Progress 06/01/04 to 05/31/08
Outputs
Progress Report Objectives (from AD-416) Isolate mutants of Arabidopsis thaliana with an altered sensitivity to oxalate and to clone the mutated genes in order to characterize the gene products that trigger oxalate sensitivity and manipulate them to enhance oxalate tolerance and Sclerotinia resistance. Approach (from AD-416) Screen more than 62,000 Arabidopsis mutant lines and isolate mutated genes. Screens for oxalate-tolerant mutants will identify seedlings that have the ability to germinate in the presence of high concentrations of oxalate. Seeds will be subjected to a secondary screen to eliminate false positives which will indicated the number of genes responsible for phenotypic change. Mutants will be exposed to white mold disease Once genes have been identified, it will be possible to deploy them for protection against Sclerotinia sclerotiorum.to determine differences in oxalate sensitivity. DNA will then be isolated from mutants. Significant Activities that Support Special Target Populations This project was initiated on June 1, 2004 and continued through May 31, 2008. The overall objective was to identify genes transcribed during Sclerotinia growth conditions and developmental states that are critical for disease development, as well as to determine which of the identified genes are uniquely expressed or significantly up-regulated in the examined tissue. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. Budding yeast was demonstrated to be a valid model system for dissecting the genetic basis of eukaryotic oxalate sensitivity. The yeast screen provided important information for analyzing homologous candidate genes in Arabidopsis thaliana for oxalate sensitivity and defense against Sclerotinia sclerotiorum. Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. Genotypic differences in susceptibility of Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme susceptibility of this cruciferous plant. To overcome this limitation, inoculation conditions were established that enable the evaluation of differences in susceptibility to S. sclerotiorum among Arabidopsis mutants and ecotypes. Two coi1 mutant alleles conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2 and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants, respectively. Actigard, a commercial formulation of the systemic acquired resistance inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a decrease in hydrogen peroxide production, but no detectable changes in plant superoxide production or gene expression. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 � Plant Diseases.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Isolate mutants of Arabidopsis thaliana with an altered sensitivity to oxalate and to clone the mutated genes in order to characterize the gene products that trigger oxalate sensitivity and manipulate them to enhance oxalate tolerance and Sclerotinia resistance. Approach (from AD-416) Screen more than 62,000 Arabidopsis mutant lines and isolate mutated genes. Screens for oxalate-tolerant mutants will identify seedlings that have the ability to germinate in the presence of high concentrations of oxalate. Seeds will be subjected to a secondary screen to eliminate false positives which will indicated the number of genes responsible for phenotypic change. Mutants will be exposed to white mold disease Once genes have been identified, it will be possible to deploy them for protection against Sclerotinia sclerotiorum.to determine differences in oxalate sensitivity. DNA will then be isolated from mutants. Significant Activities that Support Special Target Populations This report documents research conducted under a specific cooperative agreement between ARS and Oregon State University. Additional details of research can be found in the report for the in-house associated project 5442-21220-023-00D, �Sclerotinia Diseases.� This project was initiated on June 1, 2004, and research is ongoing. The overall objective is to identify genes transcribed during Sclerotinia growth conditions and developmental states that are critical for disease development, and to determine which of the identified genes are uniquely expressed or significantly up-regulated in the examined tissue. We demonstrated that budding yeast is a valid model system for dissecting the genetic basis of eukaryotic oxalate sensitivity. The yeast screen provided important information for analyzing homologous candidate genes in Arabidopsis thaliana for oxalate sensitivity and defense against Sclerotinia sclerotiorum. Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. Genotypic differences in susceptibility of Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme susceptibility of this cruciferous plant. To overcome this limitation, we have established inoculation conditions that enable evaluation of differences in susceptibility to S. sclerotiorum among Arabidopsis mutants and ecotypes. Two coi1 mutant alleles conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2 and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants, respectively. Actigard, a commercial formulation of the systemic acquired resistance inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a decrease in hydrogen peroxide production, but no detectable changes in plant superoxide production or gene expression. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. Publications: Cheng, V., Stotz, H.U., Hippchen, K., Bakalinsky, A.T. 2007. Genome- wide screen for oxalate-sensitive mutants of Saccharomyces cerevisiae. Applied and Environmental Microbiology (in press). Guo, X., Stotz, H.U. 2007. Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on JA, SA, and ethylene signaling. Molecular Plant-Microbe Interactions (in press).
