Establishing an optimized virus-induced gene silencing (VIGS) assay and functional validation of disease resistance genes in wheat and barley

Sustainable control of fungal diseases in wheat and barley requires efficient functional validation of candidate resistance and susceptibility genes. Virus-induced gene silencing (VIGS) provides a rapid alternative to stable transformation systems; however, its reliability in cereals depends on optimized cloning efficiency, viral amplification, and phenotyping consistency. This study aimed to optimize a Barley stripe mosaic virus (BSMV)-based VIGS system and apply it to the functional characterization of candidate genes involved in powdery mildew, yellow rust, and leaf rust resistance in barley and wheat. Ligation-independent cloning (LIC) efficiency for BSMV-γ constructs was improved by optimizing the insert-to-vector ratio and incorporating a controlled annealing and EDTA stabilization step, increasing cloning success from approximately 2% to 60%. ELISA-based viral quantification identified the second and third systemic leaves as the most suitable tissues for reliable viral amplification. Functional validation using mlo5 confirmed successful transient gene silencing, with significantly reduced Blumeria graminis f. sp. hordei colonization in the susceptible barley cultivar Ingrid. The optimized system was subsequently applied to several candidate genes. Silencing of a wheat MLO-like gene associated with a yellow rust GWAS peak did not significantly affect Puccinia striiformis infection. Functional analysis of FPA1, a barley candidate gene linked to non-host resistance, suggested a role in quantitative susceptibility by reducing fungal colony expansion, but not penetration, following Blumeria graminis f. sp. tritici infection in the Black Hulless genotype. Similarly, VIGS of three F-box-containing CREA candidate genes for leaf rust resistance in Triticum turgidum subsp. dicoccoides revealed no statistically significant phenotypic effects under the tested conditions. A major focus was the barley subtilisin-like protease RNR9, previously implicated in non-host resistance. Bioinformatic prediction of a C-terminal insertion variant could not be experimentally validated, indicating an annotation artifact. Transient overexpression of GFP-tagged RNR9 revealed weak basal protein accumulation that increased following pathogen challenge, suggesting tight regulation of protein abundance, although subcellular localization remained inconclusive. Overall, this work establishes an optimized BSMV-VIGS workflow for functional gene validation in cereals and provides new insights into the roles of candidate resistance genes, particularly the pathogen-responsive behavior of RNR9. The study contributes a practical platform for accelerating functional genomics and supporting durable disease resistance breeding in wheat and barley.