate
genes with similar function or different members belonging to the
same gene family. When classifying candidate target genes into
different groups by molecular function using the gene ontology
categorization from The Arabidopsis Information Resource world wide
web at Arabidopsis.org, we discovered a high proportion of genes
associated with other enzyme activity, binding activity,
transferase activity, hydrolase activity, and transcription factor
activity (FIG. 6D) for both GRF1 and GRF3. When these genes were
grouped by associated biological processes, the most abundant
groups corresponded to metabolism and other cellular processes
while response to stress, response to abiotic or biotic stimuli,
and protein metabolism also represented significant groups (FIG.
6E). These data provide strong evidence for the functional overlap
between GRF1 and GRF3 in the regulation of gene expression both
during normal development and in response to nematode infection.
Furthermore, these data provide valuable insight into the molecular
functions of GRF 1 and GRF3 as transcriptional regulators.
A Master Switch for Gene Expression in the Syncytium
[0246] If in fact the candidate GRF1 and GRF3 target genes are
regulated by these transcription factors and have a role in
mediating syncytium induction/formation, these genes should exhibit
differential regulation in the syncytium when compared with other
root tissues because we have documented differential regulation of
GRF1 and GRF3 in the syncytium. Therefore, we next compared the
candidate targets of GRF1 and GRF3 with the 7,225 genes
differentially expressed in Arabidopsis syncytia reported by
Szakasits et al. (2009). Intriguingly, out of the 1,098 genes
identified as potential targets of GRF1, we found 610 genes (55.6%,
.chi..sup.2=289.91, p-value=5.19E-65) that are differentially
expressed in the syncytium. Also, out of the 600 genes identified
as candidate targets of GRF3, we found 324 genes (54%,
.chi..sup.2=134.45, p-value=4.35E-31) that are differentially
expressed in the syncytium. In cumulo, when comparing the 1,434
unigenes of GRF1/GRF3 candidate target genes, we found that 796
(55.5%, .chi..sup.2=383.49, p-value=2.16E-85) are differentially
expressed in the syncytium. These data provide strong support for
the validity of these genes as candidate target genes of GRF 1 and
GRF3.
[0247] More interestingly, analyses of our microarray comparisons
were also extended to determine the percentage of the 7,225
syncytium-regulated genes (Szakasits et al., 2009) that could be
explained by the GRF modulations performed by us, i.e., by
comparing all genes identified as differentially expressed in the
rGRF1-overexpressing (2,293 genes) and rGRF3-overexpressing (2,410
genes) plants as well as in the grf1/grf2/grf3 mutant (3,944
genes), i.e., not just the putative target genes. We found 1,131
(49.32%, .chi..sup.2=346.13, p-value=2.95E-77) and 1,165 (48.34%,
.chi..sup.2=325.27, p-value=1.03E-72) genes as overlapping between
the 7,224 syncytium-regulated genes and those of rGRF1 and rGRF3,
respectively (FIG. 6F). After eliminating duplicates between both
cohorts, the resultant 1,965 unique genes were found to account for
27.2% (.chi..sup.2=605.47, p-value=1.08E-133) of the total number
of syncytium-regulated genes (FIG. 6F). Furthermore, 2,073 genes
overlapped between syncytium-regulated genes and those found to be
differentially regulated in the grf1/grf2/grf3 triple mutant (FIG.
6F), which means that 28.7% (.chi..sup.2=916.26, p-value=2.87E-201)
of the total number of syncytium-regulated genes change expression
in the triple mutant. The 1,965 unique syncytial genes identified
in rGRF1 and rGRF3 overexpression lines along with the 2,073
syncytial genes identified in the triple mutant make up a unigene
set of 3,160 syncytial genes (FIG. 6F). This number represents an
astonishing 44% (.chi..sup.2=1234.13, p-value=2.33E-270) of all
syncytial genes reported by Szakasits et al. (2009). In other
words, the modulations of GRFs performed by us account for almost
half of the reported syncytial gene expression changes in
Arabidopsis. GRFs, thus, play tremendously important roles in
syncytium induction/formation. Considering that GRF1 and GRF3
change expression in the syncytium as a function of miRNA396, as we
have shown above, this miRNA, thus, represents a bona fide master
switch of syncytial gene expression changes.
Discussion
[0248] Formation of functional syncytia requires a tightly
fine-tuned coordination of multiple developmental and cellular
processes to achieve the redifferentiation of hundreds of fused
root cells into a functional new organ. The mechanisms and
underlying regulatory networks that control the integration of
these processes remain poorly understood. In this paper, we report
on the biological role of miR396 in syncytium formation and
function. In response to H. schachtii, miR396, GRF1 and GRF3 are
regulated transcriptionally.
[0249] miR396 and its target genes GRF1 and GRF3 showed opposite
expression patterns in the early developing syncytium at the
parasitic J2 and early J3 stages when miR396 was down regulated and
GRF1 and GRF3 showed up regulation. At later stages, we established
that up regulation of miR396 at 8 and 14 dpi is accompanied by a
posttranscriptional down regulation of GRF1 and GRF3 (FIG. 3).
miR396, therefore, has a stage-specific function in the spatial
activation/restriction of GRF1 and GRF3 expression in the
syncytium. The fact that miRNA396 up regulation and GRF modulations
lead to smaller syncytia and reduced susceptibilities shows that
the coordinated regulation of miR396 and GRF1 and GRF3 is required
for correct cell fate specification and differentiation in the
developing syncytium.
[0250] Recent studies have shown examples of miRNA expression being
positively or negatively regulated by the transcription factors
they target through negative or positive feedback loops (Gutierrez
et al., 2009; Wu et al., 2009; Wang et al, 2009; Yant et al., 2010;
Marin et al., 2010). Similarly, the miR396/GRF1 and GRF3 regulatory
module is under a tightly fine-tuned regulation to ensure adequate
expression of GRF1 and GRF3 and their negative regulator miR396.
Our data suggest that maintenance of the homeostasis of miR396 and
the target genes at specific threshold levels is critical for
syncytium development. This suggestion is supported by our finding
that down regulation of GRFs through overexpression of miR396a/b,
or overexpression of wild-type or miR396-resistant versions of
GRF1/GRF3 resulted in reduced nematode susceptibility.
[0251] Our results further show that the homeostasis between miR396
and the target genes GRF1 and GRF3 is established through a
reciprocal feedback regulation, in which the expression of
GRF1/GRF3 and miR396 negatively regulate each other's expression.
The complexity of the miR396/GRF regulatory module was further
demonstrated by our data showing that constitutive expression of
GRF1 or GRF3 lowers the mRNA abundance of other GRFs as well as
their own endogenous transcripts. Cross-regulation among
transcription factor gene family members targeted by miRNAs also
has been reported by others (Gutierrez et al., 2009). It is most
likely that GRF1 and GRF3 are part of a highly interconnected
network of GRF transcription factors that fine tune downstream
signaling pathways in the syncytium, and that disturbance of this
interconnected network impacts normal differentiation and
developmental processes in the syncytium.
[0252] We propose that during the early stage of syncytium
development inactivation of miR396 activity in the syncytium
increases GRF1 and GRF3 expression to a defined threshold that
enables these transcription factors to regulate gene expression
reprogramming events that direct the differentiation and formation
of the nematode feeding site. Once the syncytium is established,
miR396 expression is induced to high levels in the feeding site,
which post-transcriptionally reduces the expression of GRF1 and
GRF3, thereby ending the induction/formation phase of the syncytium
and leading syncytial cells to enter the maintenance phase after
the differentiation events have been completed. The opposite
expression patterns of miR396 during syncytium initiation/formation
and maintenance stages are similar to those of Arabidopsis miR156
and miR172 during the juvenile-to-adult phase transition where
miR156 is expressed at high levels during shoot development and
then decreases with time, while miR172 has an inverse expression
pattern (Aukerman and Sakai, 2003; Jung et al., 2007; Wu and
Poethig, 2006).
