U.S. patent application number 09/776491 was filed with the patent office on 2001-08-09 for methods for controlling cell death in plants.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Briggs, Steven P., Gray, John, Johal, Gurmukh S..
Application Number | 20010013135 09/776491 |
Document ID | / |
Family ID | 25202742 |
Filed Date | 2001-08-09 |
United States Patent
Application |
20010013135 |
Kind Code |
A1 |
Briggs, Steven P. ; et
al. |
August 9, 2001 |
Methods for controlling cell death in plants
Abstract
The present invention is drawn to methods and compositions for
suppressing cell death in plants. Specifically, novel proteins and
genes are provided for use in plant transformation. The proteins
and genes are useful for activating disease resistance, enhancing
plant cell transformation efficiency, engineering herbicide
resistance, genetically targeting cell ablations, and other methods
involving the regulation of cell death in plants.
Inventors: |
Briggs, Steven P.; (Des
Moines, IA) ; Johal, Gurmukh S.; (Columbia, MO)
; Gray, John; (Cork City, IE) |
Correspondence
Address: |
ALSTON & BIRD LLP
PIONEER HI-BRED INTERNATIONAL, INC.
BANK OF AMERICA PLAZA
101 SOUTH TYRON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
25202742 |
Appl. No.: |
09/776491 |
Filed: |
February 2, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09776491 |
Feb 2, 2001 |
|
|
|
08810009 |
Mar 4, 1997 |
|
|
|
6211437 |
|
|
|
|
Current U.S.
Class: |
800/279 |
Current CPC
Class: |
C12N 15/8207 20130101;
C12N 15/8282 20130101; C12N 9/0071 20130101; C12N 15/8289
20130101 |
Class at
Publication: |
800/279 |
International
Class: |
C12N 015/82 |
Goverment Interests
[0002] The invention was made by an agency of the United States
Government or under a contract with an agency of the United States
Government. Accordingly, the United States Government may have
rights to said invention.
Claims
What is claimed is:
1. A method for suppressing cell death in a plant, said method
comprising transforming a plant with an expression cassette
comprising a chimeric gene, said gene comprising a nucleotide
sequence that encodes a protein that suppresses cell death in
plants operably linked to a promoter operable in a plant cell,
wherein said nucleotide sequence is selected from the group
consisting of: a) a nucleotide sequence that has at least 70%
identity to the nucleotide sequence set forth in SEQ ID NO: 1, and
b) a nucleotide sequence that has at least 85% identity to the
nucleotide sequence set forth in SEQ ID NO: 1.
2. The method of claim 1, wherein said protein comprises a Rieske
iron-coordinating motif.
3. The method of claim 2, wherein said protein further comprises a
mononuclear iron-binding site.
4. The method of claim 1, wherein said protein comprises the amino
acid sequence set forth in residues 261-520 of SEQ ID NO:2.
5. The method of claim 1, wherein said protein comprises the amino
acid sequence set forth in SEQ ID NO:2.
6. A method for suppressing cell death in a plant, said method
comprising transforming a plant with an expression cassette
comprising a chimeric gene, said gene comprising a nucleotide
sequence operably linked with a promoter operable in a plant cell,
wherein said nucleotide sequence encodes a protein set forth in SEQ
ID NO:2.
7. A method for suppressing cell death in a plant, said method
comprising transforming a plant with an expression cassette
comprising a chimeric gene, said gene comprising a nucleotide
sequence operably linked with a promoter operable in a plant cell,
wherein said nucleotide sequence is selected from the group
consisting of: a) a nucleotide sequence that has at least 85%
identity to the nucleotide sequence set forth in SEQ ID NO: 1, and
b) a nucleotide sequence that is set forth in SEQ ID NO: 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 08/810,009 filed on Mar. 4, 1997, which is hereby incorporated
herein in its entirety by reference.
FIELD OF THE INVENTION
[0003] The invention relates to the genetic manipulation of plants,
particularly to novel genes and proteins and their uses in
regulating cell death and disease resistance in plants.
BACKGROUND OF THE INVENTION
[0004] A host of cellular processes enable plants to defend
themselves from disease caused by pathogenic agents. These
processes apparently form an integrated set of resistance
mechanisms that is activated by initial infection and then limits
further spread of the invading pathogenic microorganism.
[0005] Subsequent to recognition of a potentially pathogenic
microbe, plants can activate an array of biochemical responses.
Generally, the plant responds by inducing several local responses
in the cells immediately surrounding the infection site. The most
common resistance response observed in both nonhost and
race-specific interactions is termed the "hypersensitive response"
(HR). In the hypersensitive response, cells contacted by the
pathogen, and often neighboring cells, rapidly collapse and dry in
a necrotic fleck. Other responses include the deposition of
callose, the physical thickening of cell walls by lignification,
and the synthesis of various antibiotic small molecules and
proteins. Genetic factors in both the host and the pathogen
determine the specificity of these local responses which can be
very effective in limiting the spread of infection.
[0006] Many environmental and genetic factors cause general leaf
necrosis in maize and other plants. In addition, numerous recessive
and dominant genes have been reported which cause discreet necrotic
lesions to form. These lesion mutants mimic disease lesions caused
by various pathogenic organisms of maize. For example, Les 1, a
temperature-sensitive conditional lethal mutant, mimics the
appearance of Helminthosporium maydis on susceptible maize.
[0007] Many genes causing necrotic lesions have been reported. The
pattern of lesion spread on leaves is a function of two factors:
lesion initiation and individual lesion enlargement.
[0008] The lethal leaf spot-1 (lls1) mutation of maize is inherited
in a recessive monogenic fashion and is characterized by the
formation of scattered, necrotic leaf spots (lesions) that expand
continuously to engulf the entire tissue. Since lls1 spots show
striking resemblance to lesions incited by race 1 of Cochiobolus
(Helminthosporium) carbonum on susceptible maize, this mutation has
been grouped among the class of genetic defects in maize called
"disease lesion mimics."
[0009] Lesion mimic mutations of maize have been shown to be
specified by more than forty independent loci. These lesion mimic
plants produce discreet disease-like symptoms in the absence of any
invading pathogens. It is intriguing that more than two thirds of
these mutations display a partially dominant, gain-of-function
inheritance, making it the largest class of dominant mutants in
maize, and suggesting the involvement of a signaling pathway in the
induction of lesions in these mutations. Similar mutations have
also been discovered in other plants including Arabidopsis and
barley.
[0010] Despite the availability of the large number of lesion mimic
mutations in plants, the mechanistic basis and significance of this
phenomenon, and the wild-type function of the genes involved, has
remained elusive. The understanding of the molecular and cellular
events that are responsible for plant disease resistance remains
rudimentary. This is especially true of the events controlling the
earliest steps of active plant defense, recognition of a potential
pathogen and transfer of the cognitive signal throughout the cell
and surrounding tissue.
[0011] Diseases are particularly destructive processes resulting
from specific causes and characterized by specific symptoms.
Generally the symptoms can be related to a specific cause, usually
a pathogenic organism. In plants, a variety of pathogenic organisms
cause a wide variety of disease symptoms. Because of the lack of
understanding of the plant defense system, methods are needed to
protect plants against pathogen attack.
SUMMARY OF THE INVENTION
[0012] Compositions and methods for suppressing cell death and
controlling disease resistance in plants are provided. The
compositions, cell death suppressing proteins and the genes
encoding such proteins, are useful for activating disease
resistance, enhancing plant cell transformation efficiency,
engineering herbicide resistance, genetically targeting cell
ablations, and other methods involving the regulation of cell death
and disease resistance in plants.
[0013] Additionally, novel promoter sequences are provided for the
expression of genes in plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 sets forth the amino acid sequence of lls1 protein
and the DNA sequence encoding the protein (SEQ ID NOs: 2 and 1
respectively).
[0015] FIG. 2 sets forth the nucleotide sequence of the lls1
promoter (SEQ ID NO: 3).
[0016] FIG. 3 sets forth a maize genomic DNA sequence comprising
the lls1 gene and promoter (SEQ ID NO: 4).
[0017] FIG. 4 sets forth the organization of (lie 3 kb EcoRI
restriction fragment containing lls sequence.
[0018] FIG. 5 shows that a single transcript was detected when mRNA
from mature leaves was probed with the lls1 transcript.
[0019] FIG. 6 shows the preferred sites for possible modification
of the protein to alter protein activity (SEQ ID NOs: 2 and 5-61,
respectively).
DETAILED DESCRIPTION OF THE INVENTION
[0020] The invention is drawn to compositions and methods for
controlling cell death and disease resistance in plant cells. The
compositions are proteins, ring-hydroxylating dioxygenases, which
act to control cell death and regulate disease resistance in
plants. The proteins and genes encoding them can be used to
regulate cell death and disease resistance in transformed plant
cells as well as a variety of other uses. The proteins are useful
in resistance to pathogens and survival of the cells particularly
after pathogen attack.
[0021] One aspect of the invention is drawn to proteins which are
involved in the degradation of plant phenolics, cell
death-suppressing and disease resistance proteins. Such proteins
are characterized by containing two consensus motifs, a Rieske-type
iron-sulfur binding site, and a mononuclear iron-binding site, and
function as aromatic ring-hydroxylating (ARH) dioxygenases. The
Rieske motif contains two cysteine and histidine residues
responsible for binding an iron atom cofactor. Plant proteins
containing at least one of the motifs have been identified and can
be used in the methods of the present invention. Alternatively,
proteins from bacteria with the Rieske motif are known in the art
and can be used in the methods of the invention. Bacterial proteins
of particular interest are ring-hydroxylating dioxygenases,
particularly those from the cyanobacterium Synechocystis. See, for
example, Gibson et al. (1984) Microbial degradation of organic
compounds, 181-252. D. T. Gibson, ed. (New York: Marcel Dekker),
pp. 181-252.
