Development of a Virus-Induced Gene-Silencing System for Hexaploid Wheat and Its Use in Functional Analysis of the <i>Lr21</i>-Mediated Leaf Rust Resistance Pathway

TLDR

Virus‑induced gene silencing (VIGS) uses engineered viruses to trigger RNA degradation of target plant transcripts, a technique common in dicots but previously unavailable for monocots until barley. The study aimed to develop and optimize BSMV‑based VIGS protocols for efficient gene silencing in hexaploid wheat. The system was first optimized by silencing phytoene desaturase, then applied to target genes involved in Lr21‑mediated leaf rust resistance. BSMV constructs carrying a 150‑bp Lr21 fragment, as well as silencing of RAR1, SGT1, and HSP90, converted wheat from resistant to susceptible, demonstrating that these genes are essential for Lr21‑mediated resistance and validating BSMV‑VIGS as a powerful tool for dissecting disease‑resistance pathways in hexaploid wheat.

Abstract

Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.