FIGURE 8.

Expression analysis of defense related genes and root colonization assay in wheat. (A) Gene expression analysis of defense response related genes in wheat roots colonized by C. rosea strains. Total RNA isolated from wheat roots 5 days post inoculation of WT and Δlysm strains and was used for RT-qPCR. Expression level of PR1 and PR4 genes was normalized to expression of the wheat β-tubulin (Marshall et al., 2011). Relative expression level based on RT-qPCR was calculated using 2^–ΔΔCt method (Livak and Schmittgen, 2001), and compared with the water inoculated control. (B) Determination of C. rosea root colonization in wheat roots, 5 days post inoculation, by quantifying DNA level using RT-qPCR. C. rosea colonization is expressed as the ratio between C. rosea DNA and wheat DNA. For DNA quantification actin and Hor1 were used as target gene for C. rosea and wheat, respectively. (C) Internal root colonization by C. rosea strains. Surface sterilized roots were homogenized in phosphate buffer and serial dilutions were plated on Rose Bengal selection plates under sterile condition at 25°C. Colony forming units (cfus) were counted 3 days post plating. Statistically significant differences (P ≤ 0.05) in gene expression between treatments were determined using Fisher’s exact test and are indicated by different letters. Error bars represent standard deviation based on five biological replicates.