[0022] The cell death-suppressing and disease resistance proteins
of the invention encompass a novel class of plant proteins. The
amino acid sequence of the lls1 protein isolated from maize is set
forth in FIG. 1. However, the proteins are conserved in plants.
Thus, as discussed below, methods are available for the
identification and isolation of genes and proteins from any plant.
Likewise, sequence similarities can be used to identify and isolate
other bacterial genes and proteins. The proteins function to
inhibit the spread of cell death and control disease resistance in
plants. Therefore, the proteins are useful in a variety of settings
involving the regulation of cell death and control of disease
resistance in plants.
[0023] The Rieske motif exhibited by the proteins of the invention
is shared by a class of enzymes known as ring-hydroxylating
dioxygenases. The motif contains two cysteine and histidine
residues responsible for binding an iron atom cofactor--residues
that are shared by other proteins termed Rieske iron-sulfur
proteins. The bacterial genes included in the proteins of the
invention are known as catabolic operons. Thus, it is predicted
that the plant proteins are related to the degradation of phenolic
compound(s). In fact, a para-coumaric ester accumulates in lls1
lesioned plants, but not in normal-type siblings or wild-type
siblings inoculated with the fungus Cochliobolus heggerostrophus.
While the present invention is not dependent upon any particular
mechanism of action, it is believed that the cell death-suppressing
function of the novel protein may be mediated by the detoxification
of a phenolic compound whose cell damaging effects are fueled by
light harvested by photosynthetically-functional pigments in the
leaf.
[0024] Modifications of such proteins are also encompassed by the
present invention. Such modifications include substitution of amino
acid residues, deletions, additions, and the like. For example, the
protein can be mutagenized in such a way that its activity is
reduced, but not completely abolished. See, for example, Jiang et
al. (1996), J. Bacterial, 178:3133-3139, where the Tyr-221 from the
mononucleate iron binding site of toluene dioxygenase was changed
to Ala. This change resulted in a reduction in activity to 42% of
the normal activity. A change of Tyr-266 to Ala reduced the
activity to 12%. In the same manner, amino acid changes,
particularly changes from Tyr to Ala, of the sequence of the
proteins of the present invention can lead to increases or
decreases in activity. FIG. 6 sets forth potential modifications
which may alter expression of the resulting protein. Such
modifications can result in dominant negative inhibitors of the
wild type protein. Using these sequences, the expression of lls1
can be regulated such that disease resistance can be obtained in
the absence of lesions.
[0025] After each modification of the protein, the resulting
protein will be tested for activity. To test for activity, plants
can be transformed with the DNA sequence and tested for their
response to a fungal pathogen. Of particular interest are changes
that result in a reduction of activity. Such changes will confer
disease resistance, yet not result in the lesion phenotype. These
modified proteins, and the corresponding genes, will be useful in
disease defense mechanisms in plants.
[0026] Accordingly, the proteins of the invention include naturally
occurring plant and bacterial proteins and modifications thereof.
Such proteins find use in preventing cell death and controlling
disease resistance. The proteins are also useful in protecting
plants against pathogens. In this manner, the plant is transformed
with a nucleotide sequence encoding the protein. The expression of
the protein in the plant prevents cell death and confers resistance
to infection by plant pathogens.
[0027] xx The nucleotide sequences encoding the novel proteins are
also provided. The lls1 gene from maize encodes the novel maize
protein which inhibits the spread of cell death from wounding or
internal stresses that occur during photosynthesis. The maize gene
call be utilized to isolate homologous genes from other plants,
including Arabidopsis, sorghum, Brassica, wheat, tobacco, cotton,
tomato, barley, sunflower, cucumber, alfalfa, soybeans, sorghum,
etc.
[0028] Methods are readily available in the art for the
hybridization of nucleic acid sequences. Coding sequences from
other plants may be isolated according to well known techniques
based on their sequence homology to the maize coding sequences set
forth herein. In these techniques all or part of the known coding
sequence is used as a probe which selectively hybridizes to other
cell death-suppressor coding sequences present in a population of
cloned genomic DNA fragments or cDNA fragments (i.e. genomic or
cDNA libraries) from a chosen organism.
[0029] For example, the entire 11sl sequence or portions thereof
may be used as probes capable of specifically hybridizing to
corresponding coding sequences and messenger RNAs. To achieve
specific hybridization under a variety of conditions, such probes
include sequences that are unique among lls1 coding sequences and
are preferably at least about 10 nucleotides in length, and most
preferably at least about 20 nucleotides in length. Such probes may
be used to amplify lls1 coding sequences from a chosen organism by
the well-know process of polymerase chain reaction (PCR). This
technique may be used to isolate additional lls1 coding sequences
from a desired organism or as a diagnostic assay to determine the
presence of lls1 coding sequences in an organism.
[0030] Such techniques include hybridization screening of plated
DNA libraries (either plaques or colonies; see, e.g. Sambrook et
al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press
(1989)) and amplification by PCR using oligonucleotide primers
corresponding to sequence domains conserved among the amino acid
sequences (see, e.g. Innis et al., PCR Protocols, a Guide to
Methods and Applications, eds., Academic Press (1990)).
[0031] For example, hybridization of such sequences may be carried
out under conditions of reduced stringency, medium stringency or
even stringent conditions (e.g., conditions represented by a wash
stringency of 35-40% Formamide with 5.times.Denhardt's solution,
0.5% SDS and Ix SSPE at 37.degree. C.; conditions represented by a
wash stringency of 40-45% Formamide with 5.times.Denhardt's
solution, 0.5% SDS, and 1.times.SSPE at 42.degree. C.; and
conditions represented by a wash stringency of 50% Formamide with
5.times.Denhardt's solution, 0.5% SDS and Ix SSPE at 42.degree. C.,
respectively), to DNA encoding the cell death suppressor genes
disclosed herein in a standard hybridization assay. See J. Sambrook
et al, Molecular Cloning, A Laboratory Manual 2d Ed. (1989) Cold
Spring Harbor Laboratory. In general, sequences which code for a
cell death suppressor and disease resistance protein and hybridize
to the maize lls1 gene disclosed herein will be at least 50%
homologous, 70% homologous, and even 85% homologous or more with
the maize sequence. That is, the sequence similarity of sequences
may range, sharing at least about 50%, about 70%, and even about
85% sequence similarity.
[0032] Generally, since leader peptides are not highly conserved
between monocots and dicots, sequences can be utilized from the
carboxyterminal end of the protein as probes for the isolation of
corresponding sequences from any plant. Nucleotide probes can be
constructed and utilized in hybridization experiments as discussed
above. In this manner, even gene sequences which are divergent in
the aminoterminal region can be identified and isolated for use in
the methods of the invention.
[0033] Also provided are mutant forms of the lls1 gene (the cell
death suppressor and disease resistance gene) and the proteins they
encode. Methods for mutagenesis and nucleotide sequence alterations
are well known in the art. See, for example, Kunkel, T. (1985)
Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods
in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and
Gaastra (eds.) Techniques in Molecular Biology, MacMillan
Publishing Company, NY (1983) and the references cited therein.
Thus, the genes and nucleotide sequences of the invention include
both the naturally occurring sequences as well as mutant forms.
Likewise, the proteins of the invention encompass both naturally
occurring proteins as well as variations and modified forms
thereof.
[0034] The nucleotide sequences encoding the proteins or
polypeptides of the invention are useful in the genetic
manipulation of plants. In this manner, the genes of the invention
are provided in expression cassettes for expression in the plant of
interest. The cassette will include 5' and 3' regulatory sequences
operably linked to the gene of interest. The cassette may
additionally contain at least one additional gene to be
co-transformed into the organism. Alternatively, the gene(s) of
interest can be provided on another expression cassette. Where
appropriate, the gene(s) may be optimized for increased expression
in the transformed plant. Where bacterial ring-hydroxylating
dioxygenases are used in the invention, they can he synthesized
using plant preferred codons for improved expression. Methods are
available in the art for synthesizing plant preferred genes. See,
for example, U.S. Pat. Nos. 5,380,831, 5,436, 391, and Murray et
al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by
reference.
[0035] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region)
(Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA,
86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco
Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic
Virus); Virology, 154:9-20), and human immunoglobulin heavy-chain
binding protein (BiP), (Macejak, D. G., and P. Sarnow (1991)
Nature, 353:90-94; untranslated leader from the coat protein mRNA
of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke,
L., (1987) Nature, 325:622-625; tobacco mosaic virus leader (TMV),
(Gallie, D. R. et al. (1989) Molecular Biology of RNA, pages
237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S.
A. et al. (1991) Virology, 81:382-385). See also, Della-Cioppa et
al. (1987) Plant Physiology, 84:965-968. Other methods known to
enhance translation can also be utilized, for example, introns, and
the like.
[0036] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Towards this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resection, ligation, PCR, or the like may be employed,
where insertions, deletions or substitutions, e.g. transitions and
transversions, may be involved.
[0037] The compositions and methods of the present invention can be
used to transform any plant. In this manner, genetically modified
plants, plant cells, plant tissue, seed, and the like can be
obtained. Transformation protocols may vary depending on the type
of plant or plant cell, i.e. monocot or dicot, targeted for
transformation. Suitable methods of transforming plant cells
include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA, 83:5602-5606, Agrobacterium mediated transformation
(Hinchee et al. (1988) Biotechnology, 6:915-921), 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; WO91/10725 and McCabe et al. (1988)
Biotechnology, 6:923-926). Also see, Weissinger et al. (1988)
Annual 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); McCabe et al. (1988)
Biotechnology, 6:923-926 (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); WO91/10725 (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; Bytebier 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, NY (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);
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 Biotechnology, 14:745-750 (maize via Agrobacterium
tumefaciens); all of which are herein incorporated by
reference.
