Abstract
BackgroundLupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV).ResultsWe identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)—a gene likely to be involved in toxic alkaloid biosynthesis—we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a ‘PDS co-silencing’ approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL.ConclusionsWe provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.
Highlights
Lupins are promising protein crops with an increasing amount of genomic and transcriptomic resources
We measured the transcript level of L. angustifolius copper amine oxidase (LaCAO) and, while average levels were higher in plants infected with apple latent spherical virus (ALSV)-LDC compared to control plants, the difference was not significant (Fig. 4B). These results provide direct evidence that L. angustifolius lysine decarboxylase (LaLDC) is a major gene involved in quinolizidine alkaloids (QAs) biosynthesis in narrow-leafed lupin (NLL)
We have demonstrated the applicability of our virus-induced gene silencing (VIGS) method to silence a target gene alone or in a ‘phytoene desaturase (PDS) co-silencing’ approach
Summary
Lupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. Lupins (Lupinus spp.) are nitrogen-fixing legumes that can grow on poor soils and accumulate high levels of protein in their seeds (up to ~ 40%) [1]. They have great potential as sustainable, alternative protein crops to meet increasing global demands for dietary protein [2]. The fourth cultivated species is the Andean lupin (L. mutabilis), which is native to South America and is recognized for its high accumulation of seed oil. Among these four species, NLL is the most widely cultivated and is grown commercially in both Australia and Europe
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