Young-Su Seo*, Maria R. Rojas*, Jung-Youn Lee†‡, Sang-Won Lee*, Jong-Seong Jeon*§, Pamela Ronald*, William J. Lucas†, and Robert L. Gilbertson*¶
*Department of Plant Pathology and †Section of Plant Biology, University of California, Davis, CA 95616 Communicated by GeorgeBruening, University of California, Davis, CA, June 19, 2006 (received for review March 1, 2006)
Genes involved in a viral resistance response in common bean (Phaseolus vulgaris cv. Othello) were identiﬁed by inoculating a geminivirus reporter (Bean dwarf mosaic virus expressing the green ﬂuorescent protein), extracting RNA from tissue undergoing the defense response, and amplifying sequenceswith degenerate R gene primers. One such gene (a TIR-NBS-LRR gene, RT4-4) was selected for functional analysis in which transgenic Nicotiana benthamiana were generated and screened for resistance to a range of viruses. This analysis revealed that RT4-4 did not confer resistance to the reporter geminivirus; however, it did activate a resistance-related response (systemic necrosis) to seven strains ofCucumber mosaic virus (CMV) from pepper or tomato, but not to a CMV strain from common bean. Of these eight CMV strains, only the strain from common bean systemically infected common bean cv. Othello. Additional evidence that RT4-4 is a CMV R gene came from the detection of resistance response markers in CMV-challenged leaves of RT4-4 transgenic plants, and the identiﬁcation of the CMV 2a geneproduct as the elicitor of the necrosis response. These ﬁndings indicate that RT4-4 functions across two plant families and is up-regulated in a non-virus-speciﬁc manner. This experimental approach holds promise for providing insights into the mechanisms by which plants activate resistance responses against pathogens.
avirulence gene Cucumber mosaic virus response systemic necrosis geminivirus hostdefense
in plant signaling processes. Thus, this complexity may reflect the mechanism by which new genes arise in response to various selection pressures. In any event, this combination of genetic conservation and complexity presents a formidable challenge in terms of the identification and characterization of any given member of an R gene family. In this study, we identified genes involved ina viral resistance response in common bean (Phaseolus vulgaris cv. Othello) using a combination of a viral reporter and reverse transcription (RT)-PCR with degenerate R gene primers. A candidate R gene, RT4-4, a member of the TNL family, was selected for detailed analysis. Screening of RT4-4 transgenic Nicotiana benthamiana lines for viral resistance revealed that, although RT4-4 did not conferresistance to the reporter virus, it activated a resistance-like response (systemic necrosis) to Cucumber mosaic virus (CMV). Further evidence that RT4-4 is a CMV R gene came from our identification of the CMV 2a gene product as the elicitor of the necrosis response. The finding that RT4-4 functions across two plant families and is up-regulated in a non-virus-specific manner, provides insights intothe mechanisms by which plants activate the pathogen resistance response. Results The general strategy developed to clone viral R genes is outlined in Fig. 1. A geminiviral-GFP reporter [Bean dwarf mosaic virus (BDMV)-GFP], (17) was used to identify tissues undergoing a defense response in the resistant common bean cv. Othello (18). Poly(A) RNA, extracted 4 days after inoculation, was used in theRT-PCR with degenerate primers to amplify domains of putative R genes expressed in these tissues. Degenerate primers were designed based on conserved NBS domains of previously characterized TNL and CNL R genes and included three forward primers and a single reverse primer. These primer pairs directed the amplification of DNA fragments of 350–500 bp. One such fragment had amino acid sequence...