[0038] The cells which have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports, 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having the
desired phenotypic characteristic identified. Two or more
generations may be grown to ensure that the subject phenotypic
characteristic is stably maintained and inherited and then seeds
harvested to ensure the desired phenotype or other property has
been achieved.
[0039] As noted earlier, the nucleotide sequences of the invention
can be utilized to protect plants from disease, particularly those
caused by plant pathogens. Pathogens of the invention include, but
are not limited to, viruses or viroids, bacteria, insects, fungi,
and the like. Viruses include tobacco or cucumber mosaic virus,
ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.
Specific fungal pathogens for the major crops include: Soybeans:
Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina,
Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum,
Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe
phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora
kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum
dematium (Colletotichum truncatum), Corynespora cassiicola,
Septoria glycines, Phyllosticta sojicola, Alternaria alternata,
Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v.
phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora
gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring
spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium
aphanidermatum, Pythium ultimum., Pythium debaryanum, Tomato
spotted wilt virus, Heterodera glycines Fusarium solani; Canola:
Albugo candida, Alternaria brassicae, Leptosphaeria maculans,
Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella
brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium
roseum, Alternaria alternata.; Alfalfa: Clavibater michiganese
subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium
splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora
megasperma, Peronospora trifoliorum, Phoma medicaginis var.
medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis,
Leptotrochila medicaginis, Fusarium oxysporum, Rhizoclonia solani,
Uromyces striatus, Colletotrichum trifolii race 1 and race 2,
Leptosphaerulina briosiana, Stemphylium botryostum, Stagonospora
meliloti, Sclerotinia trifoliorum, Alfalfa Mosaic Virus,
Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae,
Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae;
Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri,
Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v.
syringae, Alternaria alternata, Cladosporium herbarum, Fusarium
graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago
tritici, Ascochyta tritici, Cephalosporium gramineum,
Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,
Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,
Puccinia striiformis, Pyrenophora tritici-repentis, Septoria
nodorum, Septoria, tritici, Septoria avenae, Pseudocercosporella
herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis,
Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,
Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana,
Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat
Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak
Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia
tritici, Tilletia laevis, Ustilago tritici, Tilletia indica,
Rhizocionia-solani, Pythium arrhenomannes, Pythium gramicola,
Pythium aphanidermatum, High Plains Virus, European wheat striate
virus; Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum,
Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria
helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii,
Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae,
Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi,
Verticillium dahliae, Erwinia carotovorum pv. carotovora,
Cephalosporium acremonium, Phytophthora cryptogea, Albugo
tragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwinia
stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium
graminearum), Stenocarpella maydi (Diplodia maydis), Pythium
irregulare, Pythium graminicola, Pythium graminicola, Pythium
splendens, Pythium ultimum, Pythium aphanidermatunt, Aspergillus
flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus),
Helminthosporium carbonum I, II & III (Cochliobolus carbonum),
Exserohilum turicum I, II & III, Helminthosporium pedicellatum,
Physoderma maydis, Phyllosticta maydis, Kabatiella zeae,
Colletotrichum graminicola, Cercospora zeae-maydis, Cercospora
sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora,
Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae,
Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis,
Curvularia pallescens, Clavibacter michiganense subsp. nebraskense,
Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat
Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,
Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia
corotovora, Cornstunt spiroplasma, Diplodia macrospora,
Sclerophthora macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Peronosclerospora maydis,
Peronosclerospora sacchari, Spacelotheca reiliana, Physopella zeae,
Cephalosporium maydis, Caphalosporium acremonium, Maize Chlorotic
Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado
Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough
Dwarf Virus; Sorghumn: Exserohilum turcicum, Colletotrichum
graminicola (Glomerella graminicola), Cercospora sorghi,
Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae
p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas
andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia
circinata, Fusarium moniliforme, Alternaria alternate, Bipolaris
sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma
insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans),
Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari,
Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca
cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic
Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium
strictue, Sclerophthona macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Scierospora graminicola, Fusarium
graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium
graminicola, etc.
[0040] The nucleotide sequences also find use in enhancing
transformation efficiency by suppressing cell death in bombarded
cells. Thus, the sequences find particular use in transformation
methods in which programmed cell death occurs. The physical
wounding of particle bombardment triggers programmed cell death.
The expression of the cell death-suppressor gene in a bombarded
cell serves to inhibit such cell death thereby improving
transformation efficiency. By "improving efficiency" is intended
that the number of transformed plants recovered by a transformation
event is increased. Generally, the number of transformed plants
recovered is increased it least two-fold, preferably at least
five-fold, more preferably at least ten-fold.
[0041] For use in improving transformation efficiency, a cell death
suppressor gene is included in an expression cassette. Typically,
the gene will be used in combination with a marker gene. Other
genes of interest may additionally be included. The respective
genes may be contained in a single expression cassette, or
alternatively in separate cassettes. Methods for construction of
the cassettes and transformation methods have been described
above.
[0042] As noted, the cell death suppressor gene can be used in
combination with a marker gene. Selectable marker genes and
reporter genes are known in the art. See generally, G. T. Yarranton
(1992) Curr. Opin. Biotech., 3:506-511.; Christopherson et al
(1992) Proc. Natl. Acad. Sci. USA, 89:6314-6318; Yao et al. (1992)
Cell, 71:63-72; W. S. Reznikoff (1992) Mol. Microbiol.,
6:2419-2422; Barkley et al. (1980) The Operon, pp. 177-220; Hu et
al. (1987) Cell, 48:555-566; Brown et al. (1987) Cell, 49:603-612;
Figge et al. (1988) Cell, 52:713-722; and, Deuschle et al. (1989)
Proc. Natl. Acad. Aci. USA, 86:5400-5404.
[0043] Plant tissue cultures and recombinant plant cells containing
the proteins and nucleotide sequences, or the purified protein, of
the invention may also be used in an assay to screen chemicals
whose targets have not been identified to determine if they inhibit
lls1 protein. Such an assay is useful as a general screen to
identify chemicals which inhibit lls1 protein activity and which
are therefore herbicide candidates. Alternatively,
recombinantly-produced lls1 protein may be used to elucidate the
complex structure of the enzyme. Such information regarding the
structure of the lls1 protein may be used, for example, in the
rational design of new inhibitory herbicides. It is recognized that
both plant and bacterial nucleotide sequences may be utilized. The
inhibitory effect on the cell-suppressor protein may be determined
in an assay by monitoring the rate of cell death or alternatively
by monitoring the accumulation of the activating phenolic compound,
particularly the para-coumaric ester associated with lesion
mutants.
[0044] If such a chemical is found, it would be useful as a
herbicide, particularly if plant or bacterial mutant genes can be
isolated or constructed which are not inhibited by the chemical. As
indicated above, molecular techniques are available in the art for
the mutagenesis and alteration of nucleotide sequences. Those
sequences of interest can be selected based on resistance to the
chemical. Where resistant forms of lls 1 or a corresponding gene
have been identified to a chemical, the chemical is also useful as
a selection agent in transformation experiments. In these
instances, the mutant lls1 would be used as the selectable marker
gene.
[0045] The sequences of the invention also find use to genetically
target cell ablations. In this manner, dominant negative nucleotide
sequences can be utilized for cell ablation by expressing such
negative sequences with specific tissue promoters. See FIG. 6. For
example, stamen promoters can be utilized to drive the negative
alleles to achieve male sterile plants. (See, for example,
EPA0344029 and U.S. Pat. No. 5,470,359, herein incorporated by
reference). Alternatively, cell ablation can be obtained by
disrupting dominant negative oligonucleotides with a transposable
insertion. In this manner, very specific or general patterns of
cell ablations can be created. Additionally, to provide specific
cell ablation, antisense oligonucleotides for lls1 or other genes
of the invention can be expressed in target cells disrupting the
translation which produces the cell death suppressor proteins.
[0046] As discussed, the genes of the invention can be manipulated
to enhance disease resistance in plants. In this manner, the
expression or activity of lls1 or other cell death suppressor or
disease resistance gene can be altered. Such means for alteration
of the gene include co-suppression, antisense, mutagenesis,
alteration of the sub-cellular localization of the protein, etc. In
some instances, it may be beneficial to express the gene from an
inducible promoter, particularly from a pathogen inducible
promoter. Such promoters include those from pathogenesis-related
proteins (PR proteins) which are induced following infection by a
pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,
chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J.
Plant Pathol. 89:245-254; Miles et al. (1992) The Plant Cell
4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116.
[0047] A promoter which is capable of driving the expression of
genes in a plant cell is additionally provided. The promoter is
inducible. Thus, it may be manipulated to express heterologous
resistance mechanisms at the site of pathogen infection.
Accordingly, the promoter is useful for driving any gene in a plant
cell, particularly genes which are needed at the site of infection
or wounding. That is, the promoter is particularly useful for
driving the expression of disease or insect resistance genes. The
nucleotide sequence of the promoter is provided in FIG. 2.
[0048] It is recognized that the nucleotide sequence of the
promoter may be manipulated yet still retain the functional
activity. Such methods for manipulation include those discussed
above. Thus, the invention encompasses those modified promoter
sequences, as well as promoter elements retaining the functional
activity of the promoter. Such elements and modified sequences can
be assayed for activity by determining the expression of a reporter
gene operably linked to the promoter element or modified promoter
sequence.
[0049] A genomic DNA sequence comprising the lls gene and promoter
are provided in FIG. 3. The sequence can be used to construct
probes to determine the location and organization of similar
sequences in other plants, particularly to analyze the location of
other cell death suppressing and disease resistance sequences.
