Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek

Abstract

Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene functions. However, while VIGS has been utilized successfully in the diploid model leguminous species Medicago truncatula (Gaertn.) (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10–15 d through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek. The introduction of a highly homologous gene fragment from a heterologous plant species to target endogenous genes for transient VIGS-based silencing in a range of species of interest represents a potentially useful strategy for the rapid functional characterization of candidate genes in forages.

Downregulation of key genes involved in carbon metabolism in Medicago truncatula results in increased lipid accumulation in vegetative tissue

“…Agrobacterium ‐mediated infiltration of leaves and meristems was used to deliver MtSDP1 , MtAPS1 , MtTGD5 , or MtPXA1 VIGS vectors, as well as an empty vector control, into M. truncatula PI 3077453. Plants containing a 490‐bp fragment of the pCAPE‐USER viral vector sequence (Wijekoon et al., 2019) were considered positive for successful infiltration. The success rate was about 80–90% (e.g., eight to nine plants containing pCAPE‐USER viral vector out of 10 infiltrated plants).…”

“…The target genes we used in this study are likely to exist as low copy genes in forage legumes assuming all the sequences silenced were homologs or paralogs of the gene target in the same species. Orthologous genes from far-related plant species were previously used in heterologous gene silencing (Senthil-Kumar et al 2007;Wijekoon et al, 2019a). A highly homologous PDS gene fragment from field pea (Constantin et al, 2004) effectively silenced PDS genes in two perennial forage legumes (alfalfa and sainfoin) and an annual forage legume (fenugreek) by VIGS (Wijekoon et al, 2019a).…”

“…Orthologous genes from far-related plant species were previously used in heterologous gene silencing (Senthil-Kumar et al 2007;Wijekoon et al, 2019a). A highly homologous PDS gene fragment from field pea (Constantin et al, 2004) effectively silenced PDS genes in two perennial forage legumes (alfalfa and sainfoin) and an annual forage legume (fenugreek) by VIGS (Wijekoon et al, 2019a). Therefore, this approach can be extended and has a potentail to launch in the same forage legumes in order to modulate the lipid content in leaves using MtSPD1, MtAPS1, MtPXA1 and MtTGD5.…”

“…In every case, the leaves harvested for molecular analyses had developed subsequent to infiltration to avoid the possible presence of contaminating Agrobacterium . Complementary DNA was assessed for the presence of transcribed PEBV vector/coat protein sequence and the constitutive tubulin transcript (XM_013601854) using Platinum SuperFi Green PCR Master Mix and primers US2F, US2R, MTtubulinf, and MTtubulinr, respectively, as described previously (Wijekoon et al., 2019; Table 1). A thermocycling regime of 98 °C for 30 s followed by 30 cycles of 98 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min each was used.…”

“…The fatty acid composition of total acyl lipids was analysed by gas chromatography (GC)-mass spectrometry (MS). Total lipids were extracted from mature M. truncatula shoots (containing leaves and stems) from three to five plants (three technical replicates of each) exhibiting down-regulation of each target gene, respectively, as well as empty vector controls, as described previously (Singer et al 2016;Wijekoon et al 2019a) with slight modifications. In brief, powdered, oven-dried tissues (100 mg) were transmethylated using 3N methanolic HCl at 80ºC for 2hrs.…”