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Stepwise artificial evolution of a plant disease resistance gene.

ABSTRACT: Genes encoding plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins confer dominant resistance to diverse pathogens. The wild-type potato NB-LRR protein Rx confers resistance against a single strain of potato virus X (PVX), whereas LRR mutants protect against both a second PVX strain and the distantly related poplar mosaic virus (PopMV). In one of the Rx mutants there was a cost to the broad-spectrum resistance because the response to PopMV was transformed from a mild disease on plants carrying wild-type Rx to a trailing necrosis that killed the plant. To explore the use of secondary mutagenesis to eliminate this cost of broad-spectrum resistance, we performed random mutagenesis of the N-terminal domains of this broad-recognition version of Rx and isolated four mutants with a stronger response against the PopMV coat protein due to enhanced activation sensitivity. These mutations are located close to the nucleotide-binding pocket, a highly conserved structure that likely controls the "switch" between active and inactive NB-LRR conformations. Stable transgenic plants expressing one of these versions of Rx are resistant to the strains of PVX and the PopMV that previously caused trailing necrosis. We conclude from this work that artificial evolution of NB-LRR disease resistance genes in crops can be enhanced by modification of both activation and recognition phases, to both accentuate the positive and eliminate the negative aspects of disease resistance.

SUBMITTER: Harris CJ

PROVIDER: S-EPMC3876221 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Project description:BackgroundRNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects.Methodology/principal findingsIn this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA.Conclusions/significanceOur results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.

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Stepwise artificial evolution of a plant disease resistance gene.

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