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...

In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.

AvrPto from the Pseudomonas cell. To examine this, avrPto deletions C⌬25, C⌬41, and C⌬74 were placed into pBI121 and tested with the Agrobacterium transient assay. Agrobacterium EHA105 containing avrPto induced an HR in 2 days, whereas EHA105 containing the avrPto deletion C⌬25 induced an HR after 4 days; the other deletions did not elicit an HR ( X. Tang and G. Martin, unpublished results). This suggests that the carboxyl terminal 25 amino acids of AvrPto are not required for secretion from the bacterial cell; this portion of AvrPto may serve as an activation domain, interact with other components in the signaling pathway, or have a role in AvrPto stability.