The Development of an Efficient Multipurpose Bean Pod Mottle Virus Viral Vector Set for Foreign Gene Expression and RNA Silencing  

Abstract

Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.Plant virus-based vectors have been recently developed to express heterologous proteins in plants for the study of gene function, production of pharmaceuticals, analysis of plant-microbe interactions, fungicide and insecticide screening, metabolic engineering, and nutrient improvement. Plant viral gene expression vectors have many advantages over conventional transgenic technology for protein expression. They are fast, low cost, high yield, and can be used in a variety of genetic backgrounds. Plant viral vectors also have applications as virus-induced gene silencing (VIGS) tools for reverse genetic studies of gene function (Burch-Smith et al., 2004). VIGS can specifically down-regulate a single gene, members of a gene family, or sets of distinct genes (Peele et al., 2001;Turnage et al., 2002;Lu et al., 2003). Due to these advantages, many positive sense RNA plant viruses have been developed as vectors for production of recombinant proteins or as VIGS vectors for many plant species (Pogue et al., 2002;Burch-Smith et al., 2004;Constantin et al., 2004;Ding et al., 2006;Grønlund et al., 2008;Igarashi et al., 2009;Meng et al., 2009;Zhang et al., 2009). With the rapid increase in genomic information, VIGS vectors have substantial potential to advance gene function studies in both monocot and dicot plants.Bean pod mottle virus (BPMV; genus Comovirus) has a bipartite positive RNA genome consisting of RNA1 (a...

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

“…These results are consistent with those obtained using other viral systems (Constantin et al 2008;Liu and Page 2008;Zhang et al 2010). In addition to the insert size and position of target genes, the effect of the cloning orientation of the foreign gene was investigated using white lupin plants.…”

“…In addition to the insert size and position of target genes, the effect of the cloning orientation of the foreign gene was investigated using white lupin plants. No clear differences were detected between sense and antisense orientations in this study (Figure 4), although antisense fragments have been shown to be more effective for silencing in other VIGS systems (Igarashi et al 2009;Pflieger et al 2008;Zhang et al 2010). …”

Development and Application of Molecular Markers to Breed Common Bean (Phaseolus vulgaris L.) for Resistance to Common Bacterial Blight (CBB) — Current Status and Future Directions

“…2) The epistatic interaction between CBB resistance gene(s)/or QTL would make the application of MMs to assist in breeding CBB resistance bean and the cloning of the QTL more complex, and 3) the recalcitrant nature of common bean to transformation make the validation of CGs coding for CBB resistance more difficult to accomplish. So it is an urgent task for bean researchers to develop novel approaches, such as virus induced gene silencing (VIGS) (Zhang et al, 2010) for validating the functions of CGs in common bean.…”

Soybean Homologs of MPK4 Negatively Regulate Defense Responses and Positively Regulate Growth and Development

“…Seeds of soybean (Glycine max 'Williams 82') used in this study were harvested from greenhouse-grown plants previously indexed for the absence of BPMV and SMV (Zhang et al, , 2010. Soybean plants were maintained in a greenhouse or growth chamber at 22°C with a photoperiod of 16 h.…”