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Oregon State University. This project was established in June 1, 2004 and research is ongoing. The overall objective is to identify genes transcribed during Sclerotinia growth conditions and developmental states that are critical for disease development, and to determine which of the identified genes are uniquely expressed or significantly up-regulated in the examined tissue. Please refer to the report for project 5442-21220-010-00D, Sclerotinia Diseases, for additional information. Conditions were established to differentiate genotype-specific differences in resistance to Sclerotinia sclerotiorum strain 1980 among mutants and ecotypes of Arabidopsis thaliana. A critical parameter was to limit nutrient availability using a minimal medium (Cruickshank, 1983) because virulence of S. sclerotiorum is a function of nutrient supply.
Specifically, and agar medium containing 3 g/l D,L-malic acid, 1 g/l NaOH, 2 g/l NH4NO3, 0.1 g/l MgSO4 x 7 H2O was used. The jasmonic acid (JA) pathway was shown to be involved in resistance to S. sclerotiorum. Specifically, a mutation in coi1, a gene encoding an F- box protein and essential regulator of JA response, was shown to increase susceptibility to S. sclerotiorum (Fig. 1A). The involvement of the JA pathway was suggested because COS1, a suppressor of COI1 (Xiao et al., 2004), is closely related to RIB4, a yeast (Saccharomyces cerevisiae) gene we found to be involved in oxalate tolerance. Expression of the defense-related plant defensins gene PDF1.2 was blocked in coi1 mutants after inoculation with S. sclerotiorum, suggesting the absence of JA signaling (Fig. 1B). Induction of the pathogenesis-related gene PR1 expression was unchanged compared to wild type plants, suggesting that induction of salicylic acid (SA)-induced defense responses was unaltered in the coi1 mutant.
Collectively, these data strongly suggest that the JA pathway confers partial resistance to S. sclerotiorum. Similar results were previously obtained by others, suggesting that the JA pathway is involved in resistance to the closely related necrotrophic fungal pathogen Botrytis cinerea (Thomma et al., 1998; Xiao et al., 2004). The coi1 mutant was also challenged with the oxalate-deficient S. sclerotiorum strain A4. This Arabidopsis mutant was hyper-susceptible to the oxalate-deficient fungal mutant, suggesting that JA signaling is involved in both oxalate-dependent and oxalate-independent defense responses. The coi1 and cos1 mutants were tested for oxalate sensitivity using seedling growth on Murashige & Skoog (MS) agar medium containing 4 mM oxalate, pH 5.5 as an indicator. Neither mutant exhibited enhanced sensitivity to oxalate. Root length and dry weight were quantified after two weeks of growth on MS medium containing or lacking oxalic acid. Oxalate caused a clear inhibition of
growth but there were no oxalate- related genotype-specific differences. These results strongly suggest that plant defense mechanisms other than oxalate tolerance are involved in resistance to S. sclerotiorum. We detected ecotype-specific differences in susceptibility to S. sclerotiorum. Uod-7 and Pro-0 were the most resistant and susceptible Arabidopsis ecotypes, respectively. We previously reported on ethylmethane sulfonate (EMS) mutants that survive oxalate toxicity better than wild type plants. Out of 42 oxalate- tolerant lines, three lines were identified as putatively resistant to S. sclerotiorum. Publications and presentations: A manuscript entitled "Oxalate-sensitive mutants of Saccharomyces cerevisiae" by V. Cheng, H. U. Stotz, K. Hippchen, and A. T. Bakalinsky is in preparation for publication in Appl. Env. Microbiol. An NSF proposal on the "Molecular basis of resistance to an oxalate- producing pathogen" by H. U. Stotz (PI) and A. Bakalinsky, J. Myers, and Gary Tallman
(co-PIs) was submitted in preparation for submission in July 2006. Efforts are underway to make our results more broadly applicable using genome-based bioinformatics tools and the Legume Information System (Gonzales et al., 2005).
