Investigating the role of effector proteins in the rice blast fungus Magnaporthe oryzae

To cause disease in plants, microbial pathogens secrete effector proteins that can suppress basal plant immunity mechanisms and help facilitate proliferation of the pathogen in plant tissue. The rice blast fungus, Magnaporthe oryzae, causes the most serious disease of cultivated rice. During early biotrophic colonisation, this fungus evades the plant immune system via the action of secreted effector proteins, allowing penetration of individual rice cells by invasive hyphae. The ability of the Magnaporthe isolates to infect weeping lovegrass (Eragrostis curvula) is controlled by a single gene, PWL2. Pwl2, like other effector proteins and several biotrophy-associated secreted (Bas) proteins, is secreted into a structure referred to as the biotrophic interfacial complex (BIC) before translocating into the cytoplasm [1-3]. In this study, I have used comparative genomics to analyse the relationship between the effector repertoire of M. oryzae and their potential recognition as avirulence determinants in rice blast populations in Sub-Saharan Africa (SSA). During effector-triggered immunity, effectors can be recognised as avirulence gene products by rice resistance (R) proteins. Currently, 25 rice blast resistance genes have been cloned along with 10 cognate avirulence genes [4]. I have used third-generation Pacbio RSII sequencing technology to generate improved genome assemblies of two M. oryzae strains, wild type Guy11, and KE002, a Kenyan isolate that is avirulent on selected rice monogenic lines and is thought to carry many avirulence genes. I show that by using improved contiguous genomes, in combination with RNA-sequencing data, it is possible to establish a gene prediction pipeline that can identify isolate-specific or novel 2 small secreted protein encoding genes. With this approach, I have identified additional 49 and 590 genes in Guy11 and KE002 genomes respectively. Three of them, MEP13, MEP15 and MEP14, have been confirmed to encode secreted effector proteins. I also provide new insight into how effectors are secreted and delivered across the fungal plasma membrane. I report the use of high resolution microscopy and fluorescently labelled effector proteins to show that the biotrophy interfacial complex (BIC) is a plant derived structure through which the fungus continually secretes effectors in one direction into the targeted plant organelles. Using fluorescently labelled Pwl2 and Bas1, I present results suggesting that effectors translocate across the plasma membrane packaged in vesicles which are visible in the BIC, and that these effectors are sorted into distinct vesicles before crossing the plasma membrane. I then use CRISPR/Cas9 gene editing to generate pwl2 mutants in M. oryzae strain Guy11 which has three copies of the gene resulting in deletion of all copies of the effector gene and gain of virulence towards weeping lovegrass. This confirmed the function of PWL2 as a host range determinant that is conserved in most rice blast isolates. These results show that genes with multiple copies in the genome can be functionally characterised through either disruption or replacement using CRISPR/Cas9 in M. oryzae.BBSRCBill and Melinda Gates Foundation Halpin Trust Scholarshi