Molecular characterization of tomato spotted wilt virus (Orthotospovirus tomatomaculae) and development of resistant tomato (Solanum lycopersicum L.) lines using RNAi technology

Tomato spotted wilt virus (Orthotospovirus tomatomaculae, TSWV) poses a devastating threat to Solanaceous crops specially tomato and pepper. The development of resistant cultivars is one of the important management tools for incapacitating viral diseases. However, due to the widespread utilization of resistance cultivars, the resistant breaking virus isolates have emerged. In this study, symptomatic tomato and pepper samples were collected from diverse locations of Türkiye in 2020-2021, and incidence of virus was tested by both DAS-ELISA and RT-PCR. The sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers were used to screen viral resistance genes in tomato and pepper plants. Some of the cultivars were found to carry resistance genes even though they displayed severe viral infections. The extracted TSWV isolates from these plants were partially sequenced for the detection of reported mutations in the silencing suppressor gene (NSs) and movement protein (NSm) genes to test whether they were resistance-breaking (RB) isolates. Sequence comparisons and phylogenetic analyses revealed that the NSs and NSm genomic regions of virus did not show reported mutations and they were distributed in the same genetic pool while Turkish isolates of N gene were clustered in a separate clade. Moreover, in this study the prospective use of plant-mediated RNAi approach by targeting the NSs gene for the conferring resistance to orthotospoviruses was investigated. Transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region were developed. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Orthotospovirus NSs gene which contains both sense and antisense orientation in plant expression vector pFGC5941. For the transformation studies, 3 different types of explants (hypocotyls, cotyledonary nodes, and cotyledons) were used in tissue culture experiments. Co-infection was done by using Agrobacterium strain EHA-105 having viral constructs (TSWV-NSs_full (pFGC5941+sense+antisense), TSWV-NSs_sense). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers. Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. Moreover, for the confirmation, transgenic plants were inoculated with TSWV and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed which demonstrates a significant down-regulation in the gene expression levels of pathogenicity-related genes (PR-1) and NSs genes in transgenic plants harboring the TSWV-NSs_full constructs compared to control groups (non-transgenic wild type control, empty vector and the TSWV-NSs_sense constructs). For the validation of RNAi effectiveness, tobacco plants were also inoculated with viral constructs and they displayed severe symptoms of TSWV in plants containing the TSWV-NSs_sense constructs, empty vector, and in wild-type tomato plants while no any symptoms were observed with TSWV-NSs_full constructs that confirmed the hypothesis of this study by showing the efficacy of RNAi. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical significance and the potential to be a tool for controlling or mitigating the losses in tomato crops caused by TSWV around the world.