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

Abstract

White lupin (Lupinus albus L.) plants develop cluster roots and have strong resistance to phosphorus starvation. Although many expressed sequences have been identified to explain the mechanisms used by white lupin to acquire phosphorus, the lack of a stable transformation technique has made it challenging to evaluate the functions of these expressed sequences. Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are difficult to stably transform. Here, we show that the Peanut stunt virus (PSV) vector effectively induces silencing of endogenous genes in white lupin. It is unknown whether PSV is useful for VIGS; therefore, we first inoculated Nicotiana benthamiana plants with PSV harbouring fragments of the N. benthamiana phytoene desaturase gene (NbPDS). Two out of four distinct sequence fragments of NbPDS induced photo-bleaching in N. benthamiana, indicating that PSV can be used to knockdown endogenous gene sequences in a sequence-dependent manner. White lupin plants inoculated with PSV harbouring fragments of the L. albus PDS gene (LaPDS) developed photo-bleaching that was associated with a significant reduction in LaPDS mRNA accumulation. PSV spread systemically in leaves, roots, and cluster roots, and small interfering RNA of LaPDS was detected in these organs. This is the first study to demonstrate the use of VIGS by PSV, suggesting that this vector can be applied to supress endogenous gene expression in shoots and roots of white lupin and to clarify the mechanisms of phosphorus starvation resistance.

Quinolizidine Alkaloid Biosynthesis in Lupins and Prospects for Grain Quality Improvement

“…While transformation efficiencies are low due to low survival and chimeric nature of T 0 plants, this method has been successful in generating transgenic NLL ( Molvig et al, 1997 ; Pigeaire et al, 1997 ; Wijayanto et al, 2009 ; Tabe et al, 2010 ; Atkins et al, 2011 ; Barker et al, 2016 ), L. luteus ( Li et al, 2000 ; Pniewski et al, 2006 ), and L. mutabilis ( Babaoglu et al, 2000 ; Polowick et al, 2014 ) to confer various traits, with recent modifications improving this transformation method for NLL ( Nguyen et al, 2016a , b ). For L. albus , however, A. tumefaciens -mediated transformation has been unsuccessful and as such hairy root transformation using A. rhizogenes ( Uhde-Stone et al, 2005 ; Sbabou et al, 2010 ; Cheng et al, 2011 ) and VIGS using the Peanut stunt virus vector ( Yamagishi et al, 2015 ) have been used to study gene function in this species. Metabolite profiling is an additional resource that could be enhanced in lupins to provide a valuable understanding of how the QA pathway interacts with other metabolic pathways in the plant, especially under abiotic and biotic stresses.…”

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS

“…Using Potato virus X as a silencing vector in dicotyledonous species, inserts were lost with each subsequent passage to an uninfected Nicotiana benthamiana plant (Avesani et al, 2007). A Peanut stunt virus vector also displayed greater instability for maintaining larger PDS inserts (Yamagishi et al, 2015). Recently, Mei et al (2016) demonstrated a good correlation between the loss of a PDS fragment insert in a Foxtail mosaic virus silencing vector and the loss of post-transcriptional silencing.…”

Virus-induced gene silencing (VIGS) in <i>Lilium leichtlinii</i> using the <i>Cucumber mosaic virus</i> vector

“…In viral vector systems, constraints on the size of insert fragments have often been reported, where larger inserts are more susceptible to being lost from the vectors (Hsieh et al 2013;Senthil-Kumar and Mysore 2011a;Yamagishi et al 2015). In the present study, deletion of the 33-nt LlPDS fragment in the CMV-HL from the P33-1 plant was not detected in the first year, but was detected in the following year.…”

“…Short interfering RNAs (siRNAs) were transcribed into cDNA using the stemloop pulsed RT protocol, and then, end-point PCR was conducted to amplify 60-nt fragments that included the siRNA sequences (Varkonyi-Gasic et al 2007;Yamagishi et al 2015) using primers shown in Supplementary Table S2. The primers were designed based on siRNA sequences that were predicted using the algorithm of Ui-Tei et al (2004) and Reynolds et al (2004); e.g., A or U at the 5′ terminal end of the guide strand, G or C at the 5′ terminal end of the passenger strand, and base preferences at positions 3 (A), 10 (U), and 13 (A, U, or C) of the passenger strand.…”