The Role of GRF1 and GRF3 in Mediating Gene Expression In The
Syncytium
[0253] Despite ongoing efforts to identify the biological processes
regulated by GRFs during plant development, only a very limited
number of target genes has been identified and characterized to
date (Kim and Kende, 2004), thus our microarray study addresses an
important need. We retained only genes showing opposite expression
between grf1/grf2/grf3 triple mutant and rGRF1 or rGRF3 in order to
identify the most likely target gene candidates that are directly
or indirectly regulated by GRF1 or GRF3. Among these target
candidates, genes coding for transcription factors or proteins with
binding activity represent 39% and 35% of the putative GRF1 or GRF3
target genes, respectively (FIG. 6D), which documents a continuous
amplification of the GRF response by targeting regulatory genes.
I.e., the enrichment of transcription factors belonging to zinc
finger, Myb, WRKY, bHLH, AP2 domain-containing, CCAAT-binding, or
NAC domain transcription factor families among the GRF1 or GRF3
target genes represents a powerful mechanism to trigger a massive
signaling response to GRF1 or GRF3 expression. As a point in case,
syncytium formation has to be associated with a modulation of host
defense responses (Davis et al., 2004; Gheysen and Fenoll 2002;
Williamson and Kumar 2006) and we found a number of genes involved
in different aspects of plant defenses among the putative targets
of GRF1 or GRF3. Similarly, plant hormones, including auxin, have
been implicated in syncytium development (Grunewald et al., 2009),
and GRF1 or GRF3 appear to regulate a set of genes involved in
hormone biosynthesis or signaling pathways of auxin,
brassinosteroids, cytokinins, ethylene, gibberellins, and
jasmonates. Furthermore, cell wall modifications are obvious
mechanisms of syncytium formation and a high proportion of genes
with cell wall related functions also are enriched among the
putative GRF target genes. In other words, GRF1 and GRF3 likely are
impacting a very wide spectrum of physiological processes
associated with syncytium formation. This assessment becomes even
more concrete when considering our finding that almost half of the
putative GRF1 and GRF3 target genes were previously identified as
changing expression in the syncytium (Szakasits et al., 2009). This
phenomenon provides the mechanistic basis for GRF1 and GRF3 to
directly influence a variety of signaling and developmental
pathways required to govern the redifferentiation of
nematode-parasitized root cells into a functional new organ. While
it is fascinating to consider that half of the putative GRF1 and
GRF3 targets are involved in syncytial functions, as we would have
surmised from the syncytium-specific GRF expression characteristics
uncovered in this paper, the truly fascinating discovery is made
when performing this analysis in the opposite direction. Not only
are more than 55% of the GRF target genes implicated in syncytium
events, more importantly, the expression of 44% of the 7,225 genes
reported by Szkasits et al. (2009) to change expression in the
Arabidopsis syncytium, is altered by GRF1 and GRF3 and, thus, by
miRNA396. Consequently, almost half of the known syncytial gene
expression events in Arabidopsis can be modulated by miRNA396 as a
single molecular master switch. No other known mechanism is able to
exert the same powerful control over syncytial events.
Experimental Procedures
Plant Materials and Growth Conditions
[0254] Arabidopsis thaliana Wild type Columbia-0 (Col-0) was used
in all experiments except for the grf1/grf2/grf3 triple knockout
mutant, which is in the Wassilewskija (Ws) background (Kim et al.,
2003). Plants were grown in long days (16 h light/8 h dark) at
23.degree. C.
Plasmid Construction and Generation of Transgenic Arabidopsis
Plants
[0255] Procedures for plasmid construction and primer sequences
used for PCR amplification are provided in Supplemental
Experimental Procedures.
Identification of T-DNA Mutants of GRF1 and GRF3
[0256] Two independent T-DNA insertional alleles of GRF1
(Salk.sub.--069339C and Salk.sub.--078547C) or GRF3
(Salk.sub.--026786 and Salk.sub.--116709) in the Col-0 background
were obtained from the Salk T-DNA insertional mutant collection
(Alonso et al., 2003).
Histochemical Analysis of GUS Activities
[0257] The histochemical staining of GUS enzyme activity was
performed according to Jefferson et al. (1987). Tissue samples were
viewed using a Zeiss SV-11 microscope and the images were captured
using a Zeiss AxioCam MRc5 digital camera and then processed using
Zeiss Axiovision software (release 4.8).
Nematode Infection Assay
[0258] Ten-day-old seedlings were inoculated with approximately 200
surface-sterilized J2 H. schachtii nematodes per plant (see
Supplemental Experimental Procedures for details).
Nematode Development Assay
[0259] Plants were grown on modified Knop's medium in 12-well
culture plates. At 10 days, each plant was inoculated with 200
surface-sterilized J2 of H. schachtii, and plants were assessed at
5 and 21 days post infection for parasitic-stage juveniles and
females, respectively. Average numbers of developing nematodes were
calculated for each time point, and statistically significant
differences were determined in a modified test using the
statistical software package SAS.
Root Length Measurements
[0260] Arabidopsis plants were grown vertically on modified Knop's
medium for ten days and then the root length of at least 30 plants
per line was measured as the distance between the crown and the tip
of the main root in three independent experiments. Statistically
significant differences between lines were determined by unadjusted
paired t test (P<0.01).
Syncytial Measurements
[0261] Arabidopsis seeds were planted on modified Knop's medium and
10-day-old seedlings were inoculated with .about.200
surface-sterilized J2 H. schachtii. For each line, at least 20
single-female syncytia were randomly selected, photographed and
measured as previously described by Hewezi et al. (2008b).
RNA Isolation and qPCR
[0262] Total RNA was extracted from root tissues using the TRIzol
reagent (Invitrogen, Carlsbad, Calif., U.S.A.) following the
manufacturer's instructions. DNase treatment of total RNA was
carried out using Deoxyribonuclease I (Invitrogen). The treated
total RNA (5 .mu.g) was polyadenylated and reverse transcribed
using "Mir-X miRNA First-Strand Synthesis Kit" (Clontech, Mountain
View, Calif., USA) following the manufacturer's instructions. The
synthesized cDNAs then were diluted to a concentration equivalent
to 10 ng total RNA/.mu.L and used as a template in real-time RT-PCR
reactions to quantify both miRNA and GRF expression levels using
the two-step RT-PCR kit (Bio-Rad) according to the manufacturer's
protocol. PCR conditions and primer sequences are provided in the
Supplemental Experimental Procedures.
Microarray Analysis
[0263] Arabidopsis plants were grown vertically on modified Knop's
medium for 2 weeks and then root tissues were collected for RNA
extraction. Affymetrix Arabidopsis gene chips (ATH1) were used to
compare the gene expression in the wild type to gene expression in
the triple mutant and the rGRF1 or rGRF3 plants. Probe preparation
was performed as described in the GeneChip.RTM. 3' IVT Express Kit
(Affymetrix, part number 901229) technical manual. Hybridization
and washes were performed as described by Affymetrix in the
GeneChip facility at Iowa State University. Statistical analyses of
gene expression levels are detailed in the Supplemental
Experimental Procedures. Testing for the significance of gene list
overlaps was determined using Chi-square tests. See Supplemental
Experimental Procedures for details.
