A New Approach: Detection of Fungal and Wheat Genes Involved in Leaf Rust Disease By Transcript Expression Associations

We developed a methodology to narrow the gap between genomic and functional analyses of highly expressed leaf rust fungus effectors and their roles in the wheat host. A high-resolution RNA-seq experiment to compare global gene expression reprogramming of both fungal pathogen Puccinia triticina (Pt) and its wheat host Triticum aestivum (Ta) upon infection was performed. Four Pt isolates, race 1 (virulence phenotype BBBD), 12-3 (MBDS), 9 (SBDG) or 161 (FBDJ), were inoculated on susceptible cultivar Thatcher and two near-isogenic lines carrying the leaf rust resistance genes Lr2a or Lr3. Relative expression levels of both Pt and Ta genes were calculated using the race 1 reference and the International Wheat Genome Sequencing Consortium draft Ta genome sequences. In-depth analysis of Pt candidate effector protein genes revealed sequential waves of their expression across time (24 hrs to 14 days). Focusing on highly expressed effectors, we identified Pt genes whose expression varied significantly among various infection types. By correlating these expression patterns with those of the host, we detected wheat genes likely affected by and hence possibly interacting with those effectors. Fluorescent chimers of five Pt effectors, whose transcript levels were highly associated with wheat transcripts across various interactions, were found to be targeted to diverse plant organelles in a heterologous Nicotiana benthamiana plant system. Using a bimolecular fluorescent complementation assay, two Pt effectors, PTTG_08468 and PTTG_10273, were found to interact with Taphosphoethanolamine methyltransferase and glycogenin-2 like protein, respectively. Verification of these interactions by CoIP and mass spectrometry is in progress.