Doctoral Degrees (Institute for Wine Biotechnology)

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Browsing Doctoral Degrees (Institute for Wine Biotechnology) by Author "Moyo, Mukani"

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    The interaction between Vitis vinifera and fungal pathogens : a molecular approach using characterized grapevine mutants
    (Stellenbosch : Stellenbosch University, 2017-03) Moyo, Mukani; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The commercially cultivated grapevine species, Vitis vinifera, is highly susceptible to a wide range of pathogens and pests which include the fungus, Botrytis cinerea. During infection of a wide range of hosts, B. cinerea utilises a combination of cell wall degrading enzymes, phytotoxins and metabolites (amongst others) to facilitate entry into host cells, killing them in the process. Being a necrotroph, B. cinerea feeds off the dead cells and continues to proliferate. One of the lines of defence utilised by plants is through the action of cell wall associated polygalacturonase inhibiting proteins (PGIPs) whose roles include inhibiting the activity of B. cinerea endopolygalacturonases (BcPGs), prolonging the existence of longer chain cell wall fragments involved in signalling and priming the plant prior to infection. The defence roles of grapevine pgip encoding genes (Vvipgip1 from V. vinifera and non-vinifera pgips from wild vines) were previously elucidated in tobacco overexpression studies where they increased resistance to a hyper-virulent B. cinerea strain isolated from grapes. However, overexpressing two of the non-vinifera pgips in V. vinifera conferred the transgenic population with hyper-susceptibility to the same B. cinerea grape strain. This study aimed to comprehensively investigate the basis of the hyper-susceptible phenotype displayed by asking and answering important questions regarding the ability of the non-vinifera PGIPs to interact with and inhibit Botrytis ePGs on the one hand and on the other hand also investigate potentially other (non-ePG inhibition related) functions of the grapevine PGIPs. In silico structural docking simulations of grapevine PGIPs (VviPGIP1 and the two non-vinifera PGIPs) against BcPGs from the grape strain and two other B. cinerea strains (included for comparison) were conducted to gain an understanding of the inhibition interactions from a structural perspective. The predicted PGIP-BcPG interactions were highly B. cinerea strain specific with subtle PGIP genotype specificity. This prompted infection of the transgenic grapevine population with a different B. cinerea strain (B05.10) and the results complemented the in silico docking simulations. The transgenic grapevines did not display hyper-susceptibility to B05.10, indicating that it was a strain specific response. Transgenic tobacco with the same genes overexpressed, on the other hand, displayed increased resistance irrespective of B. cinerea strain used. The phenotype displayed by transgenic grapevine to B. cinerea grape strain infection was thus considered both host and strain specific. Moreover, when B. cinerea mutants in ePGs and galacturonic acid metabolism were used in infection analyses on these grapevine and tobacco populations, both host specific virulence factors and potential recognition/decoy factors could be identified. These results all confirm the importance of the specific host and pathogen and the resulting phenotype and makes it clear that interactome studies would be the most insightful in studying infection/defence. Interestingly, the transgenic grapevine population displayed partial resistance to a biotrophic pathogen, specifically in blocking initial penetration of the pathogen, indicating that the PGIP overexpression could have modulated pre-formed defences in a possible priming mechanism. Further analysis of the transgenic grapevine population confirmed that both the native and transgenic pgips were expressed during infection and active proteins, which effectively inhibited BcPGs, was produced. However, prior to infection, transgenic grapevine leaves displayed a reduction in abundance of cell wall components associated with cell wall strengthening, indicating potential weakened cell walls. Additionally, they emitted significantly lower levels of defence-related sesquiterpenes compared to the controls during B. cinerea grape strain infection. These findings were suggestive of changes in metabolic processes, brought about by overexpressing non-vinifera pgips in V. vinifera background, which favoured the pathogen over the host during infection. Thus to build on this, a whole transcriptomic study to investigate the strain specific infection strategy together with the host specific defence strategy as a dynamic interaction was conducted during the early stages of infection. B. cinerea grape strain expressed significantly higher levels of genes involved in phytotoxin synthesis on transgenic plants compared to the controls, at the local infection site. On the other hand, the transgenic plants expressed significantly lower levels of defence-related genes, also at the local infection site. Taken together, the findings of this study challenge our current understanding of the roles of PGIPs in plant defence during B. cinerea infection. It points towards the possibility that grapevine PGIPs in their native backgrounds are not primarily linked to the classical PGIP-PG fungal inhibition interactions. It also provides insight that the hyper-virulent grape strain possibly optimised mechanisms to use the plant’s defence mechanism against itself and even modulate the host-responses in its favour. The host- and pathogen specific reactions observed in this study strongly highlights the impact that the choice of host-pathogen pairing has on defining defence phenotypes. Future studies should consider strain and host specific responses and interactome approaches would be valuable to that effect. This study successfully characterised the hyper-susceptible phenotype as set out initially, but also provided several new insights as well as new testable hypothesis that can lead to further studies.