Identification of signal transduction and accessory protein requirements for resistance responses mediated by the tomato I-3 gene for Fusarium wilt resistance.

Abstract

Fusarium wilt of tomato is a vascular disease caused by the fungus Fusarium oxysporum f. sp. lycopersici (Fol). Fol secretes a number of effector proteins that facilitate host infection, including the SIX1 (Secreted In the Xylem 1) protein. Besides its role in pathogenicity, SIX1 can also trigger defence activation in tomato plants expressing the I-3 resistance protein, and so SIX1 is also designated Avr3. In this study, I explored the virulence function of the Avr3/SIX1 protein and the activation of defence signalling following its recognition by I-3. I also explored the possibility that SIX1 homologues from F. oxysporum f. sp. cubense (Foc) tropical race 4 (TR4) could restore FolSIX1 virulence on Fol-susceptible tomato plants and its avirulence on I-3 tomato plants in a SIX1 deletion mutant. Unlike other R/Avr gene combinations, co-agroinfiltration of I-3 and Avr3/SIX1 does not induce necrosis in Nicotiana tabacum or N. benthamiana. Therefore, I started my experiments by attempting to develop a rapid assay system based on a plant defence response other than necrosis/necrosis to assay I-3-mediated defence activation in N. benthamiana (Chapter 2). I made a chitinase promoter:gusA reporter construct based on an N. benthamiana orthologue of a chitinase gene that showed a 30-fold increase in expression 2 days post-inoculation of I-3 tomato plants with an isolate of Fol carrying Avr3 gene. I also checked whether this gene was suitable as a good reporter by using RT-PCR. I confirmed it was not constitutively expressed in leaves of N. benthamiana. However, it was induced by Agrobacterium, making an agroinfiltration approach impractical. I therefore generated a transgenic N. benthamiana line expressing both I-3 and the reporter, using purified Avr3/SIX1 infiltration to activate I-3-mediated signalling. In collaboration with Daniel Yu and Megan Outram from the Williams Laboratory, Research School of Biology, I expressed and purified Avr3 protein for experimental use (Chapter 3). I infiltrated purified Avr3 protein into N. benthamiana leaves to investigate potential virulence functions (Chapter 4). Surprisingly, Avr3 induced callose deposition in N. benthamiana despite suppressing flg22-induced ROS accumulation. In tomato plants, infiltration of Avr3 protein induced an I-3-dependent cell-death or necrotic response indicative of defence activation. We also attempted to express and purify the N- and C-terminal domains of Avr3 to identify which domain was responsible for these responses. Although we were able to express and purify only the N-terminal domain, this domain was sufficient to induce callose deposition, suppress ROS production in N. benthamiana, and induce I-3-dependent necrosis in tomato.

I then leveraged the ability of purified Avr3 protein to induce I-3-specific necrosis to develop an assay system based on virus-induced silencing (VIGS) to investigate downstream signalling (Chapter 5) as an alternative to the chitinase promoter:gusA reporter (Chapter 2). Silencing of downstream signalling components BAK1, SOBIR1, EDS1, SAG101 and NRG1 showed major roles, and NPR1, NPR3, and NRC1 demonstrated partial roles in I-3 mediated defence in tomato plants. Purified Avr1 protein supplied by Daniel Yu was also used as a positive control for VIGS of signalling components known to be required by the I resistance protein. I was also interested in whether the tomato I-3 resistance protein could recognise any of the SIX1 proteins encoded by three homologues of SIX1 present in Foc TR4 (Chapter 6). If so, there would be the potential to use I-3 in banana as a gene for resistance to Foc TR4. The three gene sequences were synthesised to make restoration constructs transferred into a SIX1 deletion mutant of Fol. Expression of the Foc TR4 SIX1 genes during tomato infection by the resulting Fol transformants was confirmed by RT-PCR, but none was found to restore the loss of SIX1 function in Fol. Show more