Identification and characterisation of novel components of the Pmk1 MAP kinase pathway during plant infection by the rice blast fungus Magnaporthe oryzae

Cruz Mireles, Neftaly (2022) Identification and characterisation of novel components of the Pmk1 MAP kinase pathway during plant infection by the rice blast fungus Magnaporthe oryzae. Doctoral thesis, University of East Anglia.

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Abstract

The fungus Magnaporthe oryzae causes rice blast disease which requires a series of morphogenetic transitions to develop specialised infection structures called appressoria and transpressoria. The Pmk1 MAP kinase (MAPK) signalling pathway has been reported to control appressorium development, plant penetration and host colonisation. However, the mechanisms by which the Pmk1 MAPK regulates these complex growth changes are poorly understood. In this thesis, I report two phosphoproteomic pipelines to identify direct downstream targets of the Pmk1 MAPK during plant infection. Using discovery phosphoproteomics followed by Parallel Reaction Monitoring (PRM) from a time series study of appressorium samples, I identified 55 putative direct downstream targets of Pmk1. These putative phosphorylated targets include proteins related to cellular processes such as autophagy, cytoskeleton remodelling, vesicle trafficking, and cell cycle control. One of the targets, named Vts1, is a SAM domain-containing protein of unknown function. Using in vitro and in vivo assays, I have demonstrated that Vts1 interacts with Pmk1 and contains two phosphorylation sites within a MAPK motif that depend on Pmk1 function and its kinase activity. Targeted gene replacement showed that Vts1 is necessary for efficient growth, sporulation, appressorium development and pathogenicity. Additionally, Vts1 phosphorylation-directed mutants demonstrated the importance of its phosphorylation in virulence. To understand the role of Pmk1 during rice tissue invasion, I carried out discovery phosphoproteomics analysis using a M. oryzae pmk1AS analogue sensitive mutant. I obtained 39 phosphorylated candidate proteins, most of which are non-characterised in the blast fungus. Interestingly, I identified a subset of 3 phosphorylated effector proteins and components of the secretory pathway such as Sec31. Pmk1-regulated effectors (PREs) and Sec31 functions are potentially regulated by Pmk1 during Pmk1-dependent invasive growth. When considered together, this work demonstrates the utility of quantitative phosphoproteomics to identify novel Pmk1-dependent regulators, such as Vts1, that are essential for rice blast disease

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	04 Jul 2022 09:23
Last Modified:	04 Jul 2022 09:23
URI:	https://ueaeprints.uea.ac.uk/id/eprint/85905
DOI:

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