Progress 12/15/15 to 12/14/19
Outputs
Target Audience: Target audiences included plant pathologists and plant breeders and plant and fungal geneticists and molecular biologists. Efforts included teaching through both informal mentoring and formal classroom settings and through presentation of research at meetings. In addition, knowledge was disseminated to the scientific community through research reports published in peer- reviewed journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We incorporate results in teaching for graduate level courses: Molecular Basis of Plant Pathogenesis (Bot 651) and Plant Pathology (Bot 550). We design and implement two laboratory exercises for the undergraduate level botany course, Introduction to Plant Biology (BOT220). 1. Mutagenesis and bioassays: Students screen our EMS-mutagenized seed using a victorin plate bioassay. They learn about the power of forward genetics and mutant screens. 2. Molecular systematics: Students miniprep plant DNA, run diagnostic PCR of ITS markers, prep DNA for sequencing, and analyze data by BLAST to assign unknowns to plant taxa. LSAMP = Louis Stokes Alliance for Minority Participation. We demonstrated genetic mapping techniques to approximately 60 incoming undergraduate students participating in this STEM for minorities program. We discussed breeding for disease resistance in oats, wheat, rice and barley and expose students to the plants that produce these common commodities. Parts of the described efforts were conducted by an undergraduate student for his honor college thesis. How have the results been disseminated to communities of interest?Oral/ Poster Presentations:?Center for Genome Research and Biocomputing, Fall Conference, Oregon State University, Corvallis Oregon, September 9, 2016; Genetic Analysis of Victorin Sensitivity in Phaseolus vulgaris. Thomas Wolpert, Jennifer Lorang, and Christina Hagerty 29th International Fungal Genetics Conference, Asilomar Conference Center Pacific Grove, CA March 14-19, 2017; The genetic Analysis of Victorin Sensitivity in Phaseolus vulgaris. Jennifer Lorang, Christina Hagerty and Thomas Wolpert AFRI, Agricultural Research Congressional Exhibition and Reception, Congressional Auditorium and Atrium, Washington D, C. April 5, 2017; The Plant Immune Response is Exploited by a Blight Fungus in Barley, Oat, Bean, and Rice. Jennifer Lorang and Thomas Wolpert Peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Based on our previous identification of victorin sensitivity in diverse plant species and our mapping results across several of these species identifying NB-LRRs as causal to victorin sensitivity, we hypothesize that the Victoria blight susceptibility gene in oats, (Vb) is also a NB-LRR and is the same gene as the Pc2, Puccinia coronate resistance gene. Further, we speculate that genes conferring sensitivity in other species could share a structural and likely do share a functional relationship with this oat gene. Because of Vb/'Pc2 identity, we hypothesize that P. coronata produces an effector that mimics victorin. Thus sensitivity genes identified among these different species may also recognize AvrPc2 and therefore, could have utility in conferring crown rust resistance in oats. Finally, we hypothesize that continued genetic evaluation of Arabidopsis can add additional insight into the mode-of-action of victorin and by association, the effector from the crown rust pathogen, P. coronata. This is important for understanding necrotrophic virulence and the formidable cereal rusts for which few effectors have been characterized. Completing the objectives of this proposal will provide insights into all of these issues. The work consists of 2 Objectives. Towards completion of objective 1A, we cloned both Bradi3g58937and Brachi3g58951, the NB-LRR genes that co-map with victorin sensitivity in Brachypodium. Trxh5 is the thioredoxin guardee in Arabidopsis that binds victorin and subsequently, activates LOV. Bradi3g58937 is an NB-LRR that contains a C-terminal thioredoxin h fusion suggesting it could function as an integrated decoy for victorin in Brachypodium. Also, Brachi3g58951 is positioned head to tail with Bradi3g58937, suggesting the latter NB-LRR could be co-regulated as a signaling partner. In collaboration with Dr. Matt Moscou's group, we produced several transgenic Brachypodium lines containing either Bradi58937 alone, or both genes. However, upon screening the Bradi58937 and transformants with both