Accession Numbers
[0264] Sequence data from this article can be found in the
Arabidopsis Genome Initiative or GenBank/EMBL databases under the
following accession numbers:
miR396a (AT2G10606) (SEQ ID NO:1), miR396b (AT5G35407)) (SEQ ID
NO:2), GRF1 (At2g22840) (SEQ ID NO:3), GRF2 (At4g37740)) (SEQ ID
NO:4), GRF3 (At2g36400)) (SEQ ID NO:5), GRF4 (At3g52910)) (SEQ ID
NO:6), GRF5 (At3g13960)) (SEQ ID NO:7), GRF6 (At2g06200)) (SEQ ID
NO:8), GRF7 (At5g53660) (SEQ ID NO:9), GRF8 (At4g24150) (SEQ ID
NO:10), GRF9 (At2g45480)) (SEQ ID NO:11),
and Actin8 (AT1G49240),
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TABLE-US-00001 [0301] Construct or Gene Name Primer Sequence 5'- 3'
Primer sequences for overexpression and promoter constructs
Overexpression miR396a miR396a-BamHI_F
TATAGGATCCTAGGGTTTCGTCTGCTCTACATGACCC miR396a-SacI_R
ATGATGAGCTCCGAAATTTAGAAAATCATTTGACTCT overexpression miR396b
miR396b-BamHI_F TATAGGATCCTCAGAAGAAGGAGAAGATGAAGATCC miR396b-SacI_R
ATGATGAGCTCGTGAATCAATGGAGTAAAACCCTGAAT Overexpression wtGRF1
GRF1-XbaI_F1 TATATCTAGAATGGATCTTGGAGTTCGTGTTTCTGG GRF1-SacI_R2
ATGATGAGCTCTCACAGAGAAGGAGCAGTAGCAGAAG Overexpression rGRF1 GRF1_F2
GAGGCCGCCATAGAAGCAGGAAACCGGTAGAGGGCCAAAATG GRF1_R1
CATTTTGGCCCTCTACCGGTTTCCTGCTTCTATGGCGGCCTC Overexpression wtGRF3
GRF3-XbaI_F1 TATATCTAGAATGGATTTGCAACTGAAACAATGGAG GRF3-SalI_R2
ATGATGTCGACTCAATGAAAGGCTTGTGTCGAGACAC Overexpression rGRF3 GRF3_F2
GTGGCCGCAACAGGAGCCGTAAACCGGTCGAGACTCCAACCA GRF3_R1
TGGTTGGAGTCTCGACCGGTTTACGGCTCCTGTTGCGGCCAC Promoter miR396a: GUS
pmiR396a-XbaI_F TATATCTAGACTTGATTGTTTATTTTATCGTTTTGTG
pmiR396a-BamHI_R ATGATGGATCCAGGGTCATGTAGAGCAGACGAAACCCTA Promoter
miR396b: GUS pmiR396b-XbaI_F
TATATCTAGAACCGCAACTTTCTGTTATGATATTGATGG pmiR396b-BamHI_R
ATGATGGATCCAGGATCTTCATCTTCTCCTTCTTCTGAAA Promoter GRF1: GUS
pGRF1-HindIII_F TATAAAGCTTTGTTAATTTTATCAAATGTATATTCTT pG RF1-SalI_R
ATCATGTCGACAAAAAATGGATTCAGAAGGAGACAAAG Promoter GRF3: GUS pG
RF3-SalI_F TATAGTCGACGCTGAGACTCTGTGGAAGCCGTTCGC pGRF3-BamHI_R
ATGATGGATCCTGAAGAAAGAGAGAGAGAAGTGTTGG Gene-specific primer
sequences used for qPCR Pri-miR396a Pri-miR396a_F
CAGCTTTCTTGAACTGCAAAAC Pri-miR396b Pri-miR396b_F
GGTCATACTTTTCCACAGCTTTC Mature miR396 Mature miR396_F
TTCCACAGCTTTCTTGAACTGAA wtGRF1 wtGRF1_F TCGTTCAAGAAAGCCTGTGGAAGG
wtGRF1_R GTTCCAACAGCAGCGGCAAGGC rGRF1 rGRF1_F
AGAAGCAGGAAACCGGTAGAGGG GRF2 GRF2_F CCCGAATACCGCAAAGACCT GRF2_R
GTTGTGTGTGGAGGAAGGGGA wtGRF3 wtGRF3_F CCGTTCAAGAAAGCCTGTGGAAAC
wtGRF3_R TCCTCCTTGACCAACCACTTCCT rGRF3 rGRF3_F
CAGGAGCCGTAAACCGGTCGAG GRF4 GRF4_F ACCGCCACAACCACCATCACA GRF4_R
TCCATTGCTGAATCCACTGTTAGCT GRF5 GRF5_F TGGAGGAGTTGGGGAGAGAACG GRF5_R
GTTGAACATGTCGGCGCCCAA GRF6 GRF6_F CGAGGAGAAGCAGCCGGATCGAC GRF6_R
CCTCTTGCTTCCTTGCTCTTCTTC GRF7 GRF7_F GGGCCAAGACGAAATGGGCCT GRF7_R
CCGCTAATGGTCCACCAGGTG GRF8 GRF8_F GGCTGGAGGAGGCATGGAGG GRF8_R
GGAGACACCGAGACACAGTGC GRF9 GRF9_F CGGCACATGCATAGAGGTCGT GRF9_R
CAGGATCTGGCACTAGGCAGTG Actin8, Actin8_F AGTGGTCGTACAACCGGTATTGT
Actin8_R GAGGATAGCATGTGGAACTGAGAA
Example 2
miR396 in Soybean During Cyst Nematode Infection
[0302] In order to understand the role that miR396 plays during
soybean infection by the soybean cyst nematode (Heterodera
glycines; SCN), expression analyses were performed on primary and
mature sequences for all miR396 paralogs (miR396a, miR396b, miR396c
and miR396e) and seven of its predicted target Growth Regulating
Transcription Factors (GRF8, GRF9, GRF12, GRF13, GRF15, GRF16 and
GRF19) using quantitative real-time PCR (qRT-PCR). Soybean
seedlings were infected with SCN three days after germination and
RNA was extracted 2, 4, 8 and 14 days post inoculation (dpi). RNA
from both SCN infected and mock inoculated soybean seedlings was
reverse-transcribed into cDNA for qRT-PCR.
[0303] Data were analyzed using the comparative Ct method with U6
snRNA as the reference gene for microRNA quantification and
ubiquitin for GRFs. Significance tests were performed using the
Student's t-test (p-value<0.05) and significant values are
indicated on the graph with asterisks. Error bars represent the
standard error. Three to four biological replicates were used for
each sequence at each time point as well as three technical
replicates during qRT-PCR.
[0304] In summary, steady-state RNA levels for miR396 and its
target genes in soybean during SCN infection very closely resembled
the observations made in Arabidopsis: an early downregulation of
mature miR396 with a simultaneous increase in GRF mRNA at the time
of syncytium formation. At later time points, likely coinciding
with the end of syncytium formation, abundance of mature miR396
increases and GRF target gene expression is turned off.
Consequently, there is a high probability that the manipulations we
performed in Arabidopsis and that resulted in decreased plant
susceptibility will have similar effects on susceptibility of
soybean to SCN. Results are shown in FIG. 12.
Sequence Information
[0305] Some sequences have not yet been submitted to NCBI and thus
do not have accession numbers; locus IDs obtained from Soybase.
