Progress 02/01/00 to 01/31/05
Outputs
We have developed pathogen systems for testing host gene function in Medicago truncatula. Our initial analyses have focused on Phytophthora medicaginis, Colletotricuhm trifolii and Meloidogyne incognita. From a screen of approximately 100 ecotypes of M. truncatula, we identified sources of resistance and susceptibility to each of these pathogens. We have also completed the analysis of several hundred resistance gene analog sequences, and genetically mapped a representative set of over 60 characterized RGA loci. Ultimately, the mapping of disease resistance phenotypes and the molecular RGA markers are expected to contribute to the rapid characterization of new legume resistance genes at the molecular and genetic level.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
We have been developing pathogen systems for testing host gene function. To date, we have focused on Rhizoctonia solani, Phytophthora dicaginis, Colletotrichum trifolii, Xylella fastidiosa, and Meloidogyne incognita. A collection of approximately 100 ecotypes of M. truncatula and identified sources of resistance and susceptibility for P. medicaginis, C. trifolii, and M. incognita have been screened. We are taking a molecular approach to identify resistance gene analogs (RGAs). By means of degenerate primers, we identified 10 groups of non-TIR RGAs that were sufficiently divergent that they should not cross hybridize on Southern blots. One member from each group was used to screen high density BAC library filters, yielding 91 candidate BAC clones for the NBS/LRR (non-TIR) RGAs. The combined molecular and phenotypic analysis or resistance genes is expected to reveal some correspondence between genetic mapping of resistance gene phenotypes and the map position of
sequence-characterized RGAs.
Impacts
(N/A)
Publications
- Salzer, P. Bonanomi, A., Beyer, K., Vogeli-Lange, R., Aeschbacher, R.A., Lange, J., Kim, D., Cook, D.R., Boller, T. (2000). Chitinase gene expression in mycorrhizal, nodulated and pathogen-infected roots of Medicago truncatula. MPMI, in press.
- Cook, D. 1999. Medicago truncatula - a model in the making! Current Opinion in Plant Biology, 2 (4) 301-304.
- Nam. Y-W, Penmetsa, R.V., Endre, G., Kim, D., and Cook, D.R. 1999. Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene response genes. Theor Appl Genet 98: 638-646.
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Progress 01/01/98 to 12/31/98
Outputs
We have determined that the early nodulin gene, rip1, is regulated by Rhizobium Nod factor in a highly structure specific manner, requiring sulfation at the reducing end, and a combination of wild type acylation and acetylation at the non-reducing end of the molecule. Thus, the induction of the rip1 gene exhibits the same specificity for Nod factor structure as nodulation itself. The rip1 transcription unit is preceded by a 377 bp sequence element that contains eight copies of a 17 bp repeat, and three copies of a 10 bp repeat, that resemble the OBP1 and OCS cis-elements associated with several hydrogen peroxide regulated plant genes. We initiated a series of experiments to test the hypothesis that rip1 expression is regulated by hydrogen peroxide. We have determined that rip1 is induced by exogenous hydrogen peroxide, with a dose dependence similar to hydrogen-peroxide regulated genes, such as GST. Moreover, pharmacological agents that are known to inhibit the
endogenous generation of hydrogen peroxide, such as DPI and NNDDC which block NADPH Oxidase and Superoxide Dismutase -SOD-, are effective antagonists of the Rhizobium induction of rip1. Taken together, these data support the hypothesis that hydrogen peroxide is both sufficient and necessary for rip1 up-regulation. Moreover, they suggest that an early and specific response to Nod factor involves the generation of reactive oxygen species, possibly similar to the oxidative burst associated with plant responses to pathogens. Our recent experiments have focused on monitoring superoxide production during early nodulation when rip1 is highly induced in the differentiating epidermis, and during early nodule morphogenesis when rip1 is expressed in nascent nodule promordia. We have used the chromogenic substrate nitroblue tetrazolium -NBT- to monitor superoxide production in roots treated with various Rhizobial strains or Nod factor. Initially, we determined that the distal 2mm of the root is a
