Identification, delivery, and characterisation of wheat rust pathogen effector proteins

Yellow rust of wheat, caused by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a devastating disease that poses a serious threat to global food security. Despite the losses caused by this pathogen, the infection process on the host species is poorly understood. This is largely due to the recalcitrance of this fungus to experimental manipulation. To date, there is no method for genetically transforming Pst, nor can this fungus be cultured under experimental conditions. Secreted virulence factors called effectors are a major component of pathogenicity on the wheat host. Further, some of these factors (deemed avirulence factors) can be recognised by certain host proteins, triggering an immune response. Therefore, it is critical to identify and functionally characterise these effectors to better understand the infection process on wheat host species. In this thesis, I use comparative genomics of spontaneous gain of virulence mutants to identify candidate avirulence effectors recognized by the specific host resistance protein YR2. Further, I explore different heterologous expression systems for the delivery of rust effectors in the native wheat host for subsequent characterisation. I show two previously described delivery systems involving the bacterial type III secretion system are unsuitable for screening avirulence properties of candidate rust effectors in wheat. I also develop a novel heterologous expression system using the Magnaporthe oryzae (Triticum pathotype, MoT), a different wheat infecting fungus that is experimentally tractable. This system is able to detect avirulence phenotypes of rust effectors only when sufficient mRNA levels of the transgene are produced. I obtained a single transformant which has multiple copies of the transgene that met this requirement. Using Nanopore sequencing technology I discovered this multi copy insertion occurs in a repeat rich region of the M. oryzae genome. Further, using CRISPR/Cas9 mediated targeted insertion, I explore different regions of the genome that may be suitable for optimal transgene expression. The results in this thesis therefore provide useful insight into the identification and functional characterisation of rust effectors in the native wheat host