Using RNAseq to dissect virulence factors of wheat and barley pathogen Bipolaris sorokiniana

Abstract

Bipolaris sorokiniana is a hemibiotrophic pathogen causing spot blotch (SB) and common root rot (CRR) in both wheat and barley, and is causal to significant yield and economic losses. As a hemibiotroph, B. sorokiniana displays a rapid switch in lifecycle from biotrophic to necrotrophic growth during the early stages of infection, ultimately resulting in the death of host tissue. Despite the great significance of B. sorokiniana, the molecular mechanisms underpinning disease are poorly understood. Therefore, in this study, I sought to identify host specific virulence factors that facilitate pathogenicity in the different cereal hosts and to dissect the underlying mechanisms.

A deep RNA sequencing approach was undertaken from wheat and barley leaves infected with B. sorokiniana. In this dataset, I found fungal genes encoding small secreted proteins (SSPs), secondary metabolites (SMs), and cell wall-degrading enzymes that showed strongly differentiated transcriptional profiles between the two hosts and at different stages of the infections. Based on the transcriptomic datasets and machine-learning bioinformatics prediction approaches, ten novel SSP effector candidates which were significantly differentially expressed between wheat and barley infections were identified. Further functional characterisation of these ten SSP effector candidates were employed through homologue recombination approach of protoplast transformation. Additionally, seedling pathogenicity assays were applied to confirm whether the mutants the of SSP effector candidates were required for the virulence of B. sorokiniana. Unfortunately, no significant difference in disease symptoms was observed between mutants and wild type in both wheat and barley, suggesting that all ten effector candidates were not required for the virulence of B. sorokiniana as assessed by this seedling in planta assay. The expression profile of SMs were also analysed. Among them, a siderophore synthase gene Chr04.426 was highly expressed at the early timepoint of both the barley and wheat infections. Further functional characterisation showed that the gene disruption mutants of Chr04.426 could significantly reduce disease symptoms in both wheat and barley compared to wild type. These findings indicate that Chr04.426 plays an important role in the pathogenicity of B. sorokiniana. Additionally, Chr04.426 is also crucial iron uptake ability of B. sorokinina. Overall, Chr04.426 is required for the siderophore-mediated iron uptake and virulence of B. sorokiniana. To date, the virulence determinants of B. sorokiniana have remained poorly understood. In this study, in planta transcriptome expression profile provides a broad dynamic view of the expression level of SSPs, SMs and cell wall-degrading enzymes of B. sorokiniana during infection between wheat and barley. Furthermore, in this dataset, a nonribosomal peptide synthase Chr04.426, which was highly expressed in early infection timepoint of both wheat and barley, was identified to be involved in siderophore-mediated iron uptake and virulence of B. sorokiniana. Collectively, these data have fundamentally advanced our understanding of the interaction between B. sorokiniana-wheat/ barley during infection. Show more