Strategies for wheat stripe rust pathogenicity identified by "omics" technologies

Abstract

Stripe rust is a major constraint to wheat production worldwide. The causal agent is the fungus Puccinia striiformis f.sp. tritici (Pst). During infection, the fungus creates a specialized cellular structure within host cells called the haustorium which allows Pst to obtain the nutrients necessary for development and reproduction. The haustorium is also thought to secrete virulence molecules called 'effectors', which are suspected to manipulate the physiological and immune responses of host cells. Despite this broad outline, the molecular events that underlie host colonization and the produced effectors proteins are largely unknown. In my PhD, I extensively investigated Pst using transcriptomics and proteomics techniques to obtain a better understanding of how the pathogen establishes a compatible interaction with its host, and to identify the effector proteins that are synthesised and secreted during infection. First, by the use of next generation sequencing (454 and Illumina) the transcriptomes of two contrasting pathogenic stages (germinated spores and haustoria) were generated, de novo assembled and extensively annotated. A digital gene expression analysis revealed many differentially expressed genes which highlight key metabolic differences between these cell types, and provide insight into their different roles during infection. Spores turn on the metabolic pathways to derive energy from non carbohydrate sources, required to sustain growth and development. Conversely, haustoria deploy all the necessary machinery to take advantage of the abundant nutrients derived from the host nutrients and focus on energy production and biosynthetic pathways to support fungal growth and spore production. Further analysis of the haustoria transcriptome, allowed me to identify the first set of potential effector candidate genes of Pst, comprised of 437 genes, with two thirds of these up-regulated in haustoria compared to germinated spores. Using a bacterial system to synthesise and deliver proteins encoded by effector gene candidates, a small subset of these genes was cloned and used to establish two functional characterization methods. The first one aimed to test if these proteins could be recognised by wheat resistance genes and the second one tested their capacity to inhibit cell death triggered by a necrotic toxin. From the later one two effector gene candidates were found to partially inhibit plant cell death. In parallel, I have developed a method to isolate highly purified haustoria combining density gradients and flow cytometry. Haustoria purified by this method were successfully used for proteomics analysis. Proteomics data from haustoria, germinated and ungerminated spores were generated and analyzed preliminarily to determine the presence of effector candidates as well as non-effector proteins in each tissue. More than 3,000 proteins were validated by proteomic data, including 150 effector candidates. The correlation of transcriptomic and proteomic data suggested that the synthesis and deployment of some effector proteins could occur at different spatiotemporal sites and even could have destinations other than the host cell cytoplasm. Together, these studies have substantially increased our knowledge of Pst effectors and have provided insights into the pathogenic strategies of this important organism, opening new avenues of research with immense potential in the design of novel disease control strategies.