BackgroundOver the last two years, considerable advances have been made in common bean (Phaseolus vulgaris L.) genomics, especially with the completion of the genome sequence and the availability of RNAseq data. However, as common bean is recalcitrant to stable genetic transformation, much work remains to be done for the development of functional genomics tools adapted to large-scale studies.ResultsHere we report the successful implementation of an efficient viral vector system for foreign gene expression, virus-induced gene silencing (VIGS) and genetic mapping of a BPMV resistance gene in common bean, using a “one-step” BPMV vector originally developed in soybean. With the goal of developing this vector for high-throughput VIGS studies in common bean, we optimized the conditions for rub-inoculation of infectious BPMV-derived plasmids in common bean cv. Black Valentine. We then tested the susceptibility to BPMV of six cultivars, and found that only Black Valentine and JaloEEP558 were susceptible to BPMV. We used a BPMV-GFP construct to detect the spatial and temporal infection patterns of BPMV in vegetative and reproductive tissues. VIGS of the PHYTOENE DESATURASE (PvPDS) marker gene was successfully achieved with recombinant BPMV vectors carrying fragments ranging from 132 to 391 bp. Finally, we mapped a gene for resistance to BPMV (R-BPMV) at one end of linkage group 2, in the vicinity of a locus (I locus) previously shown to be involved in virus resistance.ConclusionsThe “one-step” BPMV vector system therefore enables rapid and simple functional studies in common bean, and could be suitable for large-scale analyses. In the post-genomic era, these advances are timely for the common bean research community.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0232-4) contains supplementary material, which is available to authorized users.
Identifying the molecular basis of resistance is critical to promote chemical free cropping system. In plants, NLR constitute the largest family of disease resistance (R) genes but they can be rapidly overcome, prompting research of alternative source of resistance. Anthracnose, caused by the fungus Colletotrichum lindemuthianum, is one of the most important diseases of common bean. This study aimed to identify the molecular basis of Co-x, an anthracnose R-gene conferring total resistance to the extremely virulent C. lindemuthianum strain 100. To that end, we sequenced the Co-x 58kb target region in the resistant JaloEEP558 (Co-x) and identified KTR2/3, an additional gene encoding a truncated and chimeric CRINKLY4 kinase, located within a CRINKLY4 kinase cluster. KTR2/3 presence is strictly correlated with resistance to strain 100 in a diversity panel of common beans. Furthermore, KTR2/3 expression is upregulated 24 hours post-inoculation and its transient expression using Agrobacterium-transformation in susceptible genotype increases resistance to strain 100. Altogether, our results provide molecular evidence that Co-x encodes a truncated and chimeric CRINKLY4 kinase probably resulting from an unequal recombination event that occurred recently in the Andean domesticated gene pool. This atypical R-gene might act as a decoy involved in indirect recognition of a fungal effector.
Legume species are among the most important crops worldwide. In recent years, six legume genomes have been completely sequenced, and there is now an urgent need for reverse-genetics tools to validate genes affecting yield and product quality. As most legumes are recalcitrant to stable genetic transformation, virus-induced gene silencing (VIGS) appears to be a powerful alternative technology for determining the function of unknown genes. VIGS technology is based on the property of plant viruses to trigger a defence mechanism related to post-transcriptional gene silencing (PTGS). Infection by a recombinant virus carrying a fragment of a plant target gene will induce homology-dependent silencing of the endogenous target gene. Several VIGS systems have been developed for legume species since 2004, including those based on Bean pod mottle virus, Pea early browning virus, and Apple latent spherical virus, and used in reverse-genetics studies of a wide variety of plant biological processes. In this work, we give an overview of the VIGS systems available for legumes, and present their successful applications in functional genomics studies. We also discuss the limitations of these VIGS systems and the future challenges to be faced in order to use VIGS to its full potential in legume species.
Plant viral vectors have been developed to facilitate gene function studies especially in plant species not amenable to traditional mutational or transgenic modifications. In the Fabaceae plant family, the most widely used viral vector is derived from Bean pod mottle virus (BPMV). Originally developed for overexpression of foreign proteins and VIGS studies in soybean, we adapted the BPMV-derived vector for use in other legume species such as Phaseolus vulgaris and Pisum sativum. Here, we describe a protocol for efficient protein expression and virus-induced gene silencing (VIGS) in Pisum sativum leaves and roots using the "one-step" Bean pod mottle virus (BPMV) viral vector.
SummaryPea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus‐induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)‐derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4‐β‐D‐glucanase, respectively. The features of the ‘one‐step’ BPMV‐derived VIGS vector include (i) the ease of rub‐inoculation, without any need for biolistic or agro‐inoculation procedures, (ii) simple cost‐effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low‐ to high‐throughput VIGS studies.
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