Virus-Mediated Transient Expression Techniques Enable Functional Genomics Studies and Modulations of Betalain Biosynthesis and Plant Height in Quinoa

Quinoa (Chenopodium quinoa), native to the Andean region of South America, has been recognized as a potentially important crop in terms of global food and nutrition security since it can thrive in harsh environments and has an excellent nutritional profile. Even though challenges of analyzing the complex and heterogeneous allotetraploid genome of quinoa have recently been overcome, with the whole genome-sequencing of quinoa and the creation of genotyped inbred lines, the lack of technology to analyze gene function in planta is a major limiting factor in quinoa research. Here, we demonstrate that two virus-mediated transient expression techniques, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), can be used in quinoa. We show that apple latent spherical virus (ALSV) can induce gene silencing of quinoa phytoene desaturase (CqPDS1) in a broad range of quinoa inbred lines derived from the northern and southern highland and lowland sub-populations. In addition, we show that ALSV can be used as a VOX vector in roots. Our data also indicate that silencing a quinoa 3,4-dihydroxyphenylalanine 4,5-dioxygenase gene (CqDODA1) or a cytochrome P450 enzyme gene (CqCYP76AD1) inhibits betalain production and that knockdown of a reduced-height gene homolog (CqRHT1) causes an overgrowth phenotype in quinoa. Moreover, we show that ALSV can be transmitted to the progeny of quinoa plants. Thus, our findings enable functional genomics in quinoa, ushering in a new era of quinoa research.

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Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava

International Journal of Molecular Sciences ◽

10.3390/ijms20163976 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3976 ◽

Cited By ~ 6

Author(s):

Hongqiu Zeng ◽

Yanwei Xie ◽

Guoyin Liu ◽

Yunxie Wei ◽

Wei Hu ◽

...

Keyword(s):

Gene Silencing ◽

Functional Genomics ◽

Transient Expression ◽

Fluorescent Protein ◽

Tobacco Rattle Virus ◽

Mosaic Disease ◽

African Cassava Mosaic Virus ◽

Cassava Mosaic Disease ◽

Virus Induced Gene Silencing ◽

Transient Expression Assay

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

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VIGS technology: an attractive tool for functional genomics studies in legumes

Functional Plant Biology ◽

10.1071/fp13089 ◽

2013 ◽

Vol 40 (12) ◽

pp. 1234 ◽

Cited By ~ 25

Author(s):

Stéphanie Pflieger ◽

Manon M. S. Richard ◽

Sophie Blanchet ◽

Chouaib Meziadi ◽

Valérie Geffroy

Keyword(s):

Gene Silencing ◽

Functional Genomics ◽

Target Gene ◽

Reverse Genetics ◽

Plant Viruses ◽

Transcriptional Gene Silencing ◽

Full Potential ◽

Legume Species ◽

Virus Induced Gene Silencing ◽

Apple Latent Spherical Virus

Legume species are among the most important crops worldwide. In recent years, six legume genomes have been completely sequenced, and there is now an urgent need for reverse-genetics tools to validate genes affecting yield and product quality. As most legumes are recalcitrant to stable genetic transformation, virus-induced gene silencing (VIGS) appears to be a powerful alternative technology for determining the function of unknown genes. VIGS technology is based on the property of plant viruses to trigger a defence mechanism related to post-transcriptional gene silencing (PTGS). Infection by a recombinant virus carrying a fragment of a plant target gene will induce homology-dependent silencing of the endogenous target gene. Several VIGS systems have been developed for legume species since 2004, including those based on Bean pod mottle virus, Pea early browning virus, and Apple latent spherical virus, and used in reverse-genetics studies of a wide variety of plant biological processes. In this work, we give an overview of the VIGS systems available for legumes, and present their successful applications in functional genomics studies. We also discuss the limitations of these VIGS systems and the future challenges to be faced in order to use VIGS to its full potential in legume species.

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

Plant Methods ◽

10.1186/s13007-021-00832-4 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Davide Mancinotti ◽

Maria Cecilia Rodriguez ◽

Karen Michiko Frick ◽

Bjørn Dueholm ◽

Ditte Goldschmidt Jepsen ◽

...

Keyword(s):

Gene Silencing ◽

Gene Function ◽

Agronomic Traits ◽

Crop Improvement ◽

Marker Gene ◽

Transcript Level ◽

Alkaloid Biosynthesis ◽

Virus Induced Gene Silencing ◽

In Planta ◽

Apple Latent Spherical Virus

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.

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