[0050] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0051] Materials and Methods:
[0052] Plant Material
[0053] The original llsl mutant, containing the reference allele,
was obtained from the Maize Genetics Coop., University or Illinois,
Urbana/Champaign. Stocks containing active Mu transposons were
obtained from Dr. D. Robertson, Iowa State University. The six
transposon tagged mutant alleles, lls1-1 through lls1-6, were
previously designated as lls*-29215, lls*-42230, lls*-1127,
lls*-1424, lls*-3744, and lls*4911, respectively (Johal et al.,
(1994), A Tale of Two Mimics; Transposon Mutagenesis and
characterization of Two Disease Lesion Mimic Mutations of Maize,
Maydica 39:69-76).
[0054] DNA Extraction, RFLP Mapping and Co-segregation Analysis
[0055] DNA was isolated by a urea (Dellaporta et al. (1983), Plant
Molecular Biology Reporter 1:19-22) or CTAB (Hulbert et al. (1991)
Molecular and General Genetics 226:377-382) extraction protocol.
DNA samples (15 to 30) from either mutant or wild-type plants were
pooled and digested individually with seven restriction enzymes.
Southern blot analysis was performed as described by (Gardiner et
al. (1993) Genetics 134:917-930) except that UV crosslinking and
use of dextran sulfate were omitted. Blots were hybridized
systematically with specific probes from different Mu elements.
Mapping probes were provided either by the Maize Mapping Project at
the University of Missouri or from Pioneer Hi-Bred Int. Inc.
Pre-hybridizations and hybridization of southern blots was
performed at 65.degree. C. unless otherwise specified. A 3.0 kb
EcorR1 Mu8co-segregating DNA marker was cloned from an
lls1*-5/lls-ref plant using standard cloning procedures (Ausubel et
al. (1994) Current Protocols in Molecular Biology). The Zap
Express.TM. vector (Stratagene) was employed and packaging,
screening and in vivo excision protocols performed according to
manufacturers instructions. The primer sequences (SEQ ID NOs:
62-64, respectively) for confirmation analysis were: SP1:5' TGG GGA
ACT TGA TCG CGC ACG CCT TCG G3', GSP2:5' TCG GGC ATG GCC TGG GGG
ATC TTG G3', and GSP3:5' GGC CAC GCG TCG ACT AGT AC 3' (IDT,
Coralville Iowa). The thermocycling regime used for confirmation
analysis was 94.degree. C. for 5 min, then cycled 40 or 42 times
for 30 seconds at 94.degree. C., 1 min and 30 sec at 62.degree. C.,
and 1 min at 72.degree. C., and finally 5 min at 72.degree. C.
Mini-libraries of cloned amplified fragments using the TA Cloning4S
vector (Invitrogen) were created and individual colonies for clones
with inserts of the appropriate size. A 5' RACE fragment was used
to screen a pa405 maize seedling leaf cDNA library and 3 individual
clones were recovered and converted to the phagemid form by in vivo
excision from the Zap Express.TM. (Stratagene) vector. Primers GSP1
and GSP2 were used for 5' RACE and GSP3 was used for 3' RACE using
fig 5' and 3' RACE Kits and recommended manufacturers instructions
(GIBCO, MD). To isolate an lls1 genomic clone, a B73 partial
SauIIIA library in lambda DashII was screened using a probe from a
3' RACE product spanning the lls1 gene from GSP3 to the
polyadenylation site. A single positive clone was recovered and a
7.129 kb SacI fragment was subcloned into pBSKS+ (Stratagene) to
create the plasmid pJG201. RFLP mapping of the Arabidopsis lls1
homolog was performed using the Recombinant Inbred (RI) lines
generated from a cross between Arabidopsis ecotypes Columbia and
Landsberg erecta. 48 RI lines were scored using an EcoRV
polymorphism using an lls1 homolog cDNA as probe. The map position
was determined on MAPMAKER using the Kosambi mapping function
(Lander et al. (1987) Genetics 121:174-181).
[0056] Primer Extension Analysis
[0057] For primer extension analysis of the maize lls1 gene an
oligonucleotide complementary to the coding strand in the Us/gene
from 139-173 bases downstream of the predicted first in-frame ATG
was synthesized by DNA Technologies, Inc. (Coralville, Iowa). The
oligonucleotide (SEQ ID NO: 65) GSP17 (5' GTG CTC GGC TCC GCC TGC
TCC GCC GCT TCC CCT GG 3') was end-labeled with .sup.32P. Primer
extension analysis was performed by the method described by
McKnight et al. (1981), Analysis of Transcriptional Regulatory
Signals of the HSV Thymidine Kinase Gene: Identification an
Upstream Control Region, Cell 25:385-398, except for the following
modifications. 40 mg of total RNA from immature tassels of a B73
inbred plant and 0.2 pmol of labeled oligonucleotide were annealed
at one of either 33.degree. C., 37.degree. C., 45.degree. C., or
55.degree. C. for 4 hours. Following the extension reaction RNA in
the sample was removed by adding 2 .mu.l of 0.5M EDTA and 1 .mu.l
of mixed RNAases (0.5 mg/ml RNAase A and 10,000 units/ml RNase T1;
Ambion) and incubating at 37.degree. C. for 30 minutes. The primer
extension products were separated on a 6% denaturing polyacrylamide
sequencing gel and the size of the extension product determined by
comparison with a DNA sequence ladder.
[0058] Northern Blot Analysis
[0059] Total RNA was isolated from leaves of 10 leaf-stage
wild-type plants in a population segregating for the Les 1O1
mutation, Johal and Briggs (1992) Science 258:985-987. mRNA was
enriched from total RNA using a magnetic bead affinity protocol
(Dynal Inc. Great Neck N.Y.). mRNA was isolated from A632 inbred
plants using the MicroQuick protocol (Pharmacia, Piscataway N.J.).
Hybridizations were performed either in modified Church and
Gilberts solution at 42.degree. C. or in the following
hybridization solution at 65.degree. C.-1% casein (Technical Grade,
Sigma), 1% calf skin gelatin (225 bloom, Sigma), 0.2% SDS (Mol.
Biol Grade, Fisher), 0.1% Sarkosyl (IBI), 5.times.SSC. Transfer to
nylon membrane (Magnacharge MSI, Westboro Mass.) was performed by
standard protocols, hybridizations were carried out overnight and
blots were washed as indicated in the results section.
[0060] DNA Sequencing and Analysis
[0061] DNA sequencing was performed by a cycle sequence method
using a SequiTherm.TM. Cycle Sequencing Kit (Epicentre, Madison
Wis.) according to the manufacturers protocol. Local sequence
comparisons were performed using software including ALIGN and
MEGALIGN programs of the DNASTAR software package (DNASTAR Inc.
Madison Wis.). Algorithms such as the neighborhood search algorithm
BLAST (Autschul et al. (1990), Basic Local Alignment Search Tool,
J. Mol. Biol. 215:403-410) or BEAUTY (Worley et al. (1995), An
Enhanced BLAST-based Search Tool that Integrates Multiple
Biological Information Resources into Sequence Similarity Search
Results, Genome Res. 5:173-184) were employed. Searches of the
Genbank databases were performed using the National Center for
Biotechnology Information's BLAST WWW Server with links to Entrez
and to the Sequence Retrieval System (SRS) provided by the Human
Genome Center, Baylor College of Medicine Server at Houston Tex.
via Internet access.
[0062] Analysis of Light Requirement for Lls1 and dd Lesion
Development
[0063] To determine the spectral range of light required for lesion
formation, sections of leaves were clamped between 0.125 inch
Plexiglas GM filters held in place by a metal stand with a side arm
clamp. The following transparent filters were used: Plexiglas GM
2423 (red), 2711 (Far red), 2424 (blue), 2092 (green), 2208
(yellow), and 2422 (Amber) or Clear, (Cope Plastics Inc. St. Louis.
Mo.). Transmission spectra of filters were determined by examining
small sections of filters in a spectrophotometer. Leaf sections of
greenhouse or field-grown plants were covered in aluminum foil to
completely remove incident light. Following complete lesioning of a
leaf, filters were removed to observe if lesioning had occurred in
the covered region.
[0064] The 11s1 Mutation is Cell Autonomous and 11s1 Lesions
Exhibit Altered Phenolic Metabolism and Callose Formation.
[0065] The expression of the lls1 phenotype is developmentally
programmed: a number of round to elliptical lesions often with
concentric rings of dead and dying tissue, begin as small chlorotic
flecks near the tip of the first leaf at the three to four leaf
stage. While these lesions continue to enlarge and eventually
coalesce, new lesions initiate down the leafblade along an age
gradient and cover the whole leaf within three to four days.
Meanwhile, lesions have already started near the tip of the second
leaf. This pattern continues and the plant dies shortly after
pollen shed. Although the entire leaf tissue becomes necrotic on
lls1 plants, lesions rarely develop on stalks. The lls1 mutation is
cell autonomous (i.e., the effect of the gene is confined to the
cell in which it is expressed) as exhibited by both revertant
sectors (Johal et al. (1994) Maydica, 69-76) and forward sectors in
that the mutant phenotype does not progress into surrounding
wild-type tissue. Lls1 lesions were examined for callose deposition
which is frequently associated with response to pathogen infection,
wounding or intercellular viral movement (Hammond-Kosack et al.
(1996), Resistance Gene-dependent Plant Defense Responses, Plant
Cell 8:1773-1791). Heavy callousing of all cell types within
lesions was observed. At the edge of lesions where cells had not
yet collapsed, individual bundle sheath cells were the first cells
to exhibit callousing of the plasmodesmatal fields suggesting that
the cells were responding to some factor or signal emanating from
the dying or dead cells.
