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

[摘要] 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 highlydetrimental to the local wine industry. Antimicrobial peptides (AMPs) are small moleculesexpressed by almost all organisms as part of their non-specific defence system. Thesepeptides can offer protection against a wide variety of bacterial and fungal pathogens inplants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs areconsidered to be perfect candidates to confer resistance to this phytopathogen. The currentstudy intends to explore the in planta activity of AMPs against the grapevine pathogen asteryellows phytoplasma (AYp) through Agrobacterium-mediated transient expression.The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) wereselected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35Sexpression vectors containing four different AMP-encoding sequences were thereforeconstructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp inplanta, 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 weremade to describe the spatial distribution and pathogen titre of AYp in V. vinifera cvChardonnay‟ material. Additionally, transmission experiments were carried out to infectCatharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgeniafuscovaria. Material was screened for AYp infection by a nested-PCR procedure usinguniversal primers described by Gundersen and Lee (1996). For quantification of AYpinfection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using theSYBR 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 AYpinfection could however be detected and plantlets displayed a recovery phenotype‟. Toexamine 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-PCRprocedure. It can be concluded, that AYp was found predominantly in the nodes whencompared 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 bedetected. As AYp-infected grapevine material could not be maintained in vitro, the effect ofVvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus andN. benthamiana plants with AYp was successfully achieved by insect vector transmissionexperiments. Transient expression assays were conducted on AYp-infected N. benthamianamaterial. Quantification of phytoplasma in this material showed a decrease of AYp in boththe AMP treatment groups and the control groups.This study optimized a qPCR procedure to detect and quantify AYp in infected plantmaterial. The Agrobacterium-mediated transient expression system used during this studywas not reliable, as no significant effect of the AMPs on AYp titre could be observed. Thisstudy showed, that AYp cannot be established and maintained in in vitro cultured V. viniferacv Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYpin this cultivar. To our knowledge, this study is the first to report on the spatial distribution ofAYp in canes of an infected V. vinifera cv Chardonnay‟ vine.

[发布机构] Stellenbosch University