gma-precursor-miR396a
Accession #: MI0001785 (SEQ ID NO:12)
[0306] gma-mature-miR396a
Accession #: MIMAT0001687 (SEQ ID NO:13)
[0307] gma-precursor-miR396b
Accession #: MI0001786 (SEQ ID NO:14)
[0308] gma-mature-miR396b
Accession #: MIMAT0001688 (SEQ ID NO:15)
[0309] gma-precursor-miR396c
Accession #: MI0010572 (SEQ ID NO:16)
[0310] gma-mature-miR396c
Accession #: MIMAT0010079 (SEQ ID NO:17)
[0311] gma-precursor-miR396e (SEQ ID NO:18)
Accession #: MI0016586
[0312] gma-mature-miR396e
Accession #: MIMAT0018345 (SEQ ID NO:19)
GmGRF8
Accession #: n/a
[0313] Locus ID: Glyma10g07790 (SEQ ID NO:20)
GmGRF9
Accession #: XM.sub.--003537618 (SEQ ID NO:21)
GmGRF12
Accession #: n/a
[0314] Locus ID: Glyma13g16920 (SEQ ID NO:22)
GmGRF13
Accession #: n/a
[0315] Locus ID: Glyma13g21630 (SEQ ID NO:23)
GmGRF15
Accession #: XM.sub.--003547454 (SEQ ID NO:24)
GmGRF16
[0316] Accession #: n/a Locus ID: Glyma16g00970 (SEQ ID NO:25)
GmGRF19
Accession #: XM.sub.--003553541 (SEQ ID NO:26)
[0317] Other Sequences
[0318] Sequences for soybean miRNA396 may be found at miRBase dot
org at world wide web including accession numbers MIMAT0020922
(gma-miR3961-3p), MIMAT0001688 (gma-miR396B-5p), MIMAT0020923
(gma-miR396b-3p). MIMAT0010079 (gma-miR396c), MIMAT0018262
(gma-miR396d). Other miR396 sequences available from different
plant species include but are not limited to:
TABLE-US-00002 "miR396a" Accession ID MI0001013 ath-MIR396a
MI0001046 osa-MIR396a MI0001539 sbi-MIR396a MI0001785 gma-MIR396a
MI0001801 zma-MIR396a MI0002325 ptc-MIR396a MI0005621 mtr-MIR396a
MI0005650 ghr-MIR396a MI0005773 bna-MIR396a MI0006569 vvi-MIR396a
MI0012094 aqc-MIR396a MI0014581 aly-MIR396a MI0016122 pab-MIR396a
MI0016706 csi-MIR396a MI0016983 bgy-MIR396a MI0016987 bcy-MIR396a
MI0017511 tcc-MIR396a MI0018111 bdi-MIR396a MIMAT0001687
gma-MIR396a-5p
TABLE-US-00003 "miR396b" Accession ID MI0001014 ath-MIR396b
MI0001047 osa-MIR396b MI0001538 sbi-MIR396b MI0001786 gma-MIR396b
MI0001800 zma-MIR396b MI0002326 ptc-MIR396b MI0005622 mtr-MIR396b
MI0005651 ghr-MIR396b MI0006570 vvi-MIR396b MI0012095 aqc-MIR396b
MI10014582 aly-MIR396b MI0016123 pab-MIR396b MI0016707 csi-MIR396b
MI0016984 bgy-MIR396b MI0016988 bcy-MIR396b MI0017512 tcc-MIR396b
M10018125 bdi-MIR396b MIMAT0001688 gma-MIR396b-5p
TABLE-US-00004 "miR396c" Accession ID MI0001048 osa-MIR396c
MI0001540 sbi-MIR396c MI0002327 ptc-MIR396c MI0007955 vvi-MIR396c
MI0010569 zma-MIR396c MI0010572 gma-MIR396c MI0016124 pab-MIR396c
MI0016735 csi-MIR396c MI0017513 tcc-MIR396c MI0018101
bdi-MIR396c
TABLE-US-00005 "miR396d" Accession ID MI0001702 osa-MIR396d
MI0002328 ptc-MIR396d MI0006571 vvi-MIR396d MI0010570 zma-MIR396d
MI0010897 sbi-MIR396d MI0016503 gma-MIR396d MI0017514 tcc-MIR396c
MI0018096 bdi-MIR396d MI0001013 ath-MIR396a MI0001014 ath-MIR396b
MI0001046 Osa-MIR396a MI0001047 osa-MIR396b MI0001048
osa-MIR396c
[0319] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated by reference.
[0320] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the invention
as described in the appended claims.
Sequence CWU 1
1
281151DNAArabidopsis thaliana 1ctctgtattc ttccacagct ttcttgaact
gcaaaacttc ttcagatttt tttttttttc 60ttttgatatc tcttacgcat aaaatagtga
ttttcttcat atctctgctc gattgatttg 120cggttcaata aagctgtggg
aagatacaga c 1512135DNAArabidopsis thaliana 2ggtcatactt ttccacagct
ttcttgaact ttctttttca tttccattgt ttttttctta 60aacaaaagta agaagaaaaa
aaactttaag attaagcatt ttggaagctc aagaaagctg 120tgggaaaaca tgaca
13531593DNAArabidopsis thaliana 3atggatcttg gagttcgtgt ttctggtcat
gaaaccgttt cttctccggg tcaaactgaa 60ctcggatctg gtttcagtaa caagcaagaa
agatccggtt tcgatggtga agattgctgg 120agaagttcaa agctctcacg
aacatcaact gatggattct cttcttcccc tgcctctgct 180aaaacgctgt
cgtttcatca aggcatccct ttactgagat ctaccactat taatgatcct
240cgtaaaggac aagaacacat gcttagcttc tcttctgctt caggcaaatc
agatgtctca 300ccttatcttc agtactgtag aaactcagga tatggtttag
gaggaatgat gaacacaagc 360aacatgcatg gaaacttgtt gacaggagta
aaaggacctt tttcattgac tcagtgggca 420gagctagagc aacaggcgtt
gatctataag tatatcacag ccaatgtccc tgttccatct 480agtttacttc
tctctctcaa gaaatctttt ttcccttatg gttccttgcc tcctaattct
540tttggatggg gctcttttca tctgggcttt tccggtggta acatggatcc
cgagccaggg 600agatgtcgcc ggacagatgg aaagaaatgg cggtgctcga
gggacgctgt tcccgatcaa 660aagtactgtg aacgacatat taacagaggc
cgccatcgtt caagaaagcc tgtggaaggc 720caaaatggcc acaatactaa
tgctgccgcc gctgcttctg ctgctgccgc ttctaccgct 780gctgctgtgt
ccaaagcggc agcggggact tcagctgttg cgatgcgtgg atcagataat
840aacaatagcc ttgccgctgc tgttggaaca caacatcata ccaataatca
atctacagat 900tctttggcta acagagttca aaattctcga ggggcttcgg
tttttcctgc cacgatgaac 960ttacagtcga aggaaactca tccgaaacaa
agcaataatc cctttgaatt cggactcatc 1020tcttctgatt cgttacttaa
tccgtcgcat aaacaagcct cgtatgcaac ctcttccaaa 1080ggctttggat
cgtatcttga cttcggcaac caagccaagc acgcggggaa tcacaacaat
1140gtcgattctt ggcccgaaga gctgaaatcg gattggactc agctctcaat
gtcaatccct 1200atggctccat cttcccctgt tcaagataaa cttgcactct
cacctttaag gttatcgcgt 1260gagtttgacc ccgcgatcca catgggatta
ggcgtcaaca ccgagtttct tgaccccggg 1320aaaaagacga ataactggat
accaatctcc tggggtaata acaactccat gggaggtcca 1380ctcggcgagg
tactaaacag cacgaccaat agtcccaagt ttggttcctc tccaacaggc
1440gtcttgcaaa agtcgacatt tggttctctt tctaacagca gctcggcaag
cagcaccatc 1500attggcgata acaacaataa gaacggtgat ggaaaagatc
cgcttggccc gaccacgctg 1560atgaatactt ctgctactgc tccttctctg tga
159341608DNAArabidopsis thaliana 4atggatattg gtgttcatgt tcttgggtcg
gttactagta atgaaaatga gtcacttggt 60ctaaaagagc ttataggaac taaacaagat
agatccggat tcatcggtga ggattgcttg 120caacgaagct tgaagctagc
aagaacgaca actagagcgg aagaagaaga aaacttgtct 180tcttctgttg
