Browsing 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.
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    Molecular and phenotypic characterisation of grapevines expressing non-vinifera PGIP encoding genes
    (Stellenbosch : University of Stellenbosch, 2011-03) Moyo, Mukani; Vivier, Melane A.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the pectin component of the cell wall, thus providing an entry route, as well as nutrients for the fungus. Plants have evolved mechanisms to counteract and suppress the action of the ePGs. This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays. Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that transgene integration was successful, the transgenes were being expressed and there were at least 29 transgenic lines with independent integration events. The transgenic lines were confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from 22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs). The plant lines with positive transgene integration, expression, independent integration events and exhibiting 100% transgene-derived PGIP activity were further selected for whole plant and detached leaf antifungal assays where they were challenged with B. cinerea. The whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be explained by a quicker and stronger recognition between the pathogen and the host and the stronger activation of defence responses in the host. A more active hypersensitive response in the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed of lesion development observed on the transgenics lines versus the wild type support this possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera PGIPs during infection of grapevine tissue; the potential changes that might be caused by expressing PGIPs in a grapevine host with a native PGIP with high homology to the transgenes (including potential gene silencing) and the potential impact on defence signalling and defence responses all provides further avenues of study to elucidate this very interesting phenotype further. Overall, this study provides a comprehensively characterised population of transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence, where the host plant harbours a native copy of the PGIP encoding gene.