Location: Cereal Crops Research
Project Number: 3060-22000-051-006-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Mar 15, 2020
End Date: Mar 14, 2025
Admendment 2: 6) Functionally validate the P. teres effectors VR1 and VR2 and characterize their mode of action. The objectives of this cooperative research project are to: 1) use a genome wide association study approach to identify new necrotrophic effectors in Parastagonospora nodorum. 2) characterize the mode of action of new and validated effectors. 3) Use a similar GWAS approach as in Objective 1 to identify effector candidate genes in P. teres. 4) Validate P. teres effector candidate genes using a CRISPR Cas-9 based approach.
Amendment 2: VR1 and VR2 are Pyrenophora teres f. teres effectors that are important in causing disease on Rika barley. Candidate genes encoding for VR1 and VR2 have been identified using a biparental mapping population that segregates for both VR1 and VR2. The post-doc will use a CRISPR-Cas9 based approach to functionally validate these genes as well as functionally characterize the virulent and avirulent alleles of these genes and the corresponding proteins. Confocal microscopy will be used to characterize the role of VR1 and VR2 in virulence (disease) and yeast two-hybrid and pull-down assays will be used to identify the host (barley) interacting proteins that are targeted by VR1 and VR2. The Pyrenophora teres- barley interaction involves a diverse set of pathogen-produced effectors that target host gene products to gain entry and/or colonize barley. Our previous research on the Pyrenophora teres- barley interaction indicated that this is a complex system involving multiple virulence genes that are involved in disease. The post-doc on this project will continue the identification and characterization of new effectors using both bi-parental pathogen populations as well as a genome wide association study (GWAS) approach to identify genomic regions of the pathogen that are associated with virulence and likely harbor effector genes. Candidate genes underlying these regions will be validated using a CRISPR-Cas9 gene editing approach as well as gain of function transformation of these genes into avirulent isolates. This will allow us to identify candidate virulence genes for further study. Validated genes will be used in downstream application to further characterize this host pathogen interaction.