The efficacy of the antimicrobial peptides D4E1, VvAMP-1 and Snakin1 against the grapevine pathogen aster yellows phytoplasma

Spinas, Nicole Lotte (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Phytoplasma diseases have caused disastrous effects in vineyards around the world. Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules expressed by almost all organisms as part of their non-specific defence system. These peptides can offer protection against a wide variety of bacterial and fungal pathogens in plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are considered to be perfect candidates to confer resistance to this phytopathogen. The current study intends to explore the in planta activity of AMPs against the grapevine pathogen aster yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression. The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S expression vectors containing four different AMP-encoding sequences were therefore constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv "Sultana‟ plant material was used. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv "Chardonnay‟ material. Additionally, transmission experiments were carried out to infect Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using universal primers described by Gundersen and Lee (1996). For quantification of AYp infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the SYBR Green-based system. In total, 86 V. vinifera cv "Chardonnay‟ plantlets were screened for AYp infection two-, three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp infection could however be detected and plantlets displayed a "recovery phenotype‟. To examine the distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ plant, leaf and the corresponding node material from five canes were screened by a nested-PCR procedure. It can be concluded, that AYp was found predominantly in the nodes when compared to leaf material in the late season of the year. It is also highly unlikely for leaf material to show phytoplasma infection, if in the corresponding node no AYp could be detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and N. benthamiana plants with AYp was successfully achieved by insect vector transmission experiments. Transient expression assays were conducted on AYp-infected N. benthamiana material. Quantification of phytoplasma in this material showed a decrease of AYp in both the AMP treatment groups and the control groups. This study optimized a qPCR procedure to detect and quantify AYp in infected plant material. The Agrobacterium-mediated transient expression system used during this study was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera cv "Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ vine.

AFRIKAANSE OPSOMMING: Fitoplasma siektes veroorsaak ramspoedige gevolge in wingerde oor die hele wêreld. Dus kan die onlangse ontdekking van fitoplasma in Suid-Afrikaanse wingerde baie nadelige gevolge vir die plaaslike wynbedryf beteken. Antimikrobiese peptiede (AMPe) is klein molekules wat in amper al organismes as deel van hulle nie-spesifieke verdedegingsstelsel tot uitdruk kom. Hierdie peptiede kan beskerming aanbied teen ʼn wye verskeidenheid van bakteriële en swampatogene in plante. As gevolg van die feit dat fitoplasmas geen buitenste membraan en selwand het nie, word AMPe oorweeg as middle om weerstand te verleen teen hierdie fitopatogene. Die huidige studie beoog om die in planta aktiwiteit an AMPe teen die wingerdstok patogeen aster geel fitoplasma (AYp) deur middle van Agrobakteriumbemiddelde tydelike uitdrukkingssiteme, te ondersoek. Die AMPe, Vv-AMP1, D4E1 en Snakin1 (geïsoleer van aartappel en wingerd plante) is gekies om getoets te word vir hul in planta effek teen AYp. Blomkoolmosaïek-viruse 35S uitdrukkingsvektore met vier verskillende AMP-kodering rye, is dus ontwikkel. As ʼn aternatiewe method om die moontlike effek van Vv-AMP1 op AYp in planta in ag te neem, is oorplantings van die Vv-AMP1 transgeniese Vitis vinifera cv "Sultana‟ plantmateriaal gebruik. Om voorsiening te maak vir AMPe se doeltreffenheid in hierdie tydelike uitdrukkingsvektore, is pogings aangewend om die ruimlike verspreiding en patogeen konsentrasie van AYp in V. vinifera cv "Chardonnay‟ te beskryf. Addisioneel is transmissie eksperimente uitgevoer om Catharanthus roseus en Nicotania benthamiana te besmet met AYp deur die insekvektor, Mgenia fuscovaria. Plantmateriaal is getoets vir AYp deur van ʼn PCR protokol gebruik te maak met universele inleiers (grondlae) soos beskyf deur Grundersen en Lee (1996). Vir kwantifiseering van die AYp infeksie, is n semi-kwantitatiewe qPCR protokol geoptimaliseer, met hulp van die SYBR Groen-gebaseerde stelsel. In geheel is 86 V. vinifera cv "Chardonnay‟ planties getoets vir AYp infeksie – twee-, drie-, vier-, sewe- en elf weke na die bekendstelling aan die in vitro voorwaardes. Geen AYp infeksie kon egter opgespoor word en die plante het “herstel fenotipe‟ vertoon. Om die verspreiding van AYp in stingelknope van ʼn besemtte V. vinifera cv "Cardonnay‟ plant, blaar en ooreenstemmende stingelknope uit vyf stingels te ondersoek, is hulle getoets deur ʼn PCR protokol. Daar kon afgelei word dat AYp hoofsaaklik in die stingelknop in vergelyking met die blaarmaterial laat in die season, gevind word. Dit is hoogs oonwaarskynlik om fitoplasma infeksies in blaarmaterial te vind, as in die ooreenstemmende stingelknop daar geen AYp oopgespoor kon word nie. As gevolge daarvan dat die AYp-geinfekteerde wingerdmateriaal nie in vitro gegroei kon word nie, kon die effek van VvAMP-1 transgeniese wingerd teen AYp nie getoets word nie. Infeksies van C. roseus en N. benthamiana plante met AYp is suksesvol bereik deur transmissie eksperiemente. met ʼn insekvektor. Tydellike uitdrukkingvektore toetse is uitgevoer op die AYp besmette N. benthamiana material. Kwantifisering van fitoplasma in hierdie material het die afname van AYp in altwee, die AMP behandelings groepe en die kontrole groepe getoon. Hierdie studie het ʼn qPCR geoptimaliseer om besmette plantmaterial met AYp op te spoor en dit te kwantifiseer. Die Agrobacterium-bemiddelde tydelike uitdrukingsvektore wat in hierdie studie gebruik is, was nie vertroubaar genoeg, want geen beduidelike effek van die AMPe op AYp konsentrasie kon waargeneen word nie. Hierdie studie het bewys dat AYp nie vasgestel is en in stand gehou kan word deur in vitro aankweeking van V. vinifera cv "Chardonnay‟ material nie, en weefselkulture kan dus ʼn manier wees om AYp in hierdie kultivar uit te roei. Tot kennis, is hierdie studie die eerste studie om die ruimtelike verspreiding van AYp in stingelknope van ʼn besmette V. vinifera cv "Chardonnay‟ wingerstok te rapporteur.

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