cagctgctta ttgcaaaacg atgtcgtttc accaaggcat tcctctcatg
240agatctgctt ctcctctttc ctctgattct cgccgtcaag aacaaatgct
tagcttctca 300gataaaccag acgctcttga tttcagtaaa tatgtcggtt
tggataatag cagtaataac 360aagaactctc tctcgccgtt tcttcaccag
attcctccac cttcttactt tagaagctca 420ggaggatatg gttctggtgg
aatgatgatg aacatgagca tgcaagggaa cttcacaggt 480gttaaaggac
cttttacatt gactcaatgg gctgagttag agcaacaggc gttgatctat
540aagtacatca cagccaatgt ccctgttcct tctagtttgc tcatctctat
caagaagtct 600ttttatcctt acggatcttt gcctcctagt tccttcggat
ggggaacttt ccatctcggt 660ttcgcaggcg gtaacatgga ccctgagcca
gggagatgcc gcagaacaga tgggaagaaa 720tggcggtgct caagagacgc
cgttcctgat cagaaatact gtgaaagaca catcaacaga 780ggccgtcatc
gttcaagaaa gcctgtggaa gtccaatctg gccaaaacca aaccgccgct
840gctgcatcca aagcggttac tacaccacaa cagcctgttg tcgctggtaa
tactaacaga 900agcaatgccc gtgcatcaag caaccgcagc ctcgccattg
gaagtcaata tatcaatcct 960tctacagaat ctttacctaa caacagagga
gtttcgatat atccttccac cgtcaactta 1020caacccaagg aatctccggt
tattcatcag aaacacagaa acaacaacaa cccttttgag 1080tttggacaca
tatcctctga ttcgttactc aacccgaata ccgcaaagac ctatggatca
1140tcgttcttgg atttcagcag caaccaagag aagcattcag ggaatcacaa
tcacaattct 1200tggcctgaag agctgacatc agattggaca cagctctcaa
tgtcaattcc aatagcatca 1260tcatcccctt cctccacaca caacaacaac
aatgctcaag aaaaaacaac actctcgcct 1320ctcaggctat cccgcgagct
tgacctatcg atccaaaccg atgaaacaac aatcgagcct 1380actgtgaaaa
aggtgaatac ttggatacca atctcatggg gaaactcctt aggaggtcct
1440ctaggtgaag tactaaacag tacaacgaat agtccaacat ttggatcttc
tcctacaggg 1500gttttgcaaa agtccacatt ttgttcactc tctaacaaca
gctccgtgag cagccccatt 1560gcagagaaca acagacacaa tggcgattac
tttcattaca caacctga 160851197DNAArabidopsis thaliana 5atggatttgc
aactgaaaca atggagaagc cagcagcagc aacaacatca gacagagtca 60gaagaacaac
cttctgcagc taagatacca aaacatgtct ttgaccagat tcattctcac
120actgcaactt ctactgctct tcctctcttt acccctgagc ctacttcttc
taaactctcc 180tctttgtctc ctgattcttc ctccaggttc cccaagatgg
ggagcttctt tagctgggca 240cagtggcaag aacttgaact acaagctctg
atctacaggt acatgttggc tggtgctgct 300gttcctcagg agctcctttt
accaatcaag aaaagccttc tccatctatc tccttcctac 360tttcttcacc
atcctcttca acacctacct cattaccaac ctgcttggta tttgggaagg
420gcagcgatgg atcctgagcc aggcagatgc aggagaacgg atggtaagaa
gtggagatgt 480tcaagagacg tcttcgctgg ccacaagtat tgcgagcgcc
acatgcaccg tggccgcaac 540cgttcaagaa agcctgtgga aactccaacc
accgtcaatg caactgccac gtccatggct 600tcatcagtag cagccgcagc
caccactaca acagcaacaa caacatctac gtttgctttt 660ggtggtggtg
gtggtagtga ggaagtggtt ggtcaaggag gatctttctt cttctctggc
720tcttctaact cttcatctga acttctccac cttagtcaaa gttgttcgga
gatgaagcaa 780gaaagcaaca acatgaacaa caagaggcca tacgagtccc
acatcggatt cagtaacaac 840agatcagatg gaggacacat cctgaggccc
ttctttgacg attggcctcg ttcttcgctc 900caagaagctg acaatagttc
aagccccatg agctcagcca cttgtctctc catctccatg 960cccgggaact
cttcctcaga cgtctctctg aagctgtcca caggcaacga agagggagcc
1020cggagcaaca acaatgggag agatcagcaa aacatgagct ggtggagcgg
tggaggttcc 1080aaccaccatc atcacaacat gggcggacca ttggccgaag
ccctgagatc ttcttcctca 1140tcttccccaa ccagtgttct ccatcagctt
ggtgtctcga cacaagcctt tcattga 119761143DNAArabidopsis thaliana
6atggacttgc aactgaaaca atggagaagt cagcagcaga atgagtcaga agaacaaggc
60tctgctgcaa ctaagatatc aaactttttc tttgatcaga ttcagtccca aactgctact
120tctgctgctg cggctcctct tcctctcttt gtccctgaac ccacttcttc
ctcttctttc 180tcttgcttct ctcctgactc ttctaattct tcttcttctt
ccaggttcct caagatggga 240aacttcttca gctgggcaca gtggcaagaa
cttgagctac aagcactgat ctatagatac 300atgttggctg gtgcttctgt
tcctcaagag cttctcttac ctattaagaa aagtctcctc 360catcaatctc
ctatgcattt ccttcaccat cctcttcaac atagttttcc tcatcaccaa
420ccttcttggt attggggaag aggagcaatg gatcctgagc cagggaggtg
taagagaact 480gacggcaaga aatggagatg ttcaagggat gttgtagcgg
gccacaagta ttgtgaccgc 540cacattcacc gtggaagaaa ccgttcaaga
aagcctgtgg aaaccgccac aaccaccatc 600acaacgacag ccacaacaac
cgcatcttct tttgtcttag gtgaggagct tggtcatgga 660ccaaacaaca
accacttctt ctcctctggt tcatctcaac ctctccacct tagtcatcaa
720caaagttgtt cttcagagat gaaacaagaa agcaacaaca acaagaggcc
atatgaagct 780aacagtggat tcagcaatgg aagatcagac gatggtcaca
tcttgaggca tttctttgac 840gattggccac gatcatcaga ctctacctcc
agtccaatga gctcatccac ttgtcatctt 900tcaatctcca tgcccggtaa
caacacgtcc tcagatgttt ctctaaaact ttccacaggc 960aatgaagaag
aagaagagaa catgagaaat aacaacaatg agagggagca aatgaattgg
1020tggagcaatg gagggaatca ccacaacaat atgggaggac cattagctga
ggctttgagg 1080tcagcttctt cgacgtcaag tgttcttcat cagatgggaa
tctctactca agtttttcat 1140taa 114371194DNAArabidopsis thaliana
7atgatgagtc taagtggaag tagcgggaga acaataggaa ggcctccatt tacaccaaca
60caatgggaag aactggaaca tcaagcccta atctacaagt acatggtctc tggtgttcct
120gtcccacctg agctcatctt ctccattaga agaagcttgg acacttcctt
ggtctctaga 180ctccttcctc accaatccct tggatggggg tgttaccaga
tgggatttgg gagaaaacca 240gatccagagc caggaagatg cagaagaaca
gatggtaaga aatggagatg ctcaagagaa 300gcttacccag attcgaagta
ctgtgaaaaa cacatgcaca gaggaagaaa ccgtgccaga 360aaatctcttg
atcagaatca gacaacaaca actcctttaa catcaccatc tctctcattc
420accaacaaca acaacccaag tcccaccttg tcttcttctt cttcctctaa
ttcctcttct 480actacttatt ctgcttcttc ttcttcaatg gatgcctaca
gtaacagtaa taggtttggg 540cttggtggaa gtagtagtaa cactagaggt
tatttcaaca gccattctct tgattatcct 600tatccttcta cttcacccaa
acaacaacaa caaactcttc atcatgcttc cgctttgtca 660cttcatcaaa
atactaattc tacttctcag ttcaatgtct tagcctctgc tactgaccac
720aaagacttca ggtactttca agggattggg gagagagttg gaggagttgg
ggagagaacg 780ttctttccag aagcatctag aagctttcaa gattctccat
accatcatca ccaacaaccg 840ttagcaacag tgatgaatga tccgtaccac
cactgtagta ctgatcataa taagattgat 900catcatcaca catactcatc
ctcatcatca tctcaacatc ttcatcatga tcatgatcat 960agacagcaac
agtgttttgt tttgggcgcc gacatgttca acaaacctac aagaagtgtc
