Identification and functional characterisation of the Fusarium oxysporum Avr7 effector gene.
The fungus Fusarium oxysporum f. sp. lycopersici (Fol) causes a devastating wilt disease of tomato crops that has hitherto been managed by breeding for disease resistant cultivars. Tomato has several resistance (R) genes that enable it to recognise and respond to Fol isolates carrying corresponding avirulence (Avr) genes. Some of these resistance genes have been isolated (I-2, I-3, I-7) and several Avr genes have also been isolated (Avr1, Avr2, Avr3). The proteins encoded by these Avr genes were initially identified as small proteins secreted in the xylem sap of infected tomato. These proteins, designated SIX (secreted in the xylem) proteins, serve as disease effectors in susceptible tomato plants. The Avr2 (SIX3) protein is taken up by host plant cells and in resistant plants is recognised by the cytosolic I-2 resistance protein. In contrast, the I-3 gene encodes an S-receptor-like protein kinase (SRLK) and I-7 encodes a membrane-anchored leucine-rich-repeat receptor-like protein (LRR-RLP), both containing extracellular recognition domains, suggesting Avr3 (SIX1) and Avr7 (which has not yet been identified) are recognised at the cell surface and may not be taken up by host plant cells. Avr1 (SIX4) inhibits the detection of both Avr2 by I-2 and Avr3 by I-3, but not the detection of Avr7 by I-7. This project will identify and characterise the function of the Avr7 gene, which is present in all known races of Fol. Initially, knockout mutations will be produced in the Fol genes encoding each of the 11 remaining SIX proteins (SIX2 and SIX5-SIX14) that have been identified to date. The resulting Fol mutants will be tested for a change from avirulence to virulence on tomato plants carrying the I-7 resistance gene. If this approach is unsuccessful, knockout mutations will be generated in genes encoding small secreted proteins present in the lineage-specific regions of the Fol genome, which determine pathogenicity on tomato. Again, the resulting Fol mutants will be tested for a change from avirulence to virulence on tomato plants carrying the I-7 resistance gene. If this approach is also unsuccessful, mutations for virulence on tomato plants carrying the I-7 resistance gene will be induced by chemical mutagenesis and the mutant gene in virulent Fol mutants will be identified by genome sequencing and confirmed by generation and testing of a knockout mutant. Additional confirmation will be sought by co-expressing I-7 and Avr7 in leaves of Nicotiana benthamiana plants via agroinfiltration and looking for a necrotic response similar to that triggered by other LRR-RLP resistance proteins and their cognate Avr proteins.
Once the Avr7 gene has been identified its sequence will be analysed bioinformatically for any clues as to its function e.g. similarity to a gene of known function. If a possible function is predicted this will be tested biochemically. Other functional tests will be conducted regardless of any functional clues provided by the sequence of the gene. The knockout mutant used to identify Avr7 will be used to look at the role of Avr7 as an effector of Fol pathogenicity by testing for any reduction in virulence on susceptible tomato plants. Fol transformants carrying an Avr7 promoter:GFP reporter gene will be tested on susceptible tomato plants to examine the spatial and temporal distribution of Avr7 gene expression during infection. Finally, epitope tagged Avr7 protein will be expressed in E. coli or in the yeast Pichia pastoris and, if the yield is sufficient, the protein will be purified using the epitope tag for crystallisation and structural analysis by X-ray diffraction. Elucidating the structure of the Avr7 protein may reveal clues as to its function through structural homologies with proteins of known function and may also reveal structural features whose role in host recognition or effector function could be tested by site-directed mutagenesis. Mutations affecting recognition would be tested rapidly using the agroinfiltration approach described above, assuming co-expression of I-7 and Avr7 triggers necrosis in leaves of N. benthamiana. Mutations affecting effector function would be tested by looking for complementation of the Avr7 deficiency in the Avr7 knockout mutant.