Brachi3g58951 and Bradi3g58937 we did not identify victorin sensitivity. As only one transformant of the latter was produced, results remain ambiguous. In addition to the work conducted on Brachypodium, we also cloned Phvul05G030500 and Phvul05G031200 the two NB-LRRs that co-map with victorin sensitivity in Phaseolus vulgaris. We confirmed that one of these two genes, Phvul05G031200, confers victorin sensitivity when transiently expressed in Nicotiana benthamiana. However, transformation of Phvul05G031200 into Arabidopsis failed to generate victorin-sensitivity. Part of this sub-objective was to transform oats with either the Brachypodium gene(s) or Phaseolus gene to determine if either could confer both victorin sensitivity and rust resistance and thereby establish a causal connection between the two responses. As transformation results for Brachypodium and Arabidopsis were unsuccessful, efforts to transform oats appeared ill-advised. Subsequently, as an alternate approach to establishing the equivalency of victorin sensitivity and rust resistance, we examined effector activity isolated from Puccinia coronata infected oats. Such isolations identified victorin-like effector activity indicating we had isolated the AvrPc2 effector. Subsequent analyses demonstrated that this effector activity, identical to victorin, activated the Arabidopsis NBLRR, LOV1 in a thioredoxin5-dependent manner in transient expression assays in N. benthamiana. In addition, this same effector activity caused death in Arabidopsis ecotypes containing SSEN (At1g31540). Thus, we established equivalency between victorin and AvrPc2 and thus, by definition, the equivalency of victorin sensitivity and crown rust resistance. These efforts are still ongoing. Towards the completion of objective 1B and the identification of the Pc2/Vb gene locus we have made progress on numerous fronts. We conducted screens of our EMS-mutagenized population of Victoria oats and identified multiple independent mutants, which passed at least four separate screens demonstrating loss-of-function of the Vb gene. We conducted progeny analyses to confirm homozygousity of these mutants. Thus, we are confident that we have generated independent mutant Vb alleles for sequence analyses and rust phenotyping. We also completed GBS analysis of our F6 recombinant inbred mapping population and developed approximately 38 thousand GBS markers including 137 that map to the Pc2/Vb locus. From among these markers, we identified 34 that are homologous to markers compiled by Dr. Nick Tinker's group. This defined a map location for the Vb gene and allowed us to place the Vb locus on chromosome 21 of oats. Also, in collaboration with Dr. Moscou, we conducted RNAseq analysis of the parents of the mapping population, phenotypic pools (victorin sensitive vs. insensitive) of the mapping population, and four of the afore mentioned EMS Vb mutants. Data from RNAseq analysis of the parents was used to create an oat "NB-LRRome" of approximately 800 genes. However, bioinformatics analyses has thus far failed to identify the Vb/Pc2 gene. A major issue surrounding these analyses appeared to be the polyploid nature of oats. We are hopeful that this may be addressed in future analyses. For objective 2A we completed production of an EMS mutagenized population of the victorin "super sensitive" (SSEN) Arabidopsis ecotype, HKT2.4MPI; collected seed from pooled progeny and screened the entire population for loss-of- sensitivity mutants. We identified 19 loss of sensitivity mutants. All mutants were genetically evaluated by outcrossing to Col-0 and a partially characterized super sensitive mutant (ssen). These analyses revealed that 15 of the mutants have phenotypes due to a mutation at the SSEN gene. More interestingly, 4 of the mutants segregate as an independent gene conditioning victorin sensitivity. These 4 mutants examined by high-throughput sequence analyses but the approach failed to identify the gene(s) conferring the loss-of-function mutation. For objective 2B, based on the identification of ecotypes with a victorin sensitivity phenotype intermediate between that conferred by LOV and SSEN, we hypothesized that there may be an additional NB-LRR(s) (other than LOV and SSEN) that confers victorin sensitivity in Arabidopsis. This hypothesis was also supported by the Arabidopsis 1001 ecotypes sequencing project because sequence information indicated that some of the ecotypes with an intermediate phenotype had