title: Functional characterisation of Nudix hydrolase effectors from phytopathogenic fungi
Creators: name: McCombe, Carl
Publisher: name: The Australian National University
Year: 2024

description: Crop production is constrained by plant-pathogenic fungi, threatening the ability of society to feed a growing global population. Central to the intricate molecular interactions occurring between plants and pathogenic fungi are small pathogen-secreted proteins (effectors). Understanding the functions of effectors offers a potential pathway towards unravelling the sophisticated interplay that occurs between cropping plant species and their devastating fungal pathogens. In this thesis, I have identified and characterised effectors from pathogenic fungi that are functional Nudix hydrolase enzymes. AvrM14 is a Nudix hydrolase effector from Melampsora lini (flax rust). To elucidate AvrM14's enzymatic activity, an exhaustive substrate screen was conducted, revealing AvrM14's remarkable substrate selectivity, the effector only hydrolysed m7Gp5G (P1-(5'-7-methyl-guanosyl)-P5-(5'-guanosyl)-pentaphosphate). While m7Gp5G does not occur naturally, 7-methyl guanosine (m7G) is a distinctive molecular structure from the 5' cap of eukaryotic mRNA. I demonstrate that AvrM14 can remove the protective 5' cap from mRNA, and this activity requires a glutamate amino acid within the putative active site. Notably, homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage and mRNA decapping activity is conserved in related Nudix hydrolase effectors across the Melampsora genus. When expressed in planta, AvrM14 inhibits the immune-related reactive oxygen species burst and hypersensitive cell-death response. Significantly, the same pivotal glutamate amino acid is indispensable both for the mRNA decapping and the immune-suppressive activities. The findings support a model whereby the Nudix hydrolase effectors from Melampsora spp. decap plant mRNA to suppress immune responses and ultimately promote the infection process. Nudix hydrolase effectors from Magnaporthe and Colletotrichum spp. are more closely related to each other than the mRNA decapping effectors of Melampsora spp. Substrate screening with purified effector proteins revealed that the Magnaporthe and Colletotrichum effectors hydrolyse inositol pyrophosphate signalling molecules. I determined the crystal structure of a M. oryzae Nudix effector (MoNudix), identifying remarkable similarity to Homo sapiens diphosphoinositol polyphosphate phosphohydrolase 1 (HsDIPP1). HsDIPP1 also hydrolyses inositol pyrophosphates, and using targeted mutagenesis I demonstrate that like HsDIPP1, MoNudix utilises basic amino acids to facilitate inositol pyrophosphate binding and hydrolysis. Inositol pyrophosphates serve as plant messengers for shifts in phosphate availability: diminished inositol pyrophosphate levels activate phosphate starvation response transcription factors. Consistent with this, the production of Nudix hydrolase effectors from M. oryzae and Colletotrichum spp. in Nicotiana benthamiana elevated the expression of phosphate starvation responsive genes as measured by qPCR. Phosphate starvation induction requires amino acids involved in the hydrolysis of inositol pyrophosphates in vitro, and all qPCR data was corroborated using an innovative promoter/reporter system developed during the study. When the expression of one inositol pyrophosphate hydrolysing effector from M. oryzae was reduced using RNAi, there was a significant decline in the pathogen's virulence on rice. Collectively, the results indicate that Magnaporthe and Colletotrichum Nudix hydrolase effectors target inositol pyrophosphate signalling molecules to initiate plant phosphate starvation responses. To date, the virulence functions of fungal effectors have remained largely unidentified. Through this study, I have established that the predicted Nudix hydrolase effectors from pathogenic fungi act as enzymes, likely targeting mRNA caps and inositol pyrophosphates. All together, these data advance our understanding effector function and the complex interplay occurring between pathogenic fungi and their plant hosts.