1020cttgcaaact catcaagaca agatcaaaat caagaagaag atgagaaaga
ttcatcagag 1080tcgtccaaga agtctctaca tcacttcttt ggtgaggact
gggcacagaa caagaacagt 1140tcagattctt ggcttgacct ttcttcccac
tcaagactcg acactggtag ctaa 11948735DNAArabidopsis thaliana
8atggctacaa ggattccatt cacagaatca caatgggaag aacttgaaaa ccaagctctt
60gtgttcaagt acttagctgc aaatatgcct gttccacctc atcttctctt cctcatcaaa
120agaccctttc tcttctcttc ttcttcttct tcatcttctt cttcaagctt
cttctctccc 180actctttctc cacactttgg gtggaatgtg tatgagatgg
gaatgggaag aaagatagat 240gcagagccag gaagatgtag aagaactgat
ggcaagaaat ggagatgctc taaagaagct 300taccctgact ctaagtactg
tgagagacat atgcatagag gcaagaaccg ttcttcctca 360agaaagcctc
ctcctactca attcactcca aatctctttc tcgactcttc ttccagaaga
420agaagaagtg gatacatgga tgatttcttc tccatagaac cttccgggtc
aatcaaaagc 480tgctctggct cagcaatgga agataatgat gatggctcat
gtagaggcat caacaacgag 540gagaagcagc cggatcgaca ttgcttcatc
cttggtactg acttgaggac acgtgagagg 600ccattgatgt tagaggagaa
gctgaaacaa agagatcatg ataatgaaga agagcaagga 660agcaagaggt
tttataggtt tcttgatgaa tggccttctt ctaaatcttc tgtttctact
720tcactcttca tttga 73591098DNAArabidopsis thaliana 9atggactttc
tcaaagtttc agacaagaca acaattccat atagaagtga ttctttgttt 60agtttgaatc
agcaacaata caaagagtct tcttttggat tcagagacat ggagattcat
120ccgcatccta ctccatatgc aggaaatgga cttttgggtt gttattacta
ttaccctttc 180acaaacgcac aattgaagga gcttgagaga caagcaatga
tctacaagta catgatcgca 240tctattcctg ttcctttcga tctacttgtt
tcttcaccat cctctgcctc tccttgtaac 300aataaaaaca tcgccggaga
tttagagccg ggaagatgcc ggagaacaga cggaaagaaa 360tggagatgcg
cgaaagaagt cgtctctaat cacaaatact gtgagaaaca cttacacaga
420ggtcgtcctc gttcaagaaa gcatgtggaa cctccttatt ctcgccctaa
caacaatggt 480ggttctgtga aaaacagaga tctcaaaaag cttcctcaaa
agttatctag tagttccatc 540aaagacaaaa cacttgagcc aatggaggtt
tcatcatcaa tctcaaacta tagagactcc 600agaggaagtg agaaatttac
tgtattggca acaacagagc aagagaacaa gtatctgaat 660ttcatagatg
tatggtccga tggagtaaga tcatctgaaa aacagagtac aacttcaaca
720cctgtttctt cttccaatgg caatctctct ctttactcgc ttgatctctc
aatgggagga 780aacaacttaa tgggccaaga cgaaatgggc ctgatacaaa
tgggcttagg tgtaatcggg 840tcgggtagtg aggatcatca cgggtatggt
ccttatggtg tgacttcttc actagaggag 900atgtcaagct ggcttgctcc
gatgtctacc acacctggtg gaccattagc ggagatactg 960aggccgagta
cgaatttggc gatctctggt gatatcgaat cgtatagctt gatggagact
1020cccactccaa gctcgtcccc gtctagagtg atgaagaaga tgactagttc
agtgtccgac 1080gaaagcagcc aggtttag 1098101482DNAArabidopsis
thaliana 10atgaggatgc ttcttgggat tccttacgta gacaagtcgg ttctttccaa
ctctgttctt 60gagagaggca agcaggataa aagcaaacta ttgttagtcg acaaatgcca
ttatgagctt 120gatgttgaag aacgcaagga agattttgtt ggtgggtttg
gatttggtgt tgtagaaaat 180tcgcataaag acgttatggt gctacctcat
catcactatt atccatcata ttcatcacct 240tcctcttctt ctttgtgtta
ctgttctgct ggtgttagcg atcccatgtt ctctgtttct 300agcaatcagg
cttacacttc ttctcacagt ggtatgttca cacccgccgg ttctggttct
360gctgctgtga ctgtagcaga tccttttttc tccttgagct cttcagggga
aatgagaaga 420agtatgaacg aagatgctgg tgcagctttc agcgaagctc
aatggcatga gcttgagagg 480cagaggaata tatacaagta catgatggct
tctgttcctg ttcctccaga gcttctcaca 540ccctttccca agaaccacca
atcaaacact aacccggatg tggatacata taggagtgga 600atgtttagta
tttatgctga ttacaagaat ctgccgttgt ctatgtggat gacagtaact
660gtggcagtgg cgacaggagg ctcattgcag ctggggattg cttcaagcgc
aagcaataac 720acggctgatc tggagccatg gaggtgcaag agaacagatg
ggaagaaatg gaggtgctct 780agaaacgtga ttcctgatca gaaatactgt
gagagacaca cacacaagag ccgtcctcgt 840tcaagaaagc atgtggaatc
atctcaccaa tcatctcacc acaatgacat tcgtacggct 900aagaatgata
ctagccagct tgtgagaact tatcctcagt tttacggaca acctataagc
960cagatccctg tgctttctac tcttccgtct gcctcctctc catatgatca
ccacagagga 1020ctgaggtggt ttacgaaaga agatgatgcc attggaacct
taaacccgga gactcaagaa 1080gctgtccagc tgaaagttgg atcaagcaga
gagctcaaac ggggattcga ttatgatctg 1140aatttcaggc agaaagagcc
aatagtagac cagagctttg gagcattgca gggtctatta 1200agtctaaacc
agacaccaca acataaccaa gaaacaagac agtttgttgt agaaggaaag
1260caagatgaag cgatgggaag ctctctgaca ctctcaatgg ctggaggagg
catggaggaa 1320acagagggaa caaaccagca tcagtgggtt agccatgaag
gtccatcatg gctctattca 1380acaacaccag gtggaccatt ggctgaagca
ctgtgtctcg gtgtctccaa caacccaagt 1440tctagtacta ctactagtag
ctgcagcaga agctcaagct aa 1482111290DNAArabidopsis thaliana
11atgcagagcc ctaaaatgga gcaggaggag gttgaggagg agaggatgag gaataagtgg
60ccgtggatga aggcggcgca gttaatggag tttcggatgc aagctttggt gtatagatac
120atagaggctg gtctccgtgt gcctcatcat ctcgtggtgc ctatttggaa
cagtcttgct 180ctctcttctt cctccaatta caactatcac tcttcttctc
tgttgagtaa caagggagta 240acccatatcg acacgttgga aactgaacca
actaggtgca ggagaacaga tgggaagaaa 300tggcgctgta gcaacacggt
ccttctattc gagaagtact gtgaacggca catgcataga 360ggtcgtaaac
gttcaagaaa gcttgtggaa tcttcttctg aggttgcttc atcatcaacc
420aaatacgaca acacttatgg tttggatagg tataacgaga gtcagagtca
tcttcatggg 480acaatctcgg gttctagtaa tgcgcaggta gttaccattg
cttcactgcc tagtgccaga 540tcctgtgaaa atgtcattcg tccgtcttta
gtgatctctg aattcacaaa caaaagtgtg 600agtcacggca gaaagaacat
ggagatgagt tatgatgact ttattaatga aaaagaggcg 660agtatgtgtg
ttggagttgt tcctcttcaa ggtgatgaga gcaaaccttc ggttcaaaag
720ttcttccctg aggtatctga taaatgctta gaagctgcaa aattctcaag
caacaggaag 780aatgatataa ttgcaagaag cagagaatgg aagaatatga
atgttaatgg tggtttgttt 840catggtatcc acttttctcc agacactgtt
cttcaagaac gtggttgttt tcgtttacaa 900ggagttgaaa cagacaatga
accaggaagg tgccgaagaa cagatgggaa gaagtggaga 960tgcagcaaag
atgttttgtc tggtcagaag tactgcgata agcacatgca tagaggtatg
1020aagaagaagc atccagttga tactactaac tcacatgaga atgccgggtt
tagcccgtta 1080accgtggaaa cagctgttag atcggttgtg ccttgcaaag
atggagatga ccagaagcat 1140tctgtttcag tcatgggaat tacactgccc
cgagtttctg atgagaagag cactagcagt 1200tgcagtaccg acactaccat