[0066] Mapping of the lls1 Locus
[0067] The original lls1 allele isolated by Ullstrup and Troyer
(Ullstrup et al. (1967) Phytopathology 57:1282-1283) was used as
the reference allele (lls1-ref). Using a combination of cytogenetic
and genetic methods, the lls1 gene was initially mapped to the
short arm of chromosome 1 (1S) (Hoisington, (1984) Maize Genetics
Newsletter 58:82-84). To map the gene at a higher resolution, a new
population, in which the progeny segregated 1:1 for homozygous and
heterozygous lls1 plants, was generated. A W23 inbred plant was
fertilized with the lls1 pollen derived from an lls1-ref/lls1-ref
plant, and the resulting progeny (two plants) were backcrossed with
the lls1-ref homozygotes. DNA isolated from 16 mutant and 14
wild-type plants was used to examine the linkage with a number of
RFLP markers. Three tightly linked RFLP markers were identified
which flank the lls1 locus. The RFLP marker Php200603 is about 5 cm
distal to lls1, whereas UMC157 is about 8 cm proximal to lls1. The
linkage of lls1 with another marker, Php200689, could not be broken
with these 30 DNAs. All three of these RFLP markers were invaluable
in unequivocally classifying the mutant alleles for co-segregation
analyses.
[0068] Cloning of the lls1 Locus by Transposon Tagging
[0069] Due to the lack of biochemical information on the lls1
mutation, a transposon tagging method was employed to clone the
lls1 gene. This experimental approach allows genes to be cloned
solely on the basis of phenotype (Bennetzen et al. (1987),
Proceedings of the UCLA Symposium: Plant Gene Systems and their
Biology. ed, 183-204). Both targeted and non-targeted approaches
were employed as outlined by (Johal et al. (1994) Maydica, 69-76).
For the targeted approach, lls1-ref/lls1-ref plants were used as
male parents and crossed with wild-type plants (L1sl/Lls1) from
lines active for Mu transposition. All F1 plants were expected to
be of wild-type phenotype (L1sl/Lls1) unless a Mu insertion or some
other mechanism had inactivated the L1s allele. Such an event would
result in an lls1*lls1-ref plant (lls1* refers to a mutant allele
generated during transposon tagging) with a mutant phenotype. Three
plants from approximately 30,000 F1 progeny exhibited the mutant
phenotype and one of these died before shedding any pollen. The
remaining two plants were crossed as male parents to B73 and Pr1
inbreds and these two new mutants have been designated 1ls1*-1 and
lls1*-2 (lls*-292I5 and lls*-42230, respectively, in (Johal et al.
(1994) Maydica, 69-76).
[0070] A few of the progeny (10 plants) from the outcross of the
mutant plants with both inbreds were RFLP genotyped to identify
plants which had inherited the mutant allele (lls1*). Two plants
containing the mutant allele were self-fertilized, and the F2
progeny so derived were found to segregate for the lls1 phenotype
in a 1:3 ratio as expected for a recessive mutation. Two other
mutant allele-containing plants from the outcross progeny were
backcrossed with the lls1-ref/lls1ref mutants. The resultant
progeny segregated 1:1 for mutant (lls1*-l or -2111sl-refversus
normal plants (Llsl-B73 or -Prl/lls1-ref) and were used for
co-segregation analysis.
[0071] For non-targeted mutagenesis, Mu-active stocks were crossed
to an inbred line and the resulting progeny was self-pollinated to
generate F2 (M2) Mutator populations. With this approach, any
recessive mutation generated during the F1 cross can be detected in
the F2 generation. From more than 24,000 Mutator F2 families
screened, four independent families were identified in which
one-fourth of the plants exhibited a phenotype typical of lls1. The
four mutant alleles have been designated lls1*-3, lls1*4, lls1*-5
and lls1*-6. A number of wild-type plants from each of these four
families were pollinated with the lls1-ref/lls1-refpollen to
determine allelism between these new lls1-like mutants and the
original lls1 mutant. The segregation of lls1 mutants in the
progeny of most of these crosses confirmed allelism between lls1
and the new mutants. All of these mutants were outcrossed with B73
twice and backcrossed to the lls1ref/lls1-ref mutant to create
populations suitable for co-segregation analysis as described above
for the targeted mutants.
[0072] The next step was to confirm that the Mu elements (there are
at least nine of them for Mutator) had caused these new insertional
mutations. This step, called co-segregation analysis, involved
Southern blot analysis to detect the linkage of a Mu element with
the mutant allele in question (Bennetzen et al. (1993) Specificity
and Regulation of the Mutator Transposable Element System in Maize,
Crit. Rev. Plant Sci. 12:57-95). DNA was isolated from
phenotypically mutant and wild-type plants from the segregating
populations described above for each of the mutant alleles.
Following identification of a putative co-segregating element, the
analysis was extended employing multiple individual DNA samples
digested with an appropriate restriction enzyme. In this manner a 3
kb EcoRI restriction fragment, hybridizing with the Mu8 specific
probe was found to co-segregate with 66 DNA samples from the
lls1*-5 mutation. This co-segregating fragment was cloned and
sequenced revealing the organization indicated in FIG. 4. The DNA
sequence of the right (267 bp) flank exhibited significant homology
with an Arabidopsis EST of unknown function suggesting that an
actual gene was disrupted by the Mu8 insertion. On sequencing the
1344 bp left flanking DNA no significant homology to known DNA
sequences was detected and a Mu TIR DNA junction (terminal inverted
repeats at each end of Mu elements) was not observed. Using a Mu
TIR primer and either an M13 forward or reverse universal primer
the left flanking (1344 bp) or right flanking (267 bp) DNA was
amplified by PCR and used to probe mutant and wild-type DNA samples
of all mutant alleles. This experiment revealed single band
polymorphisms in nearly all alleles suggesting that this locus was
disrupted in several other alleles.
[0073] The occurrence of insertions in the same locus for multiple
alleles of the same mutation is considered proof that the correct
locus has been tagged. A PCR based approach was used to identify Mu
type insertions in the vicinity of the cloned region. The right
flanking DNA from the lls1*-5 clone was sequenced as described
above and primers designed for extension in each direction. These
primers were used in combination with Mu TIR primers to detect
amplification products in other mutant allele DNA samples but that
were absent in their corresponding wild-type samples. Two such PCR
polymorphisms were identified from the targeted allele lls1*-2 and
the non-targeted allele lls1*4. These products hybridized strongly
on a southern blot with (the right flanking DNA from allele lls1*-5
indicating that these amplification products were amplified from
the same locus. In addition, the amplification of a smaller (189
bp) gene specific fragment was observed in all the mutant and
wild-type DNA samples from all alleles that hybridized with the
right flanking DNA of the original lls1*-5 clone. Since all these
samples were heterozygous for the lls1-ref allele this result
indicated that the lls1-ref mutation had also resulted from a Mu
insertion. Nested PCR using a Mu TIR primer and GSP2 was performed
to isolate this fragment. All PCR products were directly sequenced
using the GSP1 or GSP2 primers as sequencing primer and allowed
identification of Mu-type insertions within 246 bp and 292 bp 5' of
the insertion site of allele lls1*-5 in allele lls1*-2 and lls1*-4
respectively. It was determined that the lls1-ref allele had a Mu
insertion at the same site of insertion as that of allele lls1*-5.
Southern analysis using the left-flanking DNA of the lls1*-5 clone
revealed that the insertion of a Mu element in the lls1-ref allele
was not accompanied by a duplication event showing that the two
alleles arose due to independent transposition events (explained
below).
[0074] The occurrence of four independent Mutator insertions in the
same locus in plants with the lls1 phenotype but not their
corresponding wild-type siblings constitutes proof that a fragment
of the lls1 locus had been isolated. It was observed that a Mu
insertion event gave rise to the lls1-ref allele which was believed
to arise in a non-Mu active background, suggesting that
cosegregation analysis should be attempted with an allele of
unknown origin before employing it in a targeted mutagenesis
strategy since the occurrence of an insertion in the same region of
the gene could obfuscate co-segregation analysis with a new
allele.
[0075] The lls1 Locus Encodes a Novel Plant Protein
[0076] To characterize the lls1 locus fully a cDNA and genomic
clone was isolated. Gene specific primers GSP1 and GSP3 were
employed along with universal primers to amplify 5' and 3'
fragments respectively of the lls1 transcript from a-cDNA library
constructed from 2 week old inbred PA405 seedlings. A 5' fragment
was then used as a probe to screen the PA405 cDNA library and 3
individual clones were recovered and the longest phagemid named
pJG200 was sequenced (Genbank Acc. # U77345). This sequence was
used to screen a maize EST database and another lls1 cDNA with an
additional 180 bp at the 5' end was recovered. The combined
sequence of these two cDNAs is shown in FIG. 1 and a 521 amino acid
continuous open reading frame can be predicted from this partial
transcript (FIG. 1). The identification of the termination codon
was supported by a similarly located predicted termination codon in
the sequence of an Arabidopsis lls1 homolog (below). A primer
designed against 139 bp to 173 bp downstream of the predicted start
codon of this sequence (GSP 17) was used for primer extension
analysis and a 454 bp long primer extension product was observed
thus predicting a 2119 bp total length transcript for the lls1
gene. In addition, the 3' ends of the cDNAs and the 3' ends of the
three PCR-amplified 3'-ends were also sequenced and three different
polyadenylation sites determined thus allowing for variation in the
size of the full length transcript (FIG. 1 and 4).
