Evaluation of two pathogen-derived resistance strategies for Grapevine leafroll-associated virus 3

[摘要] ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, isone of the most economic important viral diseases affecting grapevine. Grapevine leafrollassociated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member ofthe leafroll associated virus family. To prevent the spread of GLD, management strategiessuch as rogueing and insect vector control are required to limit crop losses. Alternative controlstrategies based on genetic modification of the grapevine genome, such as pathogen-derivedresistance (PDR), is proven to be effective in conferring resistance to several viruses.Therefore, the focus of this study was to evaluate pathogen-derived resistance strategiesfor GLRaV-3 using the following two approaches; 1) evaluation of transgenic plantsexpressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order toconfer resistance against GLRaV-3, and 2) the construction of artificial microRNAs(amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine hostand the development of an amiRNA-mediated silencing validation system.In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and#17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds ofeach onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virustitres of all grafted plants were quantified relative to two reference genes using RT-qPCR.Results were evaluated by comparing the relative virus titre of each transgenic plant line tothat of the non-modified control plant line. Results showed that resistance levels of plant line#3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be moresusceptible to the virus.The second part of the study was the construction and validation of amiRNAs targetingGLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated intomiRNA backbone vvi167b of grapevine. Moreover, target constructs were developed byincorporating corresponding GLRaV-3 target sequences into the 3' UTR of a greenfluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated withthe constructed amiRNA in Nicotiana benthamiana and GFP expression levels werequantified to determine the silencing efficiency of the amiRNAs. Results showed that theamiRNAs were successful in silencing the GFP target construct, however, they were notspecific in silencing exclusively their corresponding target. These amiRNA constructs areideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLDinfected grapevines.

[发布机构] Stellenbosch University