Transcriptome Analysis of the Durable Leaf Rust Resistance in the Wheat Variety Toropi

Abstract Background Leaf rust, caused by the fungal pathogen Puccinia triticina (Pt), is a major constraint to wheat production worldwide. The Brazilian wheat variety Toropi, released in 1965 and commercially cultivated for over 15 years, has maintained a high level of field resistance to leaf rust, while being seedling susceptible to all known Brazilian Pt isolates. Results The adult plant resistance (APR) in Toropi exhibits a prehaustorial phenotype, the pathogens development being halted at appressorial and sub-stomatal vesicle growth stages. A time-course analysis was performed to explore the resistance mechanisms behind the unique APR in Toropi, taking flag leaf samples for RNA sequencing over 24 hours following inoculation with Pt race MDT-MR. An average of 87.8% of paired-end reads aligned to the Chinese Spring IWGSC RefSeq v.1, and 0.53% to the Pt reference transcriptome, release 35. Cross-conditional different expression of wheat genes was undertaken, comparing Pt- to Mock-inoculated, and Pt- to Pt-inoculated timepoints. More genes were downregulated than upregulated, particularly at the early timepoints of 6 and 12 hours after inoculation (hai). GO enrichment analysis indicated a shutting down of RNA and protein synthesis, particularly prevalent from the downregulation of genes involved in ribosome structure and function. There was also an early effect on photosynthesis, with disruption of the electron transfer chain. Analyses of upregulated genes identified genes involved in ATP binding and protein kinase activity at 6 hai, suggesting a rapid metabolic response to Pt inoculation. There was a general upregulation of genes involved in carbohydrate metabolism and oxidoreductase activity (linked to defence through the generation of ROS), and transport of protein/peptides, nitrogen and organic compounds, indicating the possible need to relocate protein-based resources, either for the benefit of the host or the pathogen. Alignment of differentially expressed wheat genes with the genomic regions defining four QTL contributing to the leaf rust APR in Toropi identified a number of potential candidate genes, including a sugar transporter, a receptor kinase and a gene annotated as a seven transmembrane MLO family protein. In addition, 60 Pt genes were identified, six being annotated as potential effector proteins. Expression of four effectors peaked at 6 hai, one at 0 hai and the sixth at 12 hai, all well before the formation of haustoria. Conclusions Analysis of gene expression in the wheat variety Toropi at the point in Pt development at which resistance is effective indicated a strong PTI response, fully active by 6 hai.