Mancinotti Davide, Rodriguez Maria Cecilia, Frick Karen Michiko, Dueholm Bjørn, Jepsen Ditte Goldschmidt, Agerbirk Niels, Geu-Flores Fernando
Section for Plant Biochemistry and Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.
Plant Methods. 2021 Dec 28;17(1):131. doi: 10.1186/s13007-021-00832-4.
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).
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 ~ 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.
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.
羽扇豆是很有前景的蛋白作物，拥有越来越多的基因组和转录组资源。这些新资源有助于在计算机上鉴定控制重要农艺性状的候选基因。然而，羽扇豆研究和作物改良的一个主要瓶颈是基因功能的植物体内表征。在此，我们展示了一种利用苹果潜隐球形病毒（ALSV）在窄叶羽扇豆（NLL）中下调内源基因的病毒诱导基因沉默（VIGS）高效方案。
我们鉴定出ALSV是一种合适的VIGS载体，能够感染NLL且不引起明显表型。我们构建了改良的ALSV载体，以便将基因片段高效克隆到病毒基因组中，并通过农杆菌浸润本氏烟草更轻松地进行病毒繁殖。利用该系统，我们沉默了视觉标记基因八氢番茄红素去饱和酶（PDS），新产生组织的白化表明其导致了系统性、均匀性沉默。此外，通过沉默赖氨酸脱羧酶（LaLDC）——一个可能参与有毒生物碱生物合成的基因——我们证明了我们的VIGS方法适用于以“PDS共沉默”方法单独或与PDS一起沉默靶基因。共沉默方法允许直观鉴定正在发生沉默的组织，这便于组织收获和下游分析，并且在研究的性状不受PDS沉默影响时很有用。沉默LaLDC导致转录水平下降约61%或约67%，这取决于LaLDC是单独沉默还是与PDS一起沉默。总体而言，LaLDC的沉默导致生物碱水平降低，直接证明了其参与NLL中生物碱的生物合成。
我们提供了一种快速有效的VIGS方法来验证NLL中的基因功能。这将加速这种未充分利用作物的研究和改良。