[0077] A 3' fragment of the lls1 gene was utilized to screen a
partial Sau3A genomic library of the maize inbred line B73 in order
to isolate a full-length lls1 gene sequence and a single positive
clone (designated G18) was isolated. A 7129 bp SacI fragment was
subcloned from the G18 genomic clone and the resulting plasmid
named pJG201 was entirely sequenced (Genbank Acc# U77346). By
comparison with the cDNA sequence pJG201 was found to contain
almost the entire lls1 coding region and a 5' region likely to
include the entire promoter. The predicted genomic organization of
the lls1 gene (FIG. 4) includes 7 exons and 6 introns. The SacI
restriction site at bp 7129 is 45 bp upstream of the predicted stop
codon and 320 bp upstream of the polyadenylation sites. Providing
that there are no other introns in the 5' region of the gene the
predicted transcriptional start site of the lls1 gene occurs at bp
3115 of the 7129 bp subclone.
[0078] Southern hybridization suggests that the lls1 gene is single
copy in the genome of maize since only one band was observed on
Southern blots of the wild-type DNA samples of the lls1-ref allele
cut with several restriction enzymes. That a duplicate of the lls1
gene exists has not yet been determined using lower stringency
washes. Three bands were observed in lls1*-5 when the EcoRl
digested mutant samples were probed with the left flank. A 10 bp
direct repeat was not observed on each side of the Mu8 insertion in
allele lls1*5. These results suggested that a rearrangement of DNA
more complex than a simple Mu8 element insertion had occurred at
this locus and the nature of this rearrangement was determined by
comparison with the genomic sequence of the lls1 gene. The left
flanking DNA comprises a direct repeat of the lls1 genomic sequence
extending from the EcoR1 site of Intron II to bp 43 of exon 4.
[0079] The predicted lls1 protein exhibits a largely hydrophilic
protein with a pI of 7.5. No hydrophobic regions suggesting
membrane association were observed. This fact suggests a cytosolic
or plastidic subcellular location for the LLS1 protein.
[0080] The lls1 Gene Exhibits Tissue and Cell Specific
Expression
[0081] The lls1 phenotype is developmentally expressed as described
above. LLS1 appears to be needed in expanded leaves but not in very
young leaves and thus lls1 transcripts may accumulate in older
leaves if the gene is transcriptionally regulated. The expression
of lls1 in fully expanded leaves of normal plants was examined
using a partial cDNA probe that extends from the beginning of exon
2 to the end of the lls1 transcript. A weak signal was detected
using 20 pg of total RNA and a high stringency wash. There did not
appear to be a significant gradient in gene expression from three
successively older leaves. When mRNA derived from pooled total RNA
from these leaves was utilized a single transcript was readily
detected (FIG. 5). The size of this single transcript was estimated
at 1.9.+-.0.2 kb a figure which coincides closely with the
full-length size determined by primer extension analysis (1.129
kb). To further examine the developmental pattern of lls1 gene
expression, mRNA derived from various plant tissues was probed with
an 802 bp NotI/PstI fragment that extends from the end of exon 2 to
exon 7 (FIG. 4). Lowest levels of expression were seen in seedling
leaves, 3 week old embryos and in young tassels. The lls1
transcript was readily detected in more mature tassels, young and
old ear shoots and I week old embryoe. Surprisingly, the lls1
transcript was most readily detected in seedling roots where the
lls1 phenotype has not been observed. In addition, the presence of
a second larger transcript (approximately 2.4 kb) was observed that
hybridizes with the lls1 probe in seedling roots and older tassel
tissue. When observed this larger transcript seems to be expressed
in equivalent amounts to the lower transcript. Since genomic blots
have indicated that lls1 is a single copy gene, the larger
transcript may represent post-transcriptional regulation of lls1
although there is precedence for a northern blot to reveal the
existence of a second gene when a southern analysis failed to do
so. These results indicate that the lls1 gene is not expressed
constitutively in all tissues but exhibits tissue specific
transcriptional regulation.
[0082] The lls1 Gene is Conserved Between Monocot and Dicot
Plants
[0083] To determine if lls1 related genes are present in other
species or organisms the predicted lls1 protein sequence was
utilized to search public databases of sequences of both known and
unknown functions. As indicated above, significant homology (70%
nucleic acid identity) was observed between the right flanking DNA
of lls1*-5 and an expressed sequence tag (EST) from Arabidopsis
thaliana. (Genbank Acc. # T45298). Three other Arabidopsis ESTs
were identified that overlap with this EST (Genbank Acc. #s N37395,
H36617 and R30609). The four overlapping ESTs were obtained from
the ABRC (Columbus, Ohio) and further sequenced. These sequences
were organized into a single contig 1977 bp in length (Genbank Acc.
# U77347). The 3' end of this contig overlaps with the upstream
region of the rpl9 gene (a nuclear encoded plastid ribosomal
protein) ending only 109 bp upstream of the rpl9 transcriptional
start. The Arabidopsis contig that exhibits 71.6% amino acid
similarity over a 473 consensus length with the maize lls ORF from
the available maize cDNA sequence. The amino terminus of the maize
versus the Arabidopsis ORFs differ significantly indicating the
possibility that each protein has a different leader peptide or
that an alternative start codon is utilized. The maize lls1
sequence has therefore been utilized to detect a highly homologous
gene from a dicot plant. This result prompted us to map the
Arabidopsis contig and this was achieved using the recombinant
Inbred (RI) lines developed by Clare Lister and Caroline Dean at
the John Innes Center (Lister et al. (1993) Plant Journal
4:745-750). Following identification of a suitable polymorphism one
EST (Acc# T45298) was used as a probe to score 48 RI lines. The map
position was located on the lower arm of chromosome three between
GL1 and m249. Importantly, the acdl mutation, whose cell death
phenotype is reminiscent of the maize lls1, also maps in this
region (Greenberg et al. (1993) Arabidopsis Mutants Compromised for
the Control of Cellular Damage During Pathogenesis and Aging, Plant
J. 4:327-341) suggesting that these two mutations in maize and
Arabidopsis are homologous. As genomes from two divergent plant
species have been found to have related lls1 genes, it is likely
that any number of plant species will possess genes regulating cell
survival in a manner similar to the maize lls1 gene. To further
test this hypothesis we tested the linkage of maize lls1 and
flanking markers to a sorghum mutation named drop-dead-1 (dd-1)
that is an EMS induced lesion-mimic mutation with spreading lesions
highly reminiscent of lls1 lesions. A segregating mapping
population was created by crossing a dd/dd line with Shangai Red
DD/DD and the progeny were allowed to self. Plants segregating for
drop-dead were identified and DNA isolated from several mutant and
wild-type progeny. A polymorphism for the lls1 locus could not be
identified but a polymorphism for the probe PIO200640 which is
.about.33 cM distal to lls1 was identified with HindE. This
polymorphism showed complete segregation with 14 mutant and 16
wild-type progeny strongly suggesting that this mutation maps to a
region syntenic with lls1 and that lls1 and dd are homologous
mutations and possibly orthologs.
[0084] lls1 Lesions Are Induced by Wounding and in les101/lls1
Double Mutants
[0085] In addition to intrinsic, developmental signals, external
factors also affect lls1 expression. lls1 lesions normally appear
randomly on developmentally competent areas of the leaf. However,
lls1 lesions can also be triggered to initiate at any site
(provided that the tissue is developmentally competent) by killing
cells either by inducing an HR with an incompatible pathogen or by
physical means (making pin prick wounds). The additive phenotype of
the double mutant of lls1 with Les2 or Les*-101 (two dominant
mimics that can initiate numerous lesions on maize leaves before
they become developmentally competent to express lls1) further
supports these results. On the double mutants, the early phenotype
of the lesions is discrete and of the respective Les type and also
of higher density as compared to that of lls1 lesions. However, as
the tissue acquires developmental competence to be able to express
the lls1 phenotype, most, if not all, Les sites transform into lls1
lesions that expand in an uncontrolled fashion to consume the whole
leaf. Thus the internal metabolic upset and cell death events
associated with a Les*-101 lesion appear to act as a trigger for
lls1 lesions.
[0086] Light is Required for lls1 and dd Lesion Formation
[0087] These observations fully support the hypothesis that lls1
functions to contain cell death from spreading, and it appears to
be critical during late stages of plant development. Interestingly,
the expression of lls1 lesions is completely dependent on light.
The region in the center of the leaf was covered with aluminum foil
just as lesions were initiating at the tip of the leaf. The lesions
formed progressively down the leaf but not where the leaf was
protected from light. Aluminum foil also protected lesions induced
by pin-prick wounding in maize lls1 plants and also observed
clearly in sorghum drop-dead plants. Using plastic filters that
transmit different wavelengths of light, it was found that visible
light in the spectral region of 650-700 nm is sufficient for this
effect. Yellow and orange filters also transmitted some red light
in the 650-700 nm so a contribution from light in the 560 to 640 nm
range cannot be excluded. Lesions did not form when only blue,
green, or far-red light reached the leaf. This phenomenon suggested
that active photosynthesis, which harvests light pre-dominantly in
the red spectral region, is required for lesion formation. This was
addressed genetically by creating double mutants of lls1 with iojap
1 (ijl--a recessive mutation in maize that produces albino and
light green sectors on an otherwise normal green leaf) or ncs7
which also exhibits light green but not albino sectors. These
double mutants have revealed that lls1 lesions can only form in
dark green tissues. This result indicates that some activity
related to light harvest or photosynthesis may be important in the
initiation and spread of lesions. Double mutants of lls1 with oil
yellow-700 provide further support to this interpretation. Oyl- is
a dominant mutation which by virtue of its inability to convert
protoporphyrin IX to Mg-protoporphyrin, is completely devoid of
chlorophyll b and has also reduced levels of chlorophyll a. On
oyl+lls1/lls1 plants lesions initiate with a lower density and
propagate very slowly in these plants and often lethality does not
ensue. Intriguingly, the suppressible effect of oyl on lls1 is not
observed when the plants are grown in a greenhouse or growth
chamber. Also we have observed that on an lls1/ijl double mutant,
where lesions do not initiate or develop in albino tissue, the
`death` signal (that probably allows lls1 lesions to propagate) can
sometimes diffuse across (traverse) the albino tissue if the sector
is narrow. This suppression is in contrast with many other lesion
mimics such as the dominant lesion mimic Les4 which readily forms
lesions in the albino sectors of Les4/+ij/ij plants. These
observations indicate that a process or a metabolite, which is
partly diffusible and whose activity may be affected by factors
including light, wounding, and pathogen invasion, is responsible
for the initiation and spread of cell death associated with lls1
lesions.
