Development and implementation of VIGS in common bean and pea using BPMV

Team GDYNPATH / Valerie Geffroy

Despite genome sequences and other -omics resources that are now available in crop legume species such as pea and common bean, functional characterization of genes is still limited in these two species. Indeed, as most legumes, they are both recalcitrant to stable genetic transformation. In that context, VIGS is an attractive tool for functional genomics.

VIGS is a reverse-genetic tool that allows rapid and transient analysis of gene function in crops such as legumes that are difficult and time-consuming to transform genetically. VIGS exploits the natural plant anti-viral defense response i.e. post-transcriptional gene silencing (PTGS) that targets viral RNAs for sequence-specific degradation. This technology implies the generation of recombinant viral vectors carrying a fragment of sequence which is homologous to an endogenous gene of interest. After infection with this recombinant virus, transcripts of the gene of interest will become targets for degradation by the PTGS mechanism, thus knocking down expression of the target gene.

We chose to work with BPMV VIGS vectors because it is currently the most widely employed VIGS vector in legumes (Pflieger et al. 2013). In addition, it is adapted to high-throughput VIGS studies because infection of plants is performed in ‘one-step’ by mechanical inoculation of leaves using DNA plasmids carrying BPMV genomic elements under control of a CaMV 35S promoter (Zhang et al. 2010; Plant Phys). In that context, we have successfully adapted VIGS using BPMV in common bean (Pflieger et al. 2014) and pea (Meziadi et al. 2016).

VIGS in common bean (Phaseolus vulgaris)

Previous studies have shown that BPMV (Bean pod mottle virus), a widely used VIGS vector in soybean (Glycine max), is also suitable for gene silencing in Phaseolus vulgaris cv. Black Valentine (Zhang et al. 2010 ; Diaz-Camino et al. 2011).

The success of VIGS relies on the ability of viral vectors to infect the genotype of interest. In contrast to soybean for which almost all commercial cultivars are susceptible to BPMV infection, only few common bean genotypes (including Black Valentine) are susceptible to BPMV. With the recent availability of the whole genome sequence of Phaseolus vulgaris (Schmutz et al. 2014), VIGS technology opens new pastures in common bean functional genomics to assess gene functions involved in traits of agronomic importance (agronomic, disease resistance, abiotic stress tolerance).

We have developed a high-throughput protocol for VIGS in Phaseolus vulgaris using BPMV-based vectors (Pflieger et al. 2014). The susceptibility of common bean genotypes of interest was tested using BPMV-Green Fluorescent protein (GFP) vector (Figure 3). In susceptible genotypes, the efficiency of VIGS was subsequently tested using BPMV-Phytoene desaturase (BPMV-PDS) vector (Figure 4).

VIGS in pea (Pisum sativum)

In the context of the PeaMUST project (Pea MUlti-STress adaptation and biological regulations for yield improvement and stability ; project ANR-11-BTBR-0002), we are developing a high-throughput VIGS tool for rapid validation of candidate genes involved in agronomic traits (disease resistances, frost tolerance…) in pea.

We use the BPMV VIGS vector developed in Glycine max and Phaseolus vulgaris. The effectiveness of VIGS in aerial parts (leaves) is tested using BPMV-PDS vector (Figure 5) and in roots using a BPMV-KOR1 vector targeting the endogene KORRIGAN-1 previously shown to be a good reporter gene for VIGS in roots (Constantin et al. 2004) (Figure 6).

Functional validation of candidate genes in legumes using VIGS

We are using VIGS for the functional validation of genes derived from our own research programs (e.g. candidate R genes), but also to establish collaborations on selected research topics. For example, in collaboration with the group of Phil McClean (North Dakota State University; USA), we have confirmed by VIGS a candidate gene for the P (pigment)-gene (McClean et al. 2018).