Analysis of Fusarium oxysporum effectors shared between strains that infect cape gooseberry and tomato

Physalis peruviana, known as cape gooseberry, is a solanaceous plant native to tropical South America, typically growing in the Andes at 2000 m. Its economic value has grown due to its nutritional and medicinal properties. However, a vascular wilt disease caused by a newly discovered forma specialis of Fusarium oxysporum, (here designated f. sp. physali [Foph]), has become one of the limiting factors in cape gooseberry production, with losses up to 90%. The F. oxysporum species complex incudes numerous formae speciales (special forms), which are the causal agents of vascular wilt disease in a broad range of plants, including economically important crops such as banana, cotton, melon, tomato and recently, cape gooseberry. F. oxysporum f. sp. lycopersici (Fol) causes wilt disease on tomato. At least fourteen small secreted in xylem (SIX) proteins have been identified from the xylem sap of Fol-infected tomato plants. Five are associated with virulence and three are recognised by resistance proteins in the host. However, the function of most of these SIX proteins remains unclear. In this project, six homologues of Fol SIX genes (SIX1a, SIX1b, SIX7, SIX10, SIX12 and SIX15) and a homologue of Ave1 (an avirulence gene present in the broad-host-range wilt-pathogen Verticillium dahliae, with homologues in many other phytopathogens including Fol), were identified in Foph. These and other candidate effector genes were identified by mapping Foph RNAseq data against the Fol lineage-specific transcriptome and candidate F. oxysporum effector genes identified in other formae speciales in other studies. The Foph SIX gene and Ave1 homologues were found to encode proteins with 70 to 100% identity to their Fol counterparts, with the latter suggesting recent horizontal transfer of a cluster of SIX genes comprising SIX7, SIX10, SIX12 and SIX15. The Foph SIX1a and SIX1b proteins are 74% and 80% identical, respectively, to their Fol counterpart. Both homologues were tested in a ΔSIX1 strain of Fol to see if they could complement the virulence function of Fol SIX1 in tomato. The results showed no restoration of virulence for ten SIX1a and six SIX1b transformants tested, suggesting that their function might be restricted to cape gooseberry pathogenicity. Foph SIX1a and SIX1b transformants were also tested to see if they might be recognised by tomato plants carrying the I-3 resistance gene, which enables recognition of Fol SIX1 (i.e. Avr3). The results indicated that SIX1a was not recognised while SIX1b was recognised, suggesting that Foph-SIX1b may be recognised by I-3 as an avirulence factor and that the I-3 resistance gene could potentially be used in cape gooseberry plants to mediate Foph resistance. VI To investigate the function of SIX7, SIX10 and SIX12, a triple gene knockout strategy was initiated to assess their role in Fol virulence. This strategy included the use of the HSVtk (Herpes Simplex Virus thymidine kinase) gene as a counter selection marker against the ectopic insertion of transfer DNA (T-DNA) during fungal transformation by Agrobacterium tumefaciens. However, after several transformation attempts no gene knockouts were obtained. Attempts to produce single (SIX10) and double (SIX7 and SIX12) knockouts also failed. The Fol Rapid Alkalinisation Factor (RALF) gene was also subjected to gene disruption using this approach. Four RALF knockouts were obtained out of 44 transformants thereby validating the gene deletion strategy described above. Pathogenicity tests in tomato showed that these four mutants all developed disease symptoms that were not significantly different from those of wild type Fol under the assay conditions used