Barley RPG4/RPG5 Integrated Decoy Resistance to Ug99; Towards Effector Identification

The rpg4/Rpg5 locus, harbors two NLR R-genes, Rpg5 and HvRga1, that are required together for resistance against wheat stem rust Puccinia graminis f. sp. tritici (Pgt) races including TTKSK (Ug99). Rpg5 is a predicted NLR resistance-proteins with an additional C-terminal kinase (STPK) domain. The transcription factor, HvVOZ1, was identified by yeast-two-hybrid of a library constructed from RNA of the rpg4/Rpg5+ line Q21861 48 hours post inoculation utilizing the Rpg5-STPK as bait. We hypothesize that the Rpg5-STPK acts as an integrated decoy that HvVOZ1 binds to negatively regulate defense activation or binds after activation as part of a signaling complex. The second NLR protein HvRga1 may guard the VOZ1-Rpg5 interaction or the Rpg5-STPK domain from Pgt rpg4/Rpg5-Avr (r45-Avr) effector manipulation. HvRga1 detects the manipulation by r45-Avr triggering the defense responses. The r45-Avr needs to be identified to investigate these mechanisms and test our hypothesis, thus a panel of 37 rust isolates with differential reactions on rpg4/Rpg5 were genotyped using restriction site associated DNA-genotyping-by-sequencing (RAD-GBS) producing 4,919 informative SNPs. The genotyping information was used to select 24 diverse isolates (16 avrRpg4/rpg5- and 8 Avrrpg4/Rpg5+) that are being used to conduct RNAseq during the infection process 5 days post inoculation. The RNAseq data will be used to identify Puccinia graminis SNPs within genes expressed during the infection process that will be added to previous RAD-GBS SNPs to conduct association mapping. This robust genotyping and the solid phenotyping on these diverse differential isolates should provide enough power to identify candidate r45-Avr genes.