Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract.

ABSTRACT

BACKGROUND: Virus 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. RESULTS: In 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. CONCLUSIONS: This 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.