Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin

Background: Lupins 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). Results: We 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 similar to 61% or similar to 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. Conclusions: We provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

Automated summary

By utilizing the apple latent spherical virus (ALSV) for virus-induced gene silencing (VIGS), an efficient method has been established to validate gene function in narrow-leafed lupins (NLL). This approach can be used to silence target genes alone or simultaneously, resulting in decreased levels of toxic alkaloid biosynthesis, providing direct evidence for research and improvement of this underutilized crop.