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

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.Wheat is one of the most important sources of protein in the human diet. It is a staple for 35% of the human population and supplies approximately 20% of the calories consumed worldwide (http://www.cymmt. org/). Losses from pathogens and pests greatly impact wheat production. One of the most pervasive of these diseases is leaf rust, caused by Puccinia triticina, which over the course of human history has caused famines and ruined the economies of entire countries (Agrios, 1988). Currently, worldwide annual losses from leaf rust are estimated to be the equivalent of U.S. $2 billion (National Agricultural Statistics Service, http://www. usda.gov/nass).Plants have evolved potent surveillance and response systems that provide resistance to a diverse set of pathogens, including fungi, bacteria, viruses, nematodes, and insects. Despite this broad range of intruders, in most cases examined, the resistance pathways (R-pathways) that counter them are based on very similar molecular architecture. One of the bestcharacterized modes of disease resistance is known as gene-for-gene resistance (Flor, 1971), whereby resistance to a specific pathogen requires the presence of a particular allele of a plant resistance gene (R-gene) and a gene encoding its cognate elicitor in the pathogen. The vast majority of R-genes that have been isolated are kn...

RNA interference for wheat functional gene analysis

“…Similar results were extended recently to common wheat (Scofield et al 2005). Using barley PDS as the RNAi trigger (96% sequence homology to the corresponding wheat PDS region), they found photobleaching in wheat similar to that seen before in barley (Scofield et al 2005). PDS transcript levels were reduced to at least 40% of the non-infected control during the duration of the study (3-21 days after infection).…”

“…Using PDS and the visible photobleaching phenotype, Holzberg et al (2002) showed that the BSMV-VIGS system elicits target-directed gene silencing in barley in a sequence dependent manner. Similar results were extended recently to common wheat (Scofield et al 2005). Using barley PDS as the RNAi trigger (96% sequence homology to the corresponding wheat PDS region), they found photobleaching in wheat similar to that seen before in barley (Scofield et al 2005).…”

“…Successful RNAi studies in wheat have used trigger regions ranging in size from *200 to 550-bp (Table 1), although trigger dsRNA as short as 23-bp have been shown to induce the degradation of target mRNAs in Nicotiana benthamiana (Thomas et al 2001). Transient RNAi studies using VIGS showed that a trigger of 120-bp was sufficient to produce significant gene silencing (Scofield et al 2005). In the same experiment, trigger sequences of 80-bp were less effective for gene silencing and no detectable silencing was seen when using trigger sequences of 40-bp.…”

“…To further determine the usefulness of the BSMV-VIGS system for wheat functional genomics, Scofield et al (2005) targeted the leaf rust resistance gene Lr21. They reduced Lr21 transcript levels to 17% relative to the non-infected control, leading to increased susceptibility of infected wheat plants to Puccinia triticina, the causal agent of leaf rust.…”

Differential Requirement of Oryza sativa RAR1 in Immune Receptor-Mediated Resistance of Rice to Magnaporthe oryzae

“…The function of these proteins in disease resistance responses have been extensively investigated previously using mutant analyses Chandra-Shekara et al, 2004;Hubert el al., 2003;Lu et al, 2003;Shirasu et al, 1999;Takahashi et al, 2003) and by virus induced gene silencing (VIGS) analyses in several plant species (Bhattarai et al, 2007;de la Fuente van Bentem et al, 2005;Leister et al, 2005;Liu et al, 2004;Scofield et al, 2005). The data from these studies revealed differing specificities for these proteins in diverse NB-LRR-mediated resistance responses.…”