the non- functional Col-0 lov allele. We have completed genetic evaluation of all of these previously identified ecotypes and found that in all cases, sensitivity is due to LOV. Resequencing of purported lov alleles revealed sequencing errors in the Arabidopsis 1001 ecotypes database, and that these ecotypes actually retain a functional LOV allele. Thus, these data do not support the hypothesis and have revealed that genetic background effects on LOV function can be more significant than we had originally anticipated. These data suggest that evaluation of LOV or SSEN function in Arabidopsis could be an excellent tool for examining QTL that impact NB-LRR function.?For objective 2C, we obtained seed for the Arabidopsis 1001 ecotype collection which we organized and cataloged. Using data from the 1001 genome sequencing project, we initiated our screening effort by selecting ecotypes with purported non- functional LOV alleles and/or non-functional SSEN alleles. Following this priority, we directed our focus on ecotypes with < 35 day flowering times to expedite genetic analyses. While we identified numerous ecotypes with intermediate phenotypes indicative of a locus different from either LOV or SSEN, genetic analyses have indicated, similar to results described for objective e 2B, that all phenotypes could be attributed to the LOV gene.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lorang, J.M., C.H. Hagerty, R. Lee, P.E. McClean and T.J. Wolpert. 2018. Genetic Analysis of victorin Sensitivity and Identification of a Causal Nucleotide-Binding Site Leucine-Rich Repeat Gene in Phaseolus vulgaris. Molec. Plant Microbe Interac. 10:1069-1074.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Lorang, J.M. 2019. Necrotrophic Exploitation and Subversion of Plant Defense: A Lifestyle or Just a Phase, and Implications in Breeding Resistance. Phytopathology 109:332-346.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Dickman, M., B. Williams Y. Li, P. de Figueiredo and T.J. Wolpert. 2017. Reassessing Apoptosis in Plants. Nature Plants 3:773-779.
|
Progress 12/15/16 to 12/14/17
Outputs
Target Audience: Target audiences include plant pathologists and plant breeders and plant and fungal geneticists and molecular biologists. Efforts will include teaching through both informal mentoring and formal classroom settings and through presentation of research at meetings. In addition, knowledge will be disseminated to the scientific community through research reports published in peer- reviewed journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We incorporate results in teaching for graduate level courses: Molecular Basis of Plant Pathogenesis (Bot 651) and Plant Pathology (Bot 550). We design and implement two laboratory exercises for the undergraduate level botany course, Introduction to Plant Biology (BOT220). Mutagenesis and bioassays: Students screen our EMS-mutagenized seed using a victorin plate bioassay. They learn about the power of forward genetics and mutant screens. Molecular systematics: Students miniprep plant DNA, run diagnostic PCR of ITS markers, prep DNA for sequencing, and analyze data by BLAST to assign unknowns to plant taxa. LSAMP = Louis Stokes Alliance for Minority Participation We demonstrate genetic mapping techniques to approximately 60 incoming undergraduate students participating in this STEM for minorities program. We discuss breeding for disease resistance in oats, wheat, rice and barley and expose students to the plants that produce these common commodities. How have the results been disseminated to communities of interest?Oral/ Poster Presentations: Center for Genome Research and Biocomputing, Fall Conference, Oregon State University, Corvallis Oregon, September 9, 2016;Genetic Analysis of VictorinSensitivityin Phaseolus vulgaris. ThomasWolpert,Jennifer Lorang, and Christina Hagerty 29th International Fungal Genetics Conference, Asilomar Conference Center Pacific Grove, CA March 14-19, 2017; The genetic Analysis of VictorinSensitivityinPhaseolus vulgaris. Jennifer Lorang, Christina Hagerty and ThomasWolpert AFRI, Agricultural Research Congressional Exhibition and Reception, Congressional Auditorium and Atrium,WashingtonD, C. April 5, 2017; The Plant Immune Response is Exploited by a Blight Fungus in Barley, Oat, Bean, and Rice. Jennifer Lorang and Thomas Wolpert What do you plan to do during the next reporting period to accomplish the goals?We will continue to pursue the stated objectives.
