Characterization of the Barley nec3 Gene; A Negative Regulator of Pathogen Elicited Programmed Cell Death

Plant innate immunity relies on elicitation of programmed cell death (PCD) via PAMP-triggered immunity or effector-triggered immunity pathways, yet plants require regulatory mechanisms to shut PCD down.  Reverse genetics is a powerful tool to characterize these highly conserved regulatory genes. Three forms of irradiation were utilized to generate five independent barley nec3 mutants. The nec3 mutants display an unregulated PCD phenotype consisting of tan to orange expanded lesions that are observed at all growth stages following pathogen recognition, but it is not formed on unchallenged plants.  The phenotype was observed following inoculation with several ascomycete pathogens including Cochliobolus sativus, Pyrenophora teres and Blumeria graminis as well as infiltration with C. sativus culture filtrate and the bacterial pathogen Xanthomonas translucens pv undulosa. The basidiomycete fungal pathogen Puccinia graminis f sp. tritici races MCCF and QCCJ which are avirulent and virulent on the Rpg1 containing cultivar Bowman, respectively, did not elicit the runaway PCD phenotype. Bowman is the background genotype of the gamma 1 nec3 mutant. Mapping data, based on F₂ progeny derived from a cross between gamma1 and Quest, delimited the nec3 region to 5.4 cM near the centromere of chromosome 6H. Saturation of the region with markers will be achieved via a PCR-genotype-by-sequencing panel specifically designed to the 6H region and will narrow the nec3 candidate gene list generated by RNAseq analysis. RNAseq  has been performed on Bowman and gamma1 under induced and uninduced conditions by C. sativus and differentially regulated genes were identified that map to the nec3 region.