Abstract
BackgroundVirus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. Unfortunately, VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types. This is especially true in crop plants like soybean [Glycine max (L.) Merr]. To address these shortcomings, we modified the inoculation procedure of a VIGS vector based on Apple latent spherical virus (ALSV). The efficacy of this new procedure was assessed in 19 soybean genotypes using a soybean Phytoene desaturase (GmPDS1) gene as the VIGS target. Silencing of GmPDS1 was easily scored as photo-bleached leaves and/or stems.ResultsIn this report, the ALSV VIGS vector was modified by mobilizing ALSV cDNAs into a binary vector compatible with Agrobacterium tumefaciens-mediated delivery, so that VIGS-triggering ALSV variants could be propagated in agro-infiltrated Nicotiana benthamiana leaves. Homogenate of these N. benthamiana leaves was then applied directly onto the unifoliate of young soybean seedlings to initiate systemic gene silencing. This rapid inoculation method bypassed the need for a particle bombardment apparatus. Among the 19 soybean genotypes evaluated with this new method, photo-bleaching indicative of GmPDS1 silencing was observed in nine, with two exhibiting photo-bleaching in 100% of the inoculated individuals. ALSV RNA was detected in pods, embryos, stems, leaves, and roots in symptomatic plants of four genotypes.ConclusionsThis modified protocol allowed for inoculation of soybean plants via simple mechanical rubbing with the homogenate of N. benthamiana leaves agro-infiltrated with ALSV VIGS constructs. More importantly, inoculated plants showed no apparent virus disease symptoms which could otherwise interfere with VIGS phenotypes. This streamlined procedure expanded this functional genomics tool to nine soybean genotypes.
Highlights
Virus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes
Despite repeated attempts with strict adherence to the published procedures [16, 17, 28], we failed to observe any photo-bleaching or symptoms relating to silencing of GmPDS1 in the plants that emerged from the bombarded seeds, or detect Apple latent spherical virus (ALSV) RNAs in the plant tissue using RTPCR
A modified set of ALSV vectors permit the propagation of VIGS constructs in N. benthamiana via agro‐infiltration The propagation of VIGS inoculum with agro-infiltrated N. benthamiana leaves and subsequent inoculation of soybean seedlings with N. benthamiana extracts were evaluated [30]
Summary
Virus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types This is especially true in crop plants like soybean [Glycine max (L.) Merr]. The most widely used VIGS vector for soybean, based on the Bean pod mottle virus (BPMV), requires a modified BPMV cDNA to be delivered into soybean leaves or lima bean (Phaseolus lunatus) cotyledons using particle bombardment to amplify the inoculum [3, 14,15,16,17] Another limitation of the BPMV and other VIGS systems are the foliar symptoms that develop due to virus infection such as chlorosis, necrosis, and leaf distortion. Some VIGS vectors are reported to have limited movement within the leaf and stem tissue, resulting in uneven phenotypes in different plant tissues [18]
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