tactgacaca gctttaaggg gtgaagacga cgatgaggag 1260tacttgtctt
tgttttcacc aggtgtttag 129012140RNAGlycine max 12ucauggcucu
cuuuguauuc uuccacagcu uucuugaacu gcauccaaag aguuccuuug 60caugcaugcc
auggcacucu uacucccaaa ucuuguuuug cgguucaaua aagcuguggg
120aagauacaga uagggucaac 1401321RNAGlycine max 13uuccacagcu
uucuugaacu g 2114126RNAGlycine max 14cucaaguccu ggucaugcuu
uuccacagcu uucuugaacu ucuuaugcau cuuauaucuc 60uccaccucca ggauuuuaag
cccuagaagc ucaagaaagc ugugggagaa uauggcaauu 120caggcu
1261521RNAGlycine max 15uuccacagcu uucuugaacu u 2116117RNAGlycine
max 16caacaagucc uguuaugcuu uuccacagcu uucuugaacu ucuuaugccu
agugcaauua 60uugauguggc auagaaguuu aagaaaaaug uggaaaaaca ugucaaaucu
aggacuu 1171721RNAGlycine max 17uuccacagcu uucuugaacu u
2118114RNAGlycine max 18gugaucuucc acagcuuucu ugaacugugu ugugaggcuu
cucuccaaug aagguuuaua 60cccuaugcaa aagaaauucu augagcacaa uucaagauag
cuguggaaaa ucac 1141922RNAGlycine max 19uuccacagcu uucuugaacu gu
22201200DNAGlycine max 20atggacttgc agttgaagca atggagaaac
cagcatgagt cagagcaaga acaagaacat 60tattccccaa acatggcaaa atttctatct
caacaacaac acccaccacc atttccctct 120gcactccctc tctttgtacc
tgaacaaccc aacaccaaag tcagcacctt gtcagcattt 180tctgattcca
cattaccctc ttctcccaga tttcccagaa tggagagttg cttcagcttt
240gcacaatggc aagagcttga gttgcaggct ctgatattca ggtacatgct
ggccggtgct 300cctgttcctc ctgagctcct tctaccaatc aagaaaagct
tccttcaact ttatcaccct 360cctaattgta aatttctaac cccatttttc
tacttcctaa ttatttggta ctactggcga 420agagaagcac tggatccgga
gccggggcgg tgccggagga ccgacggcaa gaagtggcgg 480tgctcgaagg
acacggtggc aggtcagaag tactgcgacc gccacatgca ccgtggccgg
540aaccgttcaa gaaagcctgt ggaacaacgt gaaggatctc tttctgctat
agactctgtt 600tcttcttcac actctgcttc attcaatctc cttcacctcg
gtcaaagaga atggcttgga 660aaaggtttaa ggctcaagtc ctctatttca
tcatatcctt tgaggttgtg ctgttccgct 720gtggccaaga gtgacagcaa
gagcttgtct agaaaccgtg atcatgtgga tggggatggc 780aaatcaaatg
gccatgtctt gaggcatttc tttgatgatt ggccaaggac actgcaagag
840cctgacaatg gtgaaagcaa tggaagccag aacaacaact caggaaaatg
tctttctatg 900tcaacaccag gaatcgatcc ctcggatgtg tcgttgaaat
tgaccactgg ctatggagag 960gacgcgtgcc aggcagcttc ggtgggagga
ccacttgcag aggcattgag atcatccacc 1020accagctcca cttcttcacc
aaccataaat aggaaacaaa cattttcgga taatgaacaa 1080gttgagtttg
tagttagcaa ttgttggcat ggtcttgttt ctttaaccaa atccatactt
1140tactgccaag cacatgcaca cattacagat agagttctga aaagtcacat
gaggaggtga 1200211016DNAGlycine max 21actcaactaa actaaactaa
agaaaagaat aataaaaagg agaagatggt ttagaattgg 60tttgagaaga gaagagtggg
ggaggagaga tgagtaagtg gcctttcaca atatctcagt 120ggcaggaact
ggaacatcaa gctttaattt acaaatacat ggtggctggt cttcctgtgc
180ctcctgatct agtcattccc attcagaaca gcttccactc catttcccaa
accttcttgc 240accatccctc taccaccatg agttattgtt ccttctatgg
gaagaaggtg gacccggagc 300caggacgatg caggaggact gatgggaaaa
agtggaggtg ctccaaggaa gcctacccag 360actccaagta ctgtgagcga
cacatgcacc gtggccgcaa ccgttcaaga aagcctgtgg 420aatcacaaac
tatgacacag tcatcatcca atgtgtcatc attgactgta actgctggca
480gcagcaccag tgcaactgga aatttccaga acctttccac cacaaatgca
tatggtaatc
540cccaagggac tgcttctgga acagaccaaa cccactatca catggattcc
attccctatg 600ggatcccaag taaagaatac aggtattttc aaggatctaa
atctgaggaa catagtttct 660tgtccaaaac tttaggaagc aacagggttc
tacacatgga gccacagatg gacaacactt 720tgatgccaac cggtggagtt
gcctcattct ctacattgag atcaaataat aattccatgt 780tgcagggtga
ttatctgcag ccttctttct tatctagtga atatgcctcg gcagaaactg
840tgaagcaaga gggtcagtcc cttcgaccgt tctttgatga atggcctaaa
agcagggact 900catggtctgg tctggaagat gagagatcca atcacactca
actctcaata tccattccta 960tgtcatcgtc aaatttctct gcaactagct
ctcattcccc acatggtgag atttaa 1016221242DNAGlycine max 22atgaatggaa
ggaacgttaa cacaaacagg ttccctttca ctccttccca gtggcaagag 60cttgaacacc
aagctctcat ctacaaatac atggcttcag gtatctccat cccccctgac
120cttctcttca ccatcaaaag aacaacccac ttggactcct caagactatt
gcctcaccac 180cctcaacact ttggatggaa ctatttgcca atggggttgg
ggaggaaaat agacccggag 240ccagggaggt gtagaagaac agatggaaag
aaatggaggt gctcaaagga ggcgtatcca 300gattcaaagt actgtgagag
gcacatgcac agagggaaaa atcgttcaag aaagcctgtg 360gaagttttga
aaacaacacc aacgacagca gcagtggcaa caaacacaga tgcctcaacc
420ccaacaacaa tcttatcaat caccaaaaac agtcctgcac atgcactctc
cccaaccact 480cattctctct ctcatgacac ttaccatcat catcatcatc
accctcaccc tcagcaacat 540tcctcccact ccttcctcta tcatcattct
tcgaggccct cttccgtatt tattgagaag 600tttatactaa caagcctgct
tggcttgatg aagtatgtgt atggactgaa ggaggaggtg 660gacgagcatg
ccttcttcac agaaccttct ggaactatga gaagcttctc tgcttcctca
720atggaagatt catggcaact cacaccactg actataagct cctcttcctc
ttcgaaacag 780aggaactgct ctggtttatc caatgacaac aacgagtact
cctacttgca acttcagagc 840ctcaatggca acaactcaaa acagccacaa
caagatcaag gttgctacat atcaggcagt 900gatgtcaagt gcgaaacatt
catgaaactt gggaaagaag aacctcagaa aaccgttcat 960cgcttcttcg
atgaatggcc ccctaaaagc agaggatcgt ggcttgattt ggatgataaa
1020tcatccacca cccagctttc aatttccatt ccaacttcta ctcatgattt
tgcaactttc 1080aattcaacaa cccaacgagt agctagggaa ttgtttatga
cttgcatttt ttgtttttgc 1140agatggttga gtttagcttt caacagtggg
gtccttcagt ccttgtactt gaatcaaagg 1200ccaaaaatgt acttacatgg
tggggttcac atcatgttgt ga 1242231266DNAGlycine max 23atggacttgc
agttgaagca atggagtaac cggcatgagt cagaacaaga acattattcc 60ccaaacatgc
caaaatttct ccctcaacac cacccaccac catctccctc tgcactccct
120ctctttgtac ctgaacaacc caacaccaaa gtctgcaccc taaacaaaca
aaggatggag 180agttgcttca gctttgcaca gtggcaagag cttgagttgc
aggctctgat attcaggtac 240atgctggccg gtgctcctgt tcctcctgag
ctccttctac caatcaagaa aagcttcctt 300caactttata accctccttg
tgaaactcta tttctggttg aaaactgtgt aaaagcagct 360ataaaactgt
atttaagttt tatgctattt atagttgatt tgtctagttt tgagttactc
420cattttggtg agaaaattgt gatgaaagtt ttgatcttgg gcatgtgttt
gaggcatcag 480tgttggaatc agggtactac tggggaagag cagcgctgga
tccggagccg gggcggtgcc 540ggaggaccga cggcaagaag tggcggtgct
cgaaggacgc ggtggcgggt cagaagtact 600gcgaccgcca catgcatcgt
ggccgaaacc gttcaagaaa gcctgtggaa caacgaaacc 660ctgatcctac
ctagctgtta tgtctcatat atacttggaa aagatttaaa aactttattg
720tgcataaata ggccccaaac tttcttgtta agttgctgtt ttggtgcatt
agaatgcatt 780tcctctgggg ccaagagtga caacaagagc ttctttgaaa
accatgatca tgtggatggg 840gatggaaatt cagccaaatc tgatggccat
gtcttgaggc atttctttga tgattggcca 900aggacactgc aagagcctga
caatggtgaa agcaatggtt gccagaacaa caactcagga 960acatgtcttt
ctatgtcaac accaggaatc acttcctcgg atgtgtcgtt gaaattgtcc
1020actggccatg gagaggatgc gtgccacgcg gcctcaatgg gaggaccact
tgcagaggca 1080ttaagatcat ccaccaccag ctccacttct tcaccaacca
gtcaactatt tattgcattg 1140tctttactat tcatcccaaa gtgggaattg
ttatttccac tctcactttc tgccaccaat 1200acactcccct ttgcttggtt
ttattcaaaa ataccactga caaatacact tcccatctgt 1260cactga
1266241396DNAGlycine max 24atgaatggaa ggaacaggtt cccctttacc
ccatcacagt ggcaagagct tgaacaccaa 60gctctcatct acaagtacat ggcttcaggc
atttccattc cacctgatct tctcttcacc 120ataaaaagga gctattttga
ttcccctctg tcctcaaggc ttttgcctaa ccagccacag 180cactttggat
ggaactacct tcagatgggt ttgggaagaa aaatagaccc tgagccaggt
240aggtgtagaa gaactgatgg caagaaatgg agatgctcca aagaagcata
cccagattca 300aagtactgtg agaggcacat gcacagaggg aagaatcgtt
caagaaagcc tgtggaagtt 360ttaaaatcaa caacaacacc atcatcatca
acaacaaact caaatgcttc ttctacacaa 420caagcaatct catcaatcac
caaaattaat agcactctct cacctcttgc atcatctgag 480actcaccaac
accaccacta tcctcaacac tatggctcct ttctctatca tcatcaccct
540ccttcaaggt cctctggcat tggcttgtct tttgaagaca acagtgctcc
cttgtttctt 600gacactggct catgctctca gtccaacaca gactgcagga
gtaggtatgt ttatggagag 660aaagaggagg tggatgagca tgctttcttc
acagaacctt gtggtgttat gaaaagcttc 720tctgcttcct ctatggatga
ctcatggcaa ctcacaccat tgactatgag ctcctcatct 780tcatcttcca
agcagaggag ttcctttggc ttgtccagtg attactcttg cttgcaactt
840cagagccact caaagcagca gcagcaagag catcatcaag atcagggttg
ctacatgttt 900ggtgctggtc aagttgtgaa agaagaacct cagaaaacgg
ttcatcgctt ctttgatgaa 960tggccacaca aaggaagaga aggctcttgg
cttgatttgg atgacaaatc ttccacaacc 1020caactttcaa tttccatccc
cacatcttct catgattttt caactttcag ttccagaacc 1080caccatgatg
gttgagtgta ggtttccaat aatgggtcct ctgtacggaa atcaaaggcc
1140aaaaatgtac ctataagggt ggggtttaag atgctttggg gtcttatttc
caaccacacc 1200ctcttctttt ttcttctgcc ataaaggcac ttgaaaggga
tttctgtctc catggagaga 1260tctaatgtgt aatgctatat gatgctaatg
ctttcttagt tataagtgct tccttccaaa 1320catagtataa aaaattctct
gtgacaaagc ctgaactgtt taatatttga gctaacattc 1380aattgcacat ctatgt
1396251122DNAGlycine max 25atgatgagtg caagtgcagg tgcaagaaat
aggtctccgt tcacacaaat tcagtggcaa 60gagcttgagc aacaagctct tgtttttaag
tacatggtta caggaacacc tatcccacca 120gatctcatct actctattaa
aagaagtcta gacacttcaa tttcttcaag gctcttccca 180catcatccaa
ttgggtgggg atgttttgaa atgggatttg gcagaaaagt agacccagag
240ccagggaggt gcagaagaac agatggcaag aaatggagat gttcaaagga
ggcatatcca 300gactcaaagt actgtgaaag acacatgcac agaggcagaa
accgttcaag aaagcctgtg 360gaagtttctt cagcaacaag caccgccaca
aacacctccc aaacaatccc atcatcttat 420accagaaacc tttccttgac
caataacagt aaccccaaca taacaccacc accaccaccc 480tcttctttcc
ctttctctca tttgccctct tctatgccta ttgatcagtc ccaacccttt
540tcccaatcct accaaaactc ttctctcaat cccttcttct actcccaatc
aacctcctct 600agacccccag atgctgattt tccaccccaa gatgccacca
cccaccacct attcatggac 660tctgctggct cttattctca tgatgaaaag
aattataggc atgttcatgg aataagggaa 720gatgtggatg agagagcttt
cttcccagaa gcatcaggat cagctaggag ctatacagac 780tcgtaccaac
aactatcaat gagctcctac aagtcctatt caaactccaa ctttcagaac
840attaataatg atgccaccac caacccaaga cagcaagagc agcaactaca
acaacaacaa 900cactgttttg ttttagggac agacttcaaa tcaacaaggc
caagcaaaga gaaagaagct 960gagacaacaa caggtcagag accccttcac
cgtttctttg gggagtggcc accaaagaac 1020acaacaacag attcctggct
agatcttgct tccaactcca gaatccaaac cggtgatgat 1080cctgcttctt
cttccctact ctcattatca cacccttttt aa 1122261298DNAGlycine max
26atggacttcc atctgaagga atggagaaac cagcatgagt cagaggaaca acaacattct
60acaaagatgc caaaacttct ccctgaatcc catcatcaac agccatctgc cactgcactc
120cctttgtttg tacctgaacc caacagcagc agcaaagtca gcaccctgtc
agattcaaca 180ttagcagctg aaactgaaac aatgaccact acaaccacta
acagattatt tcccaggatg 240gggagctact tcagcttgtc tcagtggcag
gagcttgagt tgcaggcttt gatattcagg 300tacatgttgg ctggtgctgc
tgttcctcct gaactccttc aaccaatcaa gaaaagcctt 360cttcattccc
ctcactattt cctccatcac cctctccaac attaccaacc tgctgctttg
420ttgcaatcag ggtattgggg tagaggagcg atggatccgg agccagggcg
gtgccggaga 480accgacggca agaaatggcg gtgctcgagg gacgtggtgg
ctgggcaaaa gtactgtgag 540cgccacatgc atcgtggaag aaaccgttca
agaaagcctg tggaactacc cacaccaact 600agtgctaata attgtgatgg
tggatctcta ggactaggtg cttcttcatc ttccatttct 660tcaccacccc
tagcttctgc ttcactcaaa tccccatttg atcttcttcg tcttaatgaa
720cgttcctctg ggaccaagaa tgaagacgaa gaccatgtgg gtggggatgg
cagatcaggt 780ggagggggtg gccatatgct gaggcatttc ttcgatgatt
ggccacgatc actgcaagac 840tctgacaacg ttgaaaacaa tgctgctggc
cctagcctct ctatttcaat gcccggaaat 900gctgctgctg cttcctcgga
tgtgtcattg aaattgtcca cgggctatgg agaggaccca 960ggcccaagaa
atgagaatgt gggcctcgtg gcagagcagc tgcagttgaa ttgggccgga
1020ggatgggcct cgtctaatca agtggcttcc atgggaggac cactggccga
ggcactcaga 1080tcatctattt caacttcatc tcccactagt gttttgcatc
acttgcctcg tggttctgga 1140tctgagacca gcattattag cacctgaact
tagtttgtag gtgcccccaa ttaattttct 1200cttttttgtt ttgaggttaa
gttccacttt tagagcattc ttggacaacg gttatgttca 1260tatcaaacct
ctggactact tttgtttcta agtgggcg 12982720RNAArabdipsis
thalianamisc_feature(13)..(13)n is a, c, g, or u 27caaguucuuu
cgnacaccuu 202821RNAArabisopsis thalianamisc_feature(8)..(8)n is a,
c, g, or u 28aagguguncg aaagaacuug c 21
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