Impacts
What was accomplished under these goals?
Based on our previous identification of victorin sensitivity in a number of diverse plant species and our mapping results across several of these species identifying NB-LRRs as causal to victorin sensitivity, we hypothesize that the Victoria blight susceptibility gene in oats, (Vb) is also a NB-LRR and further, that this same gene is the Pc2 gene, which confers resistance to the crown rust pathogen, Puccinia coronata. Further, we speculate that genes conferring sensitivity in other species could share a structural relationship and likely do share a functional relationship with this oat gene. Because Vb and Pc2 likely share identity, we hypothesize that P. coronata produces an effector that in some manner mimics victorin. Thus the genes identified among these different species that recognize victorin may also recognize AvrPc2 and therefore, could have utility in conferring crown rust resistance in oats. Finally, we hypothesize that continued genetic evaluation of Arabidopsis can add additional insight into the mode-of-action of victorin and by association, the functions of the corresponding biotrophic effector from the crown rust pathogen, P. coronata. This is important for understanding necrotrophic virulence and also because crown rust and cereal rusts in general are formidable pathogens for which few effectors have been characterized. Completing the objectives of this proposal will provide insights into all of these issues. The work consists of 2 Objectives each with multiple sub objectives: Objective 1- Integrated Analyses of Crown Rust Resistance in Oats. 1A) Testing the equivalency of victorin sensitivity and crown rust resistance. 1B) Mapping the Vb/Pc2 gene in oats. Objective 2- Identification of Alternate Virulence Targets of Victorin. 2A) Characterization of the effector target associated with the SSEN (At1g31540) gene. 2B) Identification of NB-LRRs other than LOV(At1g20190) and SSEN (At1g31540) that confer victorin sensitivity. 2C) Characterization of the Arabidopsis 1001 ecotypes for victorin sensitivity. Towards completion of objective 1A, we have cloned both Bradi3g58937and Brachi3g58951, the NB-LRR genes that co-map with victorin sensitivity in Brachypodium. Trxh5 is the thioredoxin guardee in Arabidopsis that binds victorin and consequently, activates LOV. Bradi3g58937 is an NB-LRR that contains a C-terminal thioredoxin h fusion suggesting it could function as an integrated decoy for victorin in Brachypodium. Also, Brachi3g58951 is positioned head to tail with Bradi3g58937, suggesting the latter NB-LRR could be co-regulated as a signaling partner. In collaboration with Dr. Matt Moscou's group, we have produced several transgenic Brachypodium lines containing either Bradi58937 alone, or both genes. Thus far, we have screened the Bradi58937 transformants and did not observe any with victorin sensitivity. These data suggest, much as we had anticipated, both Brachi3g58951 and Bradi3g58937 are necessary for victorin sensitivity. We are in the process of screening transformants containing the complete gene pair. In addition, we have cloned Phvul05G030500 and Phvul05G031200 the two NB-LRRs that co-map with victorin sensitivity in Phaseolus vulgaris. We confirmed that one of these two genes, Phvul05G031200, confers victorin sensitivity when transiently expressed in Nicotiana benthamiana. Towards the completion of objective 1B and the identification of the Pc2/Vb gene locus we have made progress on numerous fronts. We continue to conduct screens of our EMS-mutagenized population of Victoria oats. We have currently identified 10 independent mutants, which have passed at least four separate screens demonstrating loss-of-function of the Vb gene. We were able to conduct progeny analyses to confirm homozygousity of these mutants. Thus, we are confident that we have generated 10 independent mutant Vb alleles for sequence analyses. We completed GBS analysis of our F6 recombinant inbred mapping population and developed approximately 38 