Functional analysis of candidate effector PTTG-01827 of wheat brown rust (Puccinia triticina Eriks) by higs method

Wheat brown/leaf rust is a fungal disease. The causative agent, Puccinia triticina Eriks (Pt), causes yield loss by reducing the plant's ability to photosynthesize. Effectors are proteins/peptide molecules secreted by pathogens to suppress host defenses and modulate host physiology to increase their virulence success. Therefore, detection of effectors is seen as one of the most promising ways to control the disease. This study aimed to functionally analyze the candidate effector gene and determine whether it is a real effector. For this, the candidate effector PTTG_01827, previously identified in the proteomic study, was silenced and functionally analyzed by HIGS method. HIGS is a method used successfully to silence phytopathogenic fungal genes using the RNAi mechanism, one of the natural defense mechanisms of plants. In the study, a 350 bp long DNA fragment specific to the PTTG_01827 effector candidate was designed and cloned into the BSMV vector. The HIGS method was optimized for silencing the PTTG_01827 gene. Seedlings of the sensitive wheat variety Morocco were inoculated with recombinant BSMV containing the PTTG_01827 fragment, and effector candidate-specific dsRNA molecules were amplified and distributed in plant cells via the virus. When BSMV symptoms were observed at 10 dpi, the same plants were inoculated with Pt race-1. Thus, expressed effector mRNAs during the infection development of Pt race-1 were targeted. The effectiveness of the HIGS mechanism and the silencing level of the candidate gene were determined by qRT-PCR. Changes of the infection type were detected by microscobic and fenotypic observations. At the end, the coorelation between changes in Pt infection severity and the gene silencing level was evaluated. As a result of HIGS application, it was determined that there was an approximately 50% decrease in the expression of PTTG_01827. It was observed that a decrease in Pt sporulation was accompanied by a slowdown in hyphae development, and the severity of infection decreased by 50%. The findings support the expectations that PTTG_01827 is a true effector. Changes in the type of infection, the effectiveness of the RNAi mechanism and the effect of silencing the candidate gene on the development of infection were analyzed and confirmed by qRT-PCR and microscopic examination. As a result of HIGS application, the expression of PTTG-01827 was an approximately 50% decrease. Pt infection on the same leaves was also decrease accompanied by a decrease in Pt sporulation and a slowdown in hyphal development. The our findings support the predictions that PTTG_01827 may be a real effector.