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

Content Provider	Springer Nature : BioMed Central
Author	Mancinotti, Davide Rodriguez, Maria Cecilia Frick, Karen Michiko Dueholm, Bjørn Jepsen, Ditte Goldschmidt Agerbirk, Niels Geu-Flores, Fernando
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.
Related Links	https://plantmethods.biomedcentral.com/counter/pdf/10.1186/s13007-021-00832-4.pdf
Ending Page	13
Page Count	13
Starting Page	1
File Format	HTM / HTML
ISSN	17464811
DOI	10.1186/s13007-021-00832-4
Journal	Plant Methods
Issue Number	1
Volume Number	17
Language	English
Publisher	BioMed Central
Publisher Date	2021-12-28
Access Restriction	Open
Subject Keyword	Plant Sciences Biological Techniques Lupinus angustifolius Grain legume VIGS Pulse Apple latent spherical virus Quinolizidine alkaloids
Content Type	Text
Resource Type	Article
Subject	Plant Science Biotechnology Genetics
Journal Impact Factor	4.7/2023
5-Year Journal Impact Factor	5.6/2023

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Quinolizidine alkaloids are transported to seeds of bitter narrow-leafed lupin

Quinolizidine Alkaloid Biosynthesis in Lupins and Prospects for Grain Quality Improvement

Quantitative and qualitative content of alkaloids in seeds of a narrow-leafed lupin (Lupinus angustifolius L.) collection

Transcriptome-derived investigation of biosynthesis of quinolizidine alkaloids in narrow-leafed lupin (Lupinus angustifolius L.) highlights candidate genes linked to iucundus locus.

Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract

A pan-genome and chromosome-length reference genome of narrow-leafed lupin (Lupinus angustifolius) reveals genomic diversity and insights into key industry and biological traits.

Quantitative and qualitative content of alkaloids in seeds of a narrow-leafed lupin (Lupinus angustifolius L.) collection

Identification and profiling of narrow-leafed lupin (Lupinus angustifolius) microRNAs during seed development

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

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

Similar Documents

Quinolizidine alkaloids are transported to seeds of bitter narrow-leafed lupin

Quinolizidine Alkaloid Biosynthesis in Lupins and Prospects for Grain Quality Improvement

Quantitative and qualitative content of alkaloids in seeds of a narrow-leafed lupin (Lupinus angustifolius L.) collection

Transcriptome-derived investigation of biosynthesis of quinolizidine alkaloids in narrow-leafed lupin (Lupinus angustifolius L.) highlights candidate genes linked to iucundus locus.

Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract

A pan-genome and chromosome-length reference genome of narrow-leafed lupin (Lupinus angustifolius) reveals genomic diversity and insights into key industry and biological traits.

Quantitative and qualitative content of alkaloids in seeds of a narrow-leafed lupin (Lupinus angustifolius L.) collection

Identification and profiling of narrow-leafed lupin (Lupinus angustifolius) microRNAs during seed development

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

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