thousand GBS markers including 137 that map to the Pc2/Vb locus. From among these markers, we identified 34 that are homologous to markers compiled by Dr. Nick Tinker's group. This defined a map location for the Vb gene and allowed us to place the Vb locus on chromosome 21 of oats. Also, in collaboration with Dr. Moscou, we are completing RNAseq analysis of the parents of the mapping population, phenotypic pools (victorin sensitive vs. insensitive) of the mapping population, and four of the afore mentioned EMS Vb mutants. Data from RNAseq analysis of the parents was used to create the oat "NB-LRRome" of approximately 800 genes. Thus, we are in a position to bioinformatically identify the Victoria parent NB-LRR(s) that co-segregates with the Vb phenotype. Through a comparison of this allele(s) with the EMS mutants we hope to unambiguously determine the identity of Vb and confirm these results with mapping data. Thus, significant progress is being made toward the identification of the Vb/Pc2 locus. For objective 2A we completed production of an EMS mutagenized population of the victorin "super sensitive" (SSEN) Arabidopsis ecotype, HKT2.4MPI; collected seed from pooled progeny and screened the entire population for loss-of-sensitivity mutants. We identified 18 loss of sensitivity mutants. All mutants were genetically evaluated by outcrossing to Col-0 and a partially characterized super sensitive mutant (ssen). These analyses revealed that 15 of the mutants have phenotypes due to a mutation at the SSEN gene. More interestingly, 3 of the mutants segregate as an independent gene conditioning victorin sensitivity. These 3 mutants are now being genetically evaluated for allelism to determine if they all express the same gene or different genes. For objective 2B, based on the identification of ecotypes with a victorin sensitivity phenotype intermediate between that conferred by LOV and SSEN, we hypothesized that there may be an additional NB-LRR(s) (other than LOV and SSEN) that confers victorin sensitivity in Arabidopsis. This hypothesis was also supported by the Arabidopsis 1001 ecotypes sequencing project because sequence information indicated that some of the ecotypes with an intermediate phenotype had the non-functional Col-0 lov allele. We have completed genetic evaluation of all of these previously identified ecotypes and found that in all cases, sensitivity is due to LOV. Resequencing of purported lov alleles revealed sequencing errors in the Arabidopsis 1001 ecotypes database, and that these ecotypes actually retain a functional LOV allele. Thus, these data do not support the hypothesis and have revealed that genetic background effects on LOV function can be more significant than we had originally anticipated. These data suggest that evaluation of LOV or SSEN function in Arabidopsis could be an excellent tool for examining QTL that impact NB-LRR function. For objective 2C, we obtained seed for the Arabidopsis 1001 ecotype collection which we organized and cataloged. Using data from the 1001 genome sequencing project, we initiated our screening effort by selecting ecotypes with purported non-functional LOV alleles and/or non-functional SSEN alleles. Following this priority, we directed our focus on ecotypes with < 35 day flowering times to expedite genetic analyses. To date, we have screened approximately one third of the entire population for victorin sensitivity phenotypes. While we identified numerous ecotypes with intermediate phenotypes indicative of a locus different from either LOV or SSEN, genetic analyses have indicated, similar to results described for objective e 2B, that all phenotypes could be attributed to the LOV gene.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Wolpert, T.J. and J.M. Lorang. 2016. Victoria Blight, defense turned upside down. Physiol. Molec. Plant Path. 95:8-13
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Dickman, M., B. Williams Y. Li, P. de Figueiredo and T.J. Wolpert. 2017. Reassessing Apoptosis in Plants. Nature Plants 3:773-779.
