The interaction between Vitis vinifera and fungal pathogens : a molecular approach using characterized grapevine mutants

Thesis (PhDAgric)--Stellenbosch University, 2017.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.AFRIKAANSE OPSOMMING: Vitis vinifera, die kommersieel-verboude wingerdstokspesie, is hoogs vatbaar vir 'n wye verskeidenheid peste en patogene, insluitend die swam Botrytis cinerea. Tydens infeksie van 'n wye verskeidenheid gashere, gebruik B. cinerea onder andere 'n kombinasie van selwandafbrekingsensieme, fitotoksiene en metaboliete om toegang tot gasheerselle te fasiliteer, ‘n proses wat ook lei tot die selle se afsterwe. As 'n nekrotroof, voed B. cinerea op hierdie dooie selle en so versprei die infeksie dan verder. Een van die verdedigingslinies wat deur plante ontplooi word, is met behulp van selwand-geassosieerde poligalakturonase-inhiberende proteïene (PGIPs) wie se rolle beperking van die aktiwiteit van B. cinerea endopoligalakturonases (BcPGs) insluit, en sodoende die teenwoordigheid van langer-ketting selwandfragmente verleng: laasgenoemde is betrokke by sein-herleiding tydens infeksie en ook die voorbereiding op weerstand nog voor infeksie plaasvind (“priming”). Die verdedigingsrolle van druifplant pgip gene (Vvipgip1 van V. vinifera en nie-vinifera pgips van wilde druifspesies) is voorheen uitgeklaar in ooruitdrukkingstudies in tabak waar hulle verhoogde weerstand veroorsaak het teen 'n hipervirulente B. cinerea ras wat van druiwe geïsoleer is. Intereeasnt genoeg het die ooruitdrukking van twee van die nie-vinifera pgips in V. vinifera gelei tot hipersensitiwiteit van die transgeniese bevolking toe dit met die druifafkomstige B. cinerea ras geinokuleer is. In hierdie studie het word gepoog om omvattend ondersoek in te stel wat die grondslag van die hipervatbare(sensitiewe) fenotipe is, deur sleutelvrae te vra en te beantwoord met betrekking tot die vermoë van die nie-vinifera PGIPs om interaksies te hê met BcPGs en hul aktiwiteit te beperk, en om ander moontlike funksies, wat nie verband hou met aktiwiteitsbeperking van BcPGs nie, te ondersoek met betrekking tot druifplant PGIPs. Rekenaargebaseerde strukturele koppelingsimulasies van druifplant PGIPs (VviPGIP1 en die twee nie-vinifera PGIPs) teenoor BcPGs van die druifafkomstige rasen twee ander B. cinerea rasse (ingesluit ter vergelyking) is uitgevoer om, vanuit ‘n struktuuroogpunt, 'n begrip van die aktiwiteitsbeperking-interaksies te verkry. Die voorspelde PGIP-BcPG interaksies was hoogs spesifiek tot die B. cinerea ras, maar PGIP spesifisiteit was subtiel beïnvloed deur genotipe. Daarvolgens is die transgeniese wingerdstokbevolking met ‘n ander B. cinerea ras (B05.10) infekteer en die resultate het die rekenaargebaseerde simulasies ondersteun. Die transgeniese druifplante het nie hipervatbaarheid teenoor die B05.10 ras vertoon nie, wat daarop dui dat dit 'n rasspesifieke reaksie was. Transgeniese tabak met dieselfde gene ooruitgedruk, aan die ander kant, vertoon verhoogde weerstand ongeag die B. cinerea stam. Die fenotipe van transgeniese druifplante wat waargeneem was tydens B. cinerea infeksie word daarom beskou as beide gasheer- en rasspesifiek. Verder, toe B. cinerea mutante in BcPGs en galakturonaatmetabolisme gebruik was tydens infeksiestudies op hierdie druifplant- en tabakbevolkings, kon beide gasheer-spesifieke virulensiefaktore en moontlike erkennings- of verwarringsfaktore geïdentifiseer word. Hierdie resultate bevestig tesame die belangrike invloed van die spesifieke gasheer en patogeen op die gevolglike fenotipe en maak dit duidelik dat studies wat die interaksie van beide komponente insluit die mees insiggewende benadering sou wees in die bestudering van infeksie en verdediging. Dis interessant dat die transgeniese druifplante gedeeltelik weerstandbiedend was teen ‘n biotrofiese patogeen, en met ‘n meganisme wat die aanvanklike kolonisasie/indringing van die patogeen keer. Die dui daarop dat PGIP ooruitdrukking moontlik vooraf-gevormde verdedigingsmeganismes kon beïnvloed. Verdere ontleding van die transgeniese druifplante het bevestig dat beide die inheemse en transgeniese pgips uitgedruk was tydens infeksie en aktiewe proteïene, wat effektief BcPG aktiwiteit kon beperk, geproduseer was. Maar voor infeksie vertoon transgeniese wingerdblare minder selwandkomponente wat verband hou met selwandversterking, wat moontlik verswakte selwande aandui. Daarbenewens, in vergelyking met die kontrole, laat hulle aansienlik laer vlakke van verdedigingsverwante seskwiterpene vry tydens infeksie met die druifafkomstige ras van B. cinerea. Hierdie bevindings dui aan dat daar moontlik veranderinge in metaboliese prosesse was, veroorsaak deur die ooruitdrukking van nie-vinifera pgips in die V. vinifera konteks, wat tydens infeksie meer gunstig was vir die patogeen as die gasheer. Om voort te bou op die bevinding is ‘n geheeltranskriptoomstudie geloods om die rasspesifieke infeksiestrategie tesame met die gasheerspesifieke verdedigingstrategie as ‘n dinamiese interaksie te ondersoek tydens die vroeë infeksiefases. B. cinerea druifafkomstige ras het op transgeniese plante, in vergelyking met die kontrole, by die plaaslike infeksiesone aansienlik hoër vlakke van gene wat betrokke is in fitotoksiensintese uitgedruk. Aan die ander kant, het die transgeniese plante aansienlik laer vlakke van verdedigingsverwante gene uitgedruk, ook by die plaaslike infeksiesone. Tesame daag die bevindinge van hierdie studie ons huidige begrip van die rolle van PGIPs in plantverdedigingsreaksies tydens B. cinerea infeksie uit. Dit dui op die moontlikheid dat druifplant PGIPs in hul inheemse konteks dalk nie primêr gekoppel kan word aan die klassieke PGIP-PG swambeperkende interaksies nie. Dit bied verder ook insig dat die hipervirulente druifafkomstige ras moontlik strategieë optimeer het om die gasheer se verdedigingsmeganismes teen die gasheer te gebruik en selfs die gasheer-reaksies in sy guns te moduleer. Die gasheer- en patogeenspesifieke reaksies waargeneem in hierdie studie beklemtoon ten sterkste die impak wat die keuse van die gasheer-patogeen paring het op die omskrywing van verdedigingsfenotipes. Toekomstige studies behoort ras- en gasheerspesifisiteit in ga te neem en interaksiestudies is veral waardevol wees in hierdie opsig. Hierdie studie het, soos aanvanklik beplan, suksesvol die hipervatbare fenotipe omskryf, maar het ook tot nuwe insigte, asook nuwe toetsbare hipotese gelei wat verdere studies kan ondersteun