locus of constitutive superoxide generation. Proximal to this root tip region, in the 2mm to 5mm zone, NBT staining is absent or reduced in uninoculated roots, but is stimulated by Rhizobium in a Nod factor dependent manner. Briefly, both wild type Rhizobium and purified Nod factor, but not the Nod factor deficient mutant SL44, elicit NBT staining in this root tip proximal zone. This NBT staining reaction is effectively blocked by SOD pretreatment, indicating that the NBT localization is a reflection of superoxide production. Moreover, the spatial localization of NBT staining is coincident with the location of rip1 transcript accumulation determined previously by root tip dissections and in situ hybridization experiments. Taken together, these data suggest that Nod factor is both necessary and sufficient for superoxide generation in the root zone where rip1 is induced. Similar results were obtained when we analyzed 60 hr nodule primordia, which stain intensely with NBT, in an
SOD-dependent manner.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/97 to 12/31/97
Outputs
We conducted a large-scale mutagenesis in Medicago truncatula and screened several EMS treated seed bulks for altered nodulation phenotypes. Most of the nodulation mutants can be grouped into one of three general phenotypic categories: those that fail to nodulate, those that form ineffective nodules (nod+/fix-), and those that exhibit an increased number of nodules. We are examining two classes of non-nodulating mutants. One class of nod-minus mutants displays a Rhizobium-induced defect in the polar growth of root hairs and does not become infected by Rhizobium. This mutant class is also defective in infection by VA mycorrhizal fungi, a phenotype that is studied by our collaborator (Dr. Maria Harrison) at the Samuel Roberts Noble Foundation. Thus, the underlying defect is apparently integral to both rhizobial and mycorrhizal infection. The second class of nod-minus mutants has numbers of infections similar to wild type, but all infections are arrested in the root
epidermis. This mutant class is pleiotropic for excessive root hair proliferation, the absence of organized nodule primordia, and short, unevenly thickened roots. We have also identified two mutants with an approximately 10-fold increase in numbers of nodules: the first mutant, referred to as sun for super numerary, and the second, referred to as skl due to its sickle-shaped zone of nodulation. Although both sun and skl mutants display increased nodule numbers, the mutants are readily distinguished based on morphological, physiological and genetic criteria. In addition to their symbiotic phenotype, sickle plants showed delayed petal senescence and decreased abscission of seed pod and leaves. Physiological and genetic analysis indicate that the skl mutation confers insensitivity to the plant hormone ethylene. In contrast to sickle, the sun mutant displays normal ethylene phenotypes, indicating that the supernodulation phenotype is unlikely to be due to altered ethylene responses.
Impacts
(N/A)
Publications
- PENMETSA, R.V., and COOK, D. 1997. A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont. Science 275:527-530.
- COOK, D.R., VANDENBOSCH, K.A., de BRUIJN, F.J., and HUGUET, T. 1997. Model legumes get the nod. Plant Cell 9:275-281.
- COOK, D.R., and VANDENBOSCH, K.A. 1997. International research efforts on Medicago truncatula: a model for legume biology. IS-MPMI Reporter May 1997, 10-15.
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Progress 01/01/96 to 12/30/96
Outputs
Identification and characterization of nodulation mutants in the model legume Medicago truncatula. From over 50 verified and partially characterized nodulation mutants, we selected six mutants for detailed study. We have characterized: 1. A recessive mutation that completely blocks colonization by both rhizobial and mycorrhizal symbionts. 2. Two recessive mutations that limit numbers of infections, or progression of infection, respectively. 3. Two mutants characterized by increased nodule number. Double mutant analysis indicates that these genes define separate genetic pathways. Each of these genes appear to block perception of a different plant hormone. 4. We have generated mapping populations for three of these mutants.