|
Progress 12/15/15 to 12/14/16
Outputs
Target Audience: Target audiences include undergraduate and graduate students, plant pathologists and plant breeders and plant and fungal geneticists and molecular biologists. Efforts will include teaching through both informal mentoring and formal classroom settings and through presentation of research at meetings. In addition, knowledge will be disseminated to the scientific community through research reports published in peer- reviewed journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?While not a specific requirement of the grant, we are committed to undergraduate training. As such, we are currently providing training opportunities to three undergraduates. Training is being provided in the areas of plant biology, genetics and plant pathology. Students are being taught microbiological techniques, DNA extraction and analyses, phenotypic screening and how to make genetic crosses. Students are also encouraged to read and understand current, relevant literature. One student is applying his research experience toward an undergraduate thesis. How have the results been disseminated to communities of interest?Research findings have been incorporated into teaching of undergraduate and graduate students including informal and formal teaching in classroom settings. Also research results were discussed at the IS MPMI meeting in Portland Oregon and the National APS meeting in Tampa Florida. What do you plan to do during the next reporting period to accomplish the goals?Efforts for the next year of the grant include completion of screening and characterization of the oat EMS loss of function mutants, initiation of screening of the F2 population, development of high-throughput marker production for fine mapping and implementation of DNA capture technology. Also, we will conduct genetic characterization of the SSEN mutants, genetic characterization of the currently identified, novel sensitivity ecotypes and continue with screening of the 1001 ecotype collection.
Impacts
What was accomplished under these goals?
One of the most cost effective and environmentally benign approaches to controlling plant disease is to introduce genes for disease resistance into the plant population of interest. These genes, especially in combinations that share a common pathogen target, can prevent or minimize the impact of many serious pathogens. However, our work has demonstrated that these same plant genes normally involved in controlling disease may have the potential to increase it. We are studying a disease interaction where plant resistance to one pathogen appears to have led to disease susceptibility to a different pathogen. Obviously, this is a problem because it means that in our efforts to control disease, there is the potential to inadvertently create a worse situation. While additional, examples of this pathogen subversion of plant resistance appear to exist, fortunately, this does not appear to be a common phenomenon. However, studies on the mechanism by which the pathogen exploits plant resistance suggests that this problem could and perhaps "should" be common. In other words, it is unclear why many pathogens do not exploit plant resistance genes to cause disease. In the work described in this effort, we are trying to unequivocally confirm that a gene known to confer resistance is the gene being exploited for susceptibility and further, define the genetic and biochemical mechanisms involved. Overall, the effort is directed toward understanding why and how plant disease resistance can be exploited by pathogens so that it can be prevented/avoided in the future and resistance genes can be safely and effectively used to control plant disease. As indicated, the effort consists of two major objectives: Objective 1, Integrated analyses of crown rust resistance in oats; and Objective 2, Identification of alternate virulence targets of victorin. Towards the completion of objective 1 and the identification and cloning of the Pc2/Vb gene locus we have made progress on numerous fronts. We are proceeding with screens of an EMS-mutagenized population of Victoria oats and have currently identified 6 independent mutants which have passed three separate screens indicating loss-of-function mutations in the Vb gene. We have completed GBS sequencing of our F6 recombinant inbred mapping population and developed an oat map with approximately 38 thousand markers including 137 that map to the Pc2/Vb locus. We are currently initiating the creation of a large segregating F2 population to employ these linked markers for fine mapping. We have also completed RNAseq of RNA extracted from phenotypic pools (victorin sensitive vs. insensitive) of the mapping population. Data from RNA seq has been used to bioinformatically create the oat "NB-LRRome" and preparations are being made for DNA capture. Thus progress is being made toward the identification of the Vb/Pc2 locus. For objective 2 we have completed production of an EMS mutagenized population of the victorin "super sensitive" (SSEN) Arabidopsis ecotype, HKT2.4MPI; collected seed from pooled progeny and screened the entire population for loss-of-sensitivity mutants. We are currently producing outcrosses from mutant plants for genetic analyses. In addition, we have obtained seed for the Arabidopsis 1001 ecotype collection. Using data from the 1001 genome sequencing project, we have prioritized our screening effort by selecting ecotypes with known non-functional LOV alleles and/or non-functional SSEN alleles. Screening is ongoing, and currently we have screened approximately one third of the entire population. We have identified three more ecotypes with phenotypes indicative of an allele different from either LOV or SSEN and are proceeding with genetic analyses.
Publications
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