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Oregon State University. This project was established in June 1, 2004 and research is ongoing. Its overall objective is to identify genes transcribed during Sclerotinia growth conditions and developmental states that are critical for disease development, and to determine which of the identified genes are uniquely expressed or significantly up-regulated in the examined tissue. Please refer to the report for project 5442-21220-010-00D, Sclerotinia Diseases, for additional information. Transgenic Arabidopsis thaliana lines expressing a wheat oxalate oxidase gene are relatively more tolerant of oxalate compared to untransformed plants, but the magnitude of this effect and the level of oxalate oxidase expression are very low. Arabidopsis ecotypes vary in oxalate sensitivity. For example, ecotype Landsberg erecta (Ler) is reproducibly more
sensitive to oxalate than ecotype Columbia (Col). Preliminary data (n = 8, P = 0.054) indicate that Ler is also more susceptible than Col to an oxalate-deficient mutant of Sclerotinia sclerotiorum. However, as previously published (Dickman and Mitra, 1992), there was no difference in susceptibility of these two ecotypes to a wild type strain of S. sclerotiorum. The enhanced oxalate sensitivity and susceptibility to the Sclerotinia mutant of Ler may be related to the mutation in the ERECTA gene and stomatal development (Masle et al., 2005; Shpak et al., 2005). Mapping of recombinant inbred lines can provide evidence for this hypothesis. Arabidopsis is more sensitive to oxalic acid than other dicarboxylic acids. An oxalate concentration of 4 mM at pH 5.5 was optimal for screening EMS mutants. An oxalate tolerance screen of approx. 10,000 EMS mutants was successful using 250 seedlings per Petri dish (15 cm in diameter). The progeny of 42 mutant lines were on average more tolerant of
oxalate (17- 62% survival on oxalic acid containing medium) than the wild type Col parent (17% survival in the presence of oxalic acid). All but eight lines were tested for their interaction with wild type S. sclerotiorum and found to be susceptible. An oxalate sensitivity screen of approx. 10,000 EMS mutants was performed using 1,000 seedlings per Petri dish (15 cm in diameter). The progeny of 8 lines performed worse on oxalate-containing medium than wild type Col. Susceptibility to Sclerotinia relative to wild type Col has not yet been assessed. We conclude that mutations cause quantitative differences in oxalate tolerance or sensitivity that are difficult to map. As an alternative, we will test Arabidopsis ecotypes because they are genetically more diverse and, thus, better for detecting and mapping differences in oxalate sensitivity and susceptibility to Sclerotinia. Oxalic acid prevents calcium signaling in response to abscisic acid (ABA) and, instead, triggers cytosolic Ca2+
increases in response to H2O. This observation confirms our previous data (Guimaraes and Stotz, 2004), suggesting antagonism between oxalic acid and ABA. Our results are in agreement with Cessna et al. (2000) because H2O2 is a mediator of Ca2+ responses to ABA and because oxalate suppresses the oxidative burst. Oxalate activates the plasma membrane H+-ATPase. Our results (Guimaraes and Stotz, 2004) suggest that oxalate induces K+ accumulation in guard cells, which leads to stomatal opening and water stress. We wanted to know whether K+ uptake was fueled by the activation of the plasma membrane H+-ATPase. Vanadate, a specific inhibitor of P-type H+-ATPases, was shown to inhibit oxalate-induced stomatal opening. A genetic screen for oxalate sensitivity in yeast (Saccharomyces cerevisiae) was successful through collaboration with the Dept. of Food Science, Oregon State University. A total of 106 genes were discovered, which confer oxalate sensitivity in yeast. Among these genes we
will identify the ones that are exclusively sensitive to oxalic acid. We will isolate the orthologous genes from S. sclerotiorum and determine whether these orthologs complement the mutant phenotype in yeast. Lastly, we will knock out these fungal genes in S. sclerotiorum because we predict that their deletion will reduce fungal virulence. Homologous plant genes may be used as candidates for mapping oxalate tolerance and resistance to Sclerotinia. In conclusion, studying cellular aspects of oxalate toxicity appears to be straightforward. On the contrary, genetic analysis of whole plants is complicated perhaps because of differential responsiveness of cells and tissues to oxalate. Little is known about the genetic basis of both susceptibility to Sclerotinia and sensitivity to oxalic acid. The genes we have identified in yeast have been compared to the Arabidopsis database and this information will be available upon request prior to publication. This knowledge can be used by the
community to identify homologous genes in different crop species, especially canola, soybean, and sunflower. We will determine whether selected plant genes are involved in oxalate tolerance by using the yeast system, followed by testing of their function during interactions of Sclerotinia with Arabidopsis. In addition to advancing marker-assisted selection, genes that are involved in oxalate tolerance can be overexpressed in transgenic plants to reduce susceptibility to Sclerotinia. Our cell biological and physiological data suggests that it may be worthwhile to exploit drought tolerance for coping with Sclerotinia. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Chipps, TJ, Gilmore, B. Myers, JR, Stotz, HU. 2005. Relationship between oxalate, oxalate oxidase activity, oxalate sensitivity, and white mold susceptibility in Phaseolus coccineus.
Phytopathology 95:292-299. Guimaraes, RL, Stotz, HU. 2004. Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection. Plant Physiology 136:3703-3711.
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