Figure 4

BSMV-mediated silencing of CYP96B22 during infection of barley with Magnaporthe . Primary leaves of barley plants were infected with BSMV without (BSMV-γMCS_new) or with a silencing construct against CYP96B22 (BSMV-γCYP). Plants showing virus symptoms were inoculated after two weeks on their third leaf with Magnaporthe isolates CD180 or TH6772 in detached leaf assay. A) Transcript abundance of CYP96B22 was monitored by RT-qPCR in plants at 24 h p.i. with isolate CD180. Values shown are mean and standard deviation of two technical replicates and were calculated relative to the reference gene EF1α (2^{(Ct(EF1α) – Ct(CYP96B22))}). For microscopic analysis two groups of plants were built, one consisting of the five plants inoculated with BSMV-γMCS_new and the other encompassing the five plants showing the highest CYP96B22 transcript reduction (in average 86%, significance according to paired t-test). Cytological evaluation was done in the nonhost (B) and host (C) interaction at 96 and 48 h p.i., respectively. Therefore each infection site was assigned to different categories of plant defense responses which are associated with the deposition of autofluorescent material (for details see [4]). For both groups of plants 100 interaction sites were evaluated on each of the five leaves (means and standard errors are shown). Each category of plant defense responses was tested for significant differences between plants with and without silencing using t-test (P < 0.05). Significant differences were marked by different letters. In addition each attacked epidermal cell was investigated for the presence of invasive hyphae, frequencies are displayed by shading of bars. For clarity, the frequency of epidermal cells with invasive hyphae was depicted seperately for the host interaction. For the nonhost interaction (B) results were reproduced in two independent experiments. Higher invasive growth of the host isolate in silenced plants was confirmed in one biological replicate (C).