Determination of the role of candidate effector PTTG_00016 in wheat–leaf rust interaction via gene silencing

Puccinia triticina (Pt) is the causal agent of leaf rust in wheat, leading to significant yield losses. Effector proteins secreted by pathogens suppress plant defense responses and manipulate hormone signaling pathways, facilitating the establishment and progression of infection. Identification of such pathogen-derived effectors is crucial for reducing disease severity, elucidating plant immune mechanisms, and developing disease-resistant cultivars. Furthermore, it supports the implementation of environmentally, economically, and biologically sustainable approaches in agriculture. In this thesis, a functional analysis was conducted on one of the candidate effectors previously identified through proteomic studies to determine whether it acts as a true effector. The candidate effector gene PTTG_00016 was selected for analysis and was silenced using the Host-Induced Gene Silencing (HIGS) method. HIGS is an RNAi-based technique in which pathogen genes are silenced through expression in the host plant. It is frequently employed in the study of biotrophic fungal pathogens to uncover their infection strategies. For this purpose, a 449 bp fragment encompassing the exonic regions of PTTG_00016 was selected for RNAi construct design. The RNAi fragment was cloned into the Barley stripe mosaic virus (BSMV) vector, and the recombinant virus was used to deliver dsRNA into the Morocco wheat cultivar. By day 10 post-inoculation, distinct viral symptoms were observed, and the success rate of BSMV inoculation was approximately 75%. Subsequently, plants were inoculated with Pt race 1. On the third day after fungal inoculation, the gene expression levels were quantified using qRT-PCR, and infection progression was examined microscopically. qRT-PCR analysis revealed that PTTG_00016 expression was silenced by approximately 25%. However, microscopic evaluation did not show a significant difference in the development of fungal infection structures compared to the control. On day 12 post-inoculation with Pt, the severity of infection in HIGS-treated and control plants was assessed based on the Cobb scale. The results indicated that rust severity did not decrease significantly; disease severity was reduced by only about 10% compared to the control. In conclusion, this study did not provide sufficient evidence to confirm that PTTG_00016 functions as a bona fide effector.