[0088] The Predicted LLS1 Protein Contains Two Structural Motifs
Highly Conserved in Bacterial Phenolic Dioxygenases
[0089] While no definite function could be ascribed to lls1 from
homology searches, analysis of the predicted amino acid sequence of
the lls1 gene product has revealed two conserved motifs, a
consensus sequence (SEQ ID NO: 6)
(Cys-X-His-X.sub.16-17-Cis-X.sub.2-His) for coordinating the
Reiske-type [2Fe-2S] cluster (Mason and Cammock (1992) The
Electron-Transport Proteins of Hydroxylating Bacterial
Dioxygenases, Annu. Rev. Microbiol. 46:277-.305) and a conserved
mononuclear non-heme Fe-binding site (SEQ ID NO: 7)
(Glu-X.sub.3-4-Asp-X2-His-X.sub.4-5-His) (Jiang et al. (1996)
Site-directed Mutagenesis of Conserved Amino Acids in the Alpha
Subunit of Toluene Dioxygenase: Potential Mononuclear Nonheme Iron
Coordination Sites, J. Bacteriol. 178:3133-3139), which are present
in the .alpha.-subunit of all aromatic ring-hydroxylating (ARII)
dioxygenases involved in the degradation of phenolic hydrocarbons.
In addition, the spacing (.about.90 amino acids) between these
motifs, which has recently been shown to be conserved in all ARII
dioxygenases, is precisely maintained in LLS1, adding further
evidence that LLS1 may encode a dioxygenase function. The ARII
dioxygenases consist of 2 or 3 soluble proteins that interact to
form an electron transport chain that transfers electrons from
NADII via flavin and iron-sulfur (2Fe--2S) redox centers to a
terminal dioxygenase. The latter, which is also a multimeric enzyme
consisting of either a homomers or .alpha. or .beta. heteromers,
catalyzes the incorporation of two hydroxyl groups on the aromatic
ring at the expense of dioxygen and NAD(P)H.
[0090] The consensus sequence of both the Rieske- and iron-binding
motifs (SEQ ID NOs: 6-7) as well as the spacing between them are
precisely conserved in a hypothetical protein (translated from an
ORF) from Synechocystis sp. PCC6803, which in addition, exhibits
66% amino acid identity to LLS1 among a stretch of more than 100
amino acids. Additionally, the Rieske center-binding site has also
been detected in the partial sequence of two seemingly related ESTs
(SEQ ID NOs: 31-32, respectively) of unknown function, one each
from rice and Arabidopsis.
[0091] lls1 and Cochliobolus carbonum
[0092] Inoculation of lls1 leaves with Cochliobolus carbonum Race 1
causes a proliferation of lls1-type necrotic lesions in the middle
to upper parts of the leaves. These lls1-type lesions superficially
resemble C. carbonum lesions but they are sterile. That is, plating
explants on carrot agar medium does not usually yield any C.
carbonum fungal growth. Spontaneous lls1 lesions occurring without
inoculation are also sterile and appear similar. Thus the lesions
induced by C. carbonum inoculation are apparently lls1-type lesions
and not susceptible C. carbonum lesions. This raises the question
as to whether these lesions indicate that the lls1 mutant is
susceptible to C. carbonum or not. It seems likely that the lls1
plants are resistant to C. carbonum, but that C. carbonum is able
to trigger lls1 lesion formation. The C. carbonum could be acting
as a stress that sets off the lls1 development. After all, even
abiotic stresses, such as needle pricking, will also induce lls1
lesion formation.
[0093] Inoculation of lls1 leaves with Cochiobolus carbonum toxin
plus or toxin minus causes few if any lesions to form in the middle
to lower parts of the inoculated leaves. This observation is
interpreted to mean that the lls1 mutation possesses induced
resistance to C. carbonum in that area of the leaf. While both
spontaneous lls1 lesions and C. carbonum lesions physically
resemble each other, neither type was seen in this area of the
leaf. In the middle transitional area there are some nascent
smaller lls1 lesions. It appears as though only the upper acropetal
areas of the leaf at this stage of development, are capable of
forming spontaneous lls1 lesions or C. carbonum induced
lesions.
[0094] In the lower-middle areas of lls1 leaves without any
pathogen inoculation, a several fold elevation of PRI and chitinase
proteins was observed on western blots over that of Lls1/lls1
wildtype heterozygotes. Upon inoculation, the PR1 and chitinase
expression in this area of the leaves was elevated slightly in lls1
and substantially in the Lls1/lls1 heterozygotes, such that after
inoculation both lls1 and the wildtype heterozygotes have similar
levels of PR1 and chitinase. Thus it appears that: 1) elevated PR
gene expression is correlated with resistance to C. carbonum in the
lower middle area of the leaves, and 2) the PR gene induction
exists prior to the resistance.
[0095] lls1 and Cochiobolus heterostrophus
[0096] As was seen with C. carbonum, inoculation of lls1 leaves
with Cochiobolus heterostrophus also causes a proliferation of
lls1-type necrotic lesions in the middle to upper parts of the
leaves. These lls1-type lesions are generally distinguishable from
C. heterostrophus necrotic lesions. These lls1-type lesions are
also sterile; that is, plating explants on carrot agar medium does
not usually yield any C. heterostrophus fungal growth. Spontaneous
lls1 lesions occurring without inoculation are also sterile and
appear similar. Thus the lesions induced by C. heterostrophus
inoculation are apparently lls1-type lesions and not susceptible C.
heterostrophus lesions. It appears that C. heterostrophus triggers
formation of lls1 lesions. C. heterostrophus appears to be acting
as a stress that sets off the lls1 lesion development. After all,
even abiotic stresses, such as needle pricking, will also induced
lls1 lesion formation.
[0097] Inoculation of lls1 leaves with Cochliobolus heterostrophus
causes few if any lesions to form in the middle to lower parts of
the inoculated leaves. This observation was interpreted to mean
that the lls1 mutation possesses induced resistance to C.
heterostrophus in that area of the leaf. Spontaneous lls1 lesions
and C. heterostrophus lesions are usually distinguishable by
appearance, yet neither type was observed in this area of the leaf.
In the middle transitional area there are some nascent smaller lls1
lesions, so it appears as though only the upper acropetal areas of
the leaf are capable of forming lls1 lesions. However, the lack of
C. heterostrophus lesions in this area of the leaf relative to
their appearance in Lls1/lls1 and Lls1/Lls1 wildtype controls,
indicates that lls1 possesses resistance to C. heterostrophus in
that area of the leaf. That the lls1 heterozygotes are not
resistant indicates that this resistance, like lls1 lesion
formation, is a recessive Mendelian trait.
[0098] In the lower-middle areas of lls1 leaves without any C.
heterostrophus inoculation, a several fold elevation of PR1 and
chitinase proteins was observed on western blots over that of
Lls1/lls1 wildtype heterozygotes. Upon inoculation with C.
heterostrophus, the PR1 and chitinase in this area of the leaves is
elevated slightly in lls1 and substantially in the Lls1/lls1
heterozygotes, such that after inoculation they have similar levels
of PR1 and chitinase. Thus it appears that elevated PR gene
expression is correlated to resistance to C. heterostrophus in the
lower middle area of the leaves, and that this elevated PR gene
expression occurs prior to the inoculation and resistance.
[0099] lls1 and Puccinia sorghi (Rust)
[0100] Rust inoculation of lls1 plants does not necessarily induce
lls1-type necrotic lesions. It was observed that rust will infect
lls1 plants and produce sporulating lesions. This indicates that
unlike C. carbonum, C. heterostrophus, and Puccinia sorghi, rust, a
biotrophic pathogen, is able to infect lls1 and Lls/lls1
heterozygote control plants. The fact that P. sorghi will infect
and form lesions indicates that P. sorghi can evade triggering lls1
lesions formation and that it can survive and grow on lls1. The
lls1 mutation is therefore not necessarily rust resistant.
Differences that may exist in rust susceptibility in the acropetal
versus basipetal regions of the leaf have not been
investigated.
[0101] Western blots revealed that mutant lls1 plants and Lls/lls1l
wildtype heterozygote plants had similar levels of chitinase
expression following rust inoculation. The expression of PR1,
however, was slightly higher in the wildtype plants than in lls1
mutants following rust inoculation. These experiments seem to
indicate that although rust is able to avoid triggering lls1-type
lesions formation in lls1, it still manages to trigger at least
chitinase expression. These results may have important
ramifications for understanding how pathogens are detected by the
plant host, and if detected, whether by the same or different
mechanisms, how the signaling pathways determine whether PR gene
expression activated.
[0102] To date no studies have isolated a protein(s) or gene(s)
ubiquitously involved in the degradation of plant phenolics.
Phenolics in plants are often sequestered in cell compartments
until needed or synthesized only when required. Some phenolics
however such as benzoic acid and salicylic acid have been proposed
to play key roles in preconditioning cells to undergo cell death
during the hypersensitive response as described by current models
for systemic acquired resistance in dicot plants.