Impacts
(N/A)
Publications
- Penmetsa, R.V. and Cook, D. 1997. A legume ethylene-insensitive mutant is hyperinfected by its rhizobial symbiont. Science 275 (in press).
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Progress 01/01/95 to 12/30/95
Outputs
From a population of chemically mutagenized (EMS) Medicago truncatula we have identified over 200 putative symbiotic mutants, including non-nodulators and those with altered nodule development. To determine whether any of these mutants were altered in control of rhizobial infection, we modified R. meliloti ABS7M to contain a constitutively expressed lacZ. This modified strain provides a simple visual assay for rhizobial infection. In wild type M. truncatula approximately 300 infections were visible within the nodulation zone; from these, an average of eight nodules were produced. Thus, the infection efficiency of wild type M. truncatula is 8/300, or approximately 3%. In subsequent experiments, we used this modified Rhizobium strain to characterize infection in the M3 generation of the previously identified nodulation mutants. We have identified mutants that allow no infection, mutants that allow 10X fewer infections than wildtype, mutants where infections intitiate as
in wildtype but the infection threads remain limited to epidermal cells and nodule primordia fail to form, mutants in which nodule morphogenesis begins but arrests prematurely, and two distinct mutants that allow a >10-fold increase in persistent infections. Presently we are initiating genetic and molecular anaylsis with several of these mutants.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 01/01/94 to 12/30/94
Outputs
Recently we initiated a project to identify genes required for normal modulationby means of EMS mutagenesis. The identification of mutants with symbiotic defects implicates key genes that are targets for cloning by map-based strategies. Plants displaying altered symbiotic properties can also be used to examine the biology of Rhizobium-legume interactions, for example we have a temporal series of Rhizobium induced genes which will potentially allow us to localize defects to specific phases of modulation. We have created and are screening a population of chemically mutagenized seed. Conservative estimates are that the population contains a minimum of 30,000 unique variants; thus, we should be able to identify most phenotypes of interest. As a byproduct of a screen for symbiotic variation we have identified putative embryo lethal mutants, chlorophyll retention mutants, leaf and root morphology mutants, phytochrome mutants, phytohormone mutants, and disease-lesion-mimic
mutants. We have also identified two M2 individuals that lack viable pollen, and thus are male sterile. The availability of male sterile lines should greatly facilitate subsequent genetic analysis. We have devised a visual screen for nodule morphogenesis that utilizes aeroponic chambers and allows us to screen up to 2,000 individuals/week. At this point we have identified numerous individuals with putative symbiotic defects, including hypernodulators (distinct from the soybean super-nodulation phenotype), non-nodulators, and delayed modulators.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 04/01/93 to 12/30/93
Outputs
The focus of this research is to characterize a peroxidase gene that is expressed during nodule initiation in the annual legume, Medicago truncatula. During the past year we have determined that, 1. the induced peroxidase gene, designated rip1, is present as a single copy within the M. truncatula genome, and that it lacks detectable homology with other M. truncatula genes when tested under stringent hybridization conditions; 2. we have determined that rip1 is induced rapidly during nodule initiation, with maximal expression detectable after six hours. This rapid induction makes rip1 one of the earliest Rhizobium-induced genes yet identified; 3. we have also determined that rip1 expression is coincident with the zone of nodule development. During nodule initiation rip1 expression can be detected near the root tip using a modified tissue printing method, and preliminary data obtained by in situ hybridization suggests that, at the root tip, rip1 expression is localized to
epidermal cells and to differentiating vascular tissue; 4. we have also conducted a preliminary analysis of peroxidase isozymes present in Rhizobium-inoculated roots. We have tentatively identified a minimum of five peroxidase isozymes in activity stained gels, with apparent molecular weights ranging from 23Kd to >70Kd, and isoelectric points ranging from 5.0 to >8.5. 5. we have made progress towards construction genomic and cDNA libraries of M. truncatula, although genomic and cDNA clones for rip1 have yet to be isolated.
Impacts
(N/A)
Publications
- No publications reported this period.
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