Research Accomplishment Reports 2007

Assessing RNAi as a Reverse Genetic Tool for Global Analysis of NBS-LRR Gene Function in Medicago truncatula

H.Zhu
Department of Plant and Soil Sciences

 

Project Description

The goal of this project was to test if gene silencing can be used for global analysis of NBS-LRR gene function in Medicago truncatula. Our strategy is to simultaneously silence multiple highly similar genes within a gene cluster in a single transgenic line using a single RNAi construct. We have chosen two NBS-LRR gene loci in the M. truncatula genome to test this strategy. One locus contains four CC-NBS-LRR genes and another contains at least five closely related TIR-NBS-LRR genes. Highly conserved NBS sequences were used to design the RNAi constructs using the binary vector pFGC5941. Silencing efficiency was tested in the transgenic plants by RT-PCR and Northern blotting analysis of small RNAs. In both cases, the target genes were not silenced.

Parallel experiments were also performed in Arabidopsis. We have selected RPP1 and RPP4 loci for targeted gene silencing. Unlike in M. truncatula where no functional resistance genes have been characterized, using Arabidopsis allows us to test the strategy by both molecular analysis of target transcripts and characterization of resistance phenotype by inoculation with pathogens. We have detected efficient silencing of the RPP1 locus but not the RPP4 locus.

Impact

Plant encodes hundreds of potential disease resistance genes, but it is difficult to identify the genes that are responsible for resistance to particular pathogens. If the RNAi strategy is successful, it will provide a new technology for global analysis of NBS-LRR gene function in plants.