Use of RNAi-mediated gene silencing in plant-pathogene interaction: Functional analysis of PTTG_02001 effector candidate

RNA interference (RNAi) is a natural gene regulation and defense mechanism in organisms that enables the controlled silencing of gene expression through a double-stranded RNA (dsRNA) trigger molecule. This technology, commonly used as a gene silencing tool, is also employed in the functional analysis of candidate genes. Wheat is a major cereal crop consumed both globally and in Turkey. Wheat leaf rust, caused by the fungal pathogen Puccinia triticina Eriks (Pt), is one of the most prevalent and yield-limiting diseases affecting wheat production. Effector proteins secreted by fungal pathogens suppress host immune responses and enhance pathogen virulence. Identifying these effectors and elucidating their roles through techniques such as RNAi is critical for developing effective disease management strategies. In this study, the functional characterization of the effector candidate gene PTTG_02001, previously identified through proteomic analysis, was conducted, yielding strong evidence that the gene is a bona fide effector. The gene was silenced using the Host-Induced Gene Silencing (HIGS) approach, and its role during infection development was investigated. A 465 bp RNAi fragment specific to PTTG_02001 (termed RNAi02001) was designed and cloned into a Barley Stripe Mosaic Virus (BSMV) vector. Seedlings of the susceptible wheat cultivar 'Morocco' were inoculated with the recombinant BSMV carrying the RNAi02001 fragment. Through virus-mediated delivery, dsRNA molecules specific to the effector candidate were introduced into plant cells, where they were amplified and systemically distributed to new cells. On the 10th day post BSMV inoculation, plants were challenged with urediniospores of Pt race 1, facilitating the delivery of dsRNAs to the pathogen during the plant-pathogen interaction. The efficiency of the RNAi mechanism and the silencing level of the candidate gene were assessed via quantitative real-time PCR (qRT-PCR). Additionally, infection development was monitored microscopically, and disease severity was evaluated using the Cobb scale. As a result of RNAi-based HIGS application, PTTG_02001 transcript levels were reduced by approximately 76% in the third leaves and 95% in the fourth leaves of treated plants. Correspondingly, disease severity in test plants was observed to be below 10%, nearly approaching zero. Microscopic observations supported these findings, showing significantly reduced development of infection structures in the test samples. These results strongly support the conclusion that PTTG_02001 is a functional effector gene involved in the virulence of Puccinia triticina.