[0103] One candidate that may fit well in this role is salicylic
acid (SA). SA, which exhibits a 10-50 fold increase during the HR
and is also triggered in response to oxidative stresses associated
with ozone or UV exposure (Hammond-Kosack and Jones (1996)
Resistance Gene-dependent Plant Defense Responses, Plant Cell
8:1773-1791); Ryals et al. (1996) Systemic Acquired Resistance,
Plant Cell 8:1809-1819), is known to cause H.sub.2O.sub.2 buildup
(Chen et al. (1993) Involvement of Reactive Oxygen Species in the
Induction of Systemic Acquired Resistance by Salicyclic Acid in
Plants, Science 242:883-886) and transmute into a cell damaging
free radical under oxidinzing conditions (Durner and Klessig (1996)
Salicylic Acid is a Modulator of Tobacco and Mammalian Catalases,
J. Biol. Chem., 271:28492-28501). These attributes of SA indicate
that it may be a mediator of cell death in lls1 mutants, a
hypothesis fully compatible with the demonstrated dependence on SA
of cell death associated with a number of Arabidopsis lsd mutants
(Dangl et al. (1996) Death Don't Have no Mercy: Cell Death Programs
in Plant-microbe Interactions, Plant Cell 8:1793-1807; Weyman et
al. (1996) Suppression and Restoration of Lesion Formation in
Arabidopsis lsd mutants, Plant Cell 12:2013-2022). However, as
noted above, the possibility nevertheless remains that a novel
compound or mechanism is responsible for lls1-associated cell
death.
[0104] The predicted association of LLS1 with an iron-sulfur
cluster suggests that it may also participate in
oxidation-reduction reactions. Proteins that use iron-sulfur
clusters as prosthetic groups often function as biosensors of
oxidants and iron (Roualt and Klausner (1996) Iron-sulfur Clusters
as Biosensors of Oxidants and Iron, Trends Biochem. Sci.
21:174-177). LLS1 may also serve as a kind of rheostat such as that
proposed for LSD1 in regulating cell death in plants (Jabs et al.
(1996) Initiation of Runaway Cell Death in an Arabidopsis Mutant by
Extracellular Superoxide, Science 271:1853-1856).
[0105] Working Model for lls1 Function
[0106] As noted earlier, the present invention is not dependent
upon a particular mode of action. However, it is predicted that the
LLS1 protein functions to inhibit the action of a cell "suicide
factor" by degrading that factor. The suicide factor is a phenolic
compound that is either a toxin or signal associated with
photosynthetic stress or wounding or due to metabolic upset in the
case of lls1/LeslOl double mutants. Phenolics can cause superoxide
production formation by donating an electron to dioxygen while in a
semiquinone form (Appel (1993) Phenolics in Ecological
Interactions: The Importance of Oxidation, J. Chem. Ecol.
19:1521-1552). Photosynthetic organisms have evolved multiple
mechanisms to dissipate excess energy and avoid the production of
reactive oxygen intermediates (ROI) during photosynthesis.
Free-radicals are scavenged by ascorbate, carotenoids, the
xanthophyll cycle, alpha-tocopherol, glutathione, and various
phenolics (Alscher et al (1993), Antioxidants in Higher Plants).
The oxidative state of a cell influences dramatically the ability
of phenolics to promote free radical formation (Appel (1993)
Phenolics in Ecological Interactions: The Importance of Oxidation,
J. Chem. Ecol. 19:1521-1552). The development of lls1 lesions could
result in cell death due to the inability to remove a toxic
phenolic or signal that has accumulated in a cell.
[0107] Whereas a toxin may directly inhibit basic metabolic
processes a signal may trigger a programmed cell death pathway that
is reminiscent of the hypersensitive response. Lesions thus spread
because the release of the contents of dying cells cause oxidative
stress in surrounding cells and result in the autocatalytic
production of the cell suicide factor. Alternatively a signal for
cell death may activate cell death programs in surrounding cells
unless it is removed. The developmental gradient of lls1 lesion
expression may reflect the accumulation of a suicide factor in
older cells. Young tissue does not form lesions when wounded and
this may reflect the lack of accumulation of a suicide factor, the
inability to yet synthesize that compound or the existence of a
juvenile lls1 homolog. Protection of the plant tissue from light
would directly reduce the concentration of the suicide factor and
avoid lesion formation. The concentric circle appearance of lls1
lesions may thus result from variation in the production of the
suicide factor due to diurnal light cycles. Revertant sectors would
be resistant to this suicide factor and the ability of lesions to
"traverse" pale green or albino sectors in lls1/lls1I io/io or
lls1/lls1 NCS7 double mutants would reflect the concentration and
diffusibility of the toxic phenolics across tissues less able or
unable to produce the suicide factor. In normal tissues functional
LLS1 limits the effect of a suicide factor released in the process
of wounding or stress. Finally it is expected that if LLS1 affects
phenolic metabolism that a change in phenolic profile would occur
in lls1 plants. Significantly, this prediction is supported by the
report that a paracoumaric ester accumulates in lls1 lesioned
plants but not in normal wild-type siblings or wild-type siblings
inoculated with the fungus Cochliobolus heterostrophus (Obanni et
al. (1994) Phenylpropanoid accumulation and Symptom Expression in
the Lethal LeafSpot Mutant of Maize, Physiol. Mol. Plant Path.
44:379-388).
[0108] lls1 May Play a Role in the Hypersensitive Response
[0109] A complex series of cellular events is envisaged to occur
during the activation of defense responses in plants
(Hammond-Kosact et al. (1996) Resistance Gene-dependent Plant
Defense Responses, Plant Cell 8:1773-1791.). Incompatible responses
will often lead to the death of an infected cell within a few hours
of infection. There is considerable evidence that this
hypersensitive response (HR) is a form of programmed cell death
activated by file plant cell. Lesion mimic mutations may cause an
uncoupling of the regulatory steps of this process. Recent evidence
has shown that control of cell death involves checkpoints that
negatively and positively modulate the decision to progress to cell
collapse. Evidence is provided by the observation that the lesion
mimic phenotype of the lsd1 and lsd6 mutations of Arabidopsis are
suppressed in the presence of the transgene nahG which degrades
salicylic acid (SA). Application of 2,6 dichlorisonicotinic acid (a
chemical inducer of systemic acquired resistance--SAR) restored
lesion phenotype of these mutants (Dangl et al (1996) Plant Cell
8:1793-1807). This result directly implicates SA in the signaling
pathway that leads to cell death in these lesion mimics and that
normally LSD1 and LSD6 would serve to negatively modulate that
pathway. acdl plants form spreading lesions in the presence of a
functional lsd1 gene suggesting that ACD1 operates downstream or
oil a separate pathway from LSD1. Also there is evidence to
indicate that SA donates an electron to catalase and in so doing
becomes a free radical which interacts with membrane lipids to
promote lipid peroxides which further promote membrane damage and
cell collapse. Collectively these results suggest that acd1
functions downstream of lsdl to inhibit a cell death pathway that
is promoted by superoxide via SA and it may be that acd1
transcription is activated by LSD1. ACD1/LLS1 may degrade SA and
thus negatively regulate a signaling pathway that could lead to
runaway promotion of cell death. ACD1/LLS1 may be positively
regulated by competing sensors of well being within the cell via
the LSD1 protein and or other activators. Thus in an lls1 mutant
what normally may constitute a minimal stress may become
exaggerated through a runaway amplification loop and cell death
pathways may be triggered resulting in lesion formation. This model
predicts that nahG in an acd1/acd1 mutant will abolish lesion
formation.
[0110] Cell Death Mechanisms in Plants Versus Animals
[0111] Lesion mimic genes are now providing insight into the kinds
of genes involved in regulating cell death in plants. Three lesion
mimic genes have now been cloned and do not have related
counterparts in animal systems. This suggests that cell death is
regulated in plants in a manner very different from models
describing cell death regulation in animals although a role for ROI
seems common to both systems. The recently cloned mlo locus from
barley has been shown to encode a membrane protein and the lls1
gene from Arabidopsis may encode a transcriptional activator. Both
of these genes may normally serve to interpret external or internal
stress signals and when mutated turn on or off other genes that
cause cell death or cell survival respectively. The lls1 gene
appears to encode an enzyme involved in suppressing the spread of
cell death through some aspect or phenolic metabolism. Phenolic
production has long been long associated with cell death in plants
but little understood at the molecular level. Studies of the cloned
lls1 gene may afford unexpected insights into this important aspect
of plant physiology.
[0112] Expression Profile of Lethal Leaf Spot 1 (lls1)
[0113] In leaves 2 and 4 of 16-days-olds wild-type seedlings (Mo17,
B73), the strongest expression of lls1 is seen in both upper and
lower epidermis and its derivatives (such as silica cells), in
sklerenchyma cells on either side of vascular bundles, and in
protoxylem elements. A weaker, but clearly discernible expression
signal is observed in bundle sheath, mesophyll cells and midrib
parenchyma. Expression is undetectable in metaxylem, phloem and
companion cells.
[0114] In 7-day-old darkgrown wild-type seedlings (B73), lls1
expression can be detected at low levels in a uniform distribution
throughout most leaf cells. Slightly elevated levels can be found
in coleoptile and midrib of the two oldest leaves.
[0115] In leaves of the dominant lesion mimic mutant Les 101, and
in the lls1 mutant itself, expression of lls1 is essentially the
same as in wild-type.
[0116] For in situ expression analysis of lls1, a 0.7 kb NotI-Ps
fragment from the middle of the cDNA was used to make labeled sense
and antisense riboprobes.
[0117] Clones comprising the genomic sequence and cDNA sequence
described herein were deposited on Nov. 14, 1996 with the American
Type Culture Collection, Rockville, Md., and given accession
numbers ATCC 97791 and ATCC 97792.
[0118] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled 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 to be incorporated by
reference.
[0119] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *