Hide/Show Apps

Bio-functional analysis of RAD23 gene using virus induced gene silencing method in response to powdery mildew attack in Barley

Download
2012
Aksoy, Yağmur
Blumeria graminis f.sp. hordei is an obligate biotrophic parasite causing powdery mildew disease in several cereals, including barley. Virus induced gene silencing (VIGS) is a widely used powerful tool in plant functional-genomic studies to determine the functions of genes in disease resistance. In this thesis study, the function of RAD23 gene in barley in plant resistance was investigated. The RAD23 is a ubiquitin (Ub) binding protein involved in programmed protein degradation by transferring of ubiquitylated substrates to the proteasome. It was identified as an induced protein responding to compatible powdery mildew-barley interaction in a previous proteomics study performed in our laboratory. Pallas-01 and Pallas-03 barley line seedlings were inoculated with Blumeria graminis f.sp. hordei race (Bgh103). Curiously, RAD23 was v observed in compatible interaction (Pallas-03/Bgh103) with 4 fold induction at 12 dpi, while in incompatible interaction RAD23 was absent. Thus, it is thought that RAD23 may play a role in disease resistance as a negative regulator. In order to confirm this hypothesis, BSMV mediated virus induced gene silencing (VIGS) was performed. To determine the effects of the silencing of HvRAD23 gene in disease resistance mechanism, BSMV:HvRAD23 treated (silenced group) and BSMV:00 treated (control group) Pallas-03 seedlings were inoculated with a virulent (Bgh103) and avirulent (Bgh95) pathogen races. The pathogen growth levels at 3 and 5-days post inoculation were analyzed under light microscope and then, hyphae lengths were measured to detect the difference in pathogen development between silenced and control groups. As a result, 21 % difference in primary hyphae lengths, 22 % difference in secondary hyphae lengths and 16 % difference in the longest hyphae lengths between two groups were observed. Then, quantitative real time PCR was performed to determine the HvRAD23 gene silencing level. The HvRAD23 silencing was only seen in 3 of the 6 samples. The average silencing of the samples is 17 %. Despite of low silencing level, microscopic analyses indicated significant difference in pathogen growth between silenced and control groups. Therefore, the results of the initial studies support that the RAD23 is a negative regulator of plant disease resistance.