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

Abstract

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 ~ 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. 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.

Biosynthesis of quinolizidine alkaloids in lupins: mechanistic considerations and prospects for pathway elucidation

“…Most notably, gene downregulation in L. angustifolius is now possible using a recently published virus-induced gene silencing (VIGS) method. 79 Downregulation of LDC using this method resulted in a marked decrease in QA accumulation in leaves. Stable transformation protocols are also available for L. angustifolius, L. luteus, and L. mutabilis; [80][81][82] however, transformation efficiencies are low.…”

The causal mutation leading to sweetness in modern white lupin cultivars

“…The samples were analyzed by LC-MS using LC method 2 (1-μl injections). The identity of the alkaloids was inferred from their m / z and MS 2 fragmentation spectrum ( 22 ). For relative quantification, QA peak areas were normalized by the area of the caffeine peak and by the weight of the sample ( n = 6 for each genotype).…”

The aerial epidermis is a major site of quinolizidine alkaloid biosynthesis in narrow-leafed lupin

“…The UHPLC was coupled to a Compact micrOTOF-Q mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive mode. Mass spectrometer conditions were as described before (Otterbach et al, 2019; Mancinotti et al, 2021).…”

“…Imaging was performed in positive ion mode using a scan range of m/z 150-1050. QAs were identified based on the accurate m/z ratios of their protonated forms in accordance to previous assignment in NLL plant organs using LC-MS (Otterbach et al, 2019;Mancinotti et al, 2021). Phosphatidylcholine (34:2)-a component of cell membranes-was chosen as a control metabolite for imaging, which could be visualized across the whole area of the tissue sections (data not shown).…”

“…RNA was extracted from ~30 mg of abaxial epidermis or the remaining leaf tissue using the Spectrum Plant Total RNA kit (Sigma-Aldrich) including on-column DNase I digestion (Sigma-Aldrich). cDNA synthesis and qPCR to determine the expression of LDC and CAO was carried out as described previously (Mancinotti et al, 2021).…”