Development of a virus-induced gene-silencing system for functional analysis of the RPS2-dependent resistance signalling pathways in Arabidopsis.
Virus-induced gene silencing (VIGS) offers a rapid and high throughput technique platform for the analysis of gene function in plants. Although routinely used in some Solanaceous species, VIGS system has not been well established in Arabidopsis thaliana (L.) Heynh. We have recently reported some factors that potentially influence tobacco rattle virus (TRV)-mediated VIGS of phytoene desaturase (PDS) and actin gene expression in Arabidopsis. In this study, we have further established that the Agrobacterium strain used for agro-inoculation significantly affects the VIGS efficiency. Strain GV3101 was highly effective; C58C1 and LBA4404 were invalid, while EHA105 was plant growth stage-dependent for TRV-induced gene silencing. Furthermore, the VIGS procedure optimised for the PDS gene was applied for the functional analysis of the disease resistance gene RPS2-mediated resistance pathway. Silencing of RPS2 led to loss of resistance to the otherwise avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene AvrRpt2. Silencing of RIN4, a RPS2 repressor gene, gave rise to conversion of compatible interaction to incompatible. Silencing of NDR1, RAR1 and HSP90, known to be required for the RPS2-mediated resistance, resulted in loss of the resistance, while silencing of EDS1 and SGT1b, which are not required for the RPS2-mediated resistance, caused no change of the resistance. These results indicate that the optimised procedure for the TRV-based VIGS is a potentially powerful tool for dissecting the signal transduction pathways of disease resistance in Arabidopsis.
Cai
Xin-Zhong
XZ
Institute of Biotechnology, and Department of Plant Protection, Zhejiang University, 268 Kai Xuan Road, Hangzhou 310029, P.R. China. xzhcai@zju.edu.cn
Xu
Qiu-Fang
QF
Wang
Chang-Chun
CC
Zheng
Zhong
Z
D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development / Q000821:virology*; D029681:Arabidopsis Proteins / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D010935:Plant Diseases / Q000235:genetics / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029681:Arabidopsis Proteins; C089426:RPS2 protein, Arabidopsis