Agrodrench: a novel and effective agroinoculation method for virus‐induced gene silencing in roots and diverse Solanaceous species

Abstract

SummaryVirus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…The characteristic photobleached phenotype of PDSsilenced plants (Liu et al, 2002) was consistently observed 10 d after infiltration, with phenotypes increasing in severity in plants 3 weeks postinfiltration (Supplemental Figure 4). Similarly, ACTINsilenced plants displayed a stunted phenotype compared with the TRV2 empty vector controls similar to that previously described in Nicotiana benthamiana (Ryu et al, 2004) (Supplemental Figure 4). In addition, VIGS was performed to silence PMT and H6H, which are involved in catalyzing early and late steps in the biosynthesis of scopolamine, respectively Matsuda et al, 1991;Biastoff et al, 2009; Figure 1).…”

Comprehensive Transcriptome Profiling in Tomato Reveals a Role for Glycosyltransferase in Mi-Mediated Nematode Resistance

“…To further investigate the possibility that the glycosyltransferase gene plays a functional role in Mimediated resistance, we used VIGS (Liu et al, 2002a(Liu et al, , 2002bRyu et al, 2004) to down-regulate its expression in susceptible ('Moneymaker') and resistant ('Motelle') tomato plants that were subsequently challenged with M. incognita. Because Mi is known to function only during a narrow temporal window corresponding to the period of GC initiation in a susceptible plant (Dropkin, 1969), we performed a calibration experiment to gauge when effective silencing in roots was occurring.…”

“…Approximately 2 weeks after germination, CaMV35STGFP transformants (verified for GFP expression) were planted into 1:1 soil:sand. Seedlings were agroinoculated by leaf infiltration and agrodrench (Ryu et al, 2004) with appropriate VIGS constructs. Two and 3 weeks after agroinoculation, when GFP fluorescence was no longer detected in 'Rutgers Large Red' CaMV35STGFP roots, all plants were inoculated with 500 to 1,000 M. incognita J2.…”

Airborne Induction and Priming of Plant Defenses against a Bacterial Pathogen

“…RT-PCR (RETROscript; Ambion) analysis was performed according to the manufacturer's instructions. RT-PCR and qRT-PCR were conducted as described previously (Ryu et al, 2004a). To detect expression levels of PR-2, LOX, and Actin genes, adequate primers were obtained from Edington et al (1991;PR-2), Meier et al (1993;LOX), and Maffei et al (2006;Actin) and were as follows: PvPR-2 (forward, 5#-GCCACAAATGCCGACACTGC-3#; reverse, 5#-GGACT-CACTTCATTGCCAACTGC-3#), PvLOX (forward, 5#-GTGAGAGGCGATG-GAAGTGGAG-3#; reverse, 5#-TGCGAGGGTAAGGTAAGGTAGAAC-3#), and PlActin (forward, 5#-AGGCTCCTCTTAACCCCAAG-3#; reverse, 5#-GTGGGAGAGCATAACCCTCA-3#).…”