A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease

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.

Download Full-text

Faculty Opinions recommendation of High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1003609.201391 ◽

2003 ◽

Author(s):

Jen Sheen

Keyword(s):

Disease Resistance ◽

Heat Shock ◽

Heat Shock Protein ◽

Gene Silencing ◽

High Throughput ◽

Plant Disease Resistance ◽

Plant Disease ◽

Heat Shock Protein 90 ◽

Virus Induced Gene Silencing

Download Full-text

Faculty Opinions recommendation of Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.8177956.8591054 ◽

2011 ◽

Author(s):

Keith Davis ◽

Nobuyuki Matoba

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Virus Induced Gene Silencing

Download Full-text

Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves

BIO-PROTOCOL ◽

10.21769/bioprotoc.1506 ◽

2015 ◽

Vol 5 (12) ◽

Author(s):

Lokanadha Gunupuru ◽

Shahin Ali ◽

Fiona Doohan ◽

Steven Scofield

Keyword(s):

Gene Silencing ◽

Virus Induced Gene Silencing ◽

Barley Seedling

Download Full-text

Cucumber mosaic virus-induced gene silencing in banana

Scientific Reports ◽

10.1038/s41598-019-47962-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Yuh Tzean ◽

Ming-Chi Lee ◽

Hsiao-Hsuan Jan ◽

Yi-Shu Chiu ◽

Tsui-Chin Tu ◽

...

Keyword(s):

Gene Silencing ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Virus Induced Gene Silencing

Download Full-text

Effects of Vector Maturation Time on the Dynamics of Cassava Mosaic Disease

Bulletin of Mathematical Biology ◽

10.1007/s11538-021-00921-4 ◽

2021 ◽

Vol 83 (8) ◽

Author(s):

F. Al Basir ◽

Y. N. Kyrychko ◽

K. B. Blyuss ◽

S. Ray

Keyword(s):

Time Delay ◽

Plant Density ◽

Plant Viruses ◽

Mosaic Disease ◽

Plant Diseases ◽

Cassava Mosaic Disease ◽

Maturation Time ◽

Negative Effect ◽

Dynamical Regimes

AbstractMany plant diseases are caused by plant viruses that are often transmitted to plants by vectors. For instance, the cassava mosaic disease, which is spread by whiteflies, has a significant negative effect on plant growth and development. Since only mature whiteflies can contribute to the spread of the cassava mosaic virus, and the maturation time is non-negligible compared to whitefly lifetime, it is important to consider the effects this maturation time can have on the dynamics. In this paper, we propose a mathematical model for dynamics of cassava mosaic disease that includes immature and mature vectors and explicitly includes a time delay representing vector maturation time. A special feature of our plant epidemic model is that vector recruitment is negatively related to the delayed ratio between vector density and plant density. We identify conditions of biological feasibility and stability of different steady states in terms of system parameters and the time delay. Numerical stability analyses and simulations are performed to explore the role of various parameters, and to illustrate the behaviour of the model in different dynamical regimes. We show that the maturation delay may stabilise epidemiological dynamics that would otherwise be cyclic.

Download Full-text

A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Plant Methods ◽

10.1186/s13007-021-00775-w ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Decai Tuo ◽

Peng Zhou ◽

Pu Yan ◽

Hongguang Cui ◽

Yang Liu ◽

...

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Gene Function ◽

Viral Vector ◽

Function Analysis ◽

Systemic Infection ◽

Cdna Clones ◽

Virus Induced Gene Silencing ◽

Efficient Gene ◽

Gene Function Analysis

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Download Full-text

Virus-induced gene silencing as a tool for analysis of gene functions in plants

Uirusu ◽

10.2222/jsv.60.155 ◽

2010 ◽

Vol 60 (2) ◽

pp. 155-162 ◽

Cited By ~ 2

Author(s):

Noriko YAMAGISHI ◽

Nobuyuki YOSHIKAWA

Keyword(s):

Gene Silencing ◽

Virus Induced Gene Silencing ◽

Gene Functions

Download Full-text

Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

Virology Journal ◽

10.1186/s12985-021-01572-6 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Saengsoon Charoenvilaisiri ◽

Channarong Seepiban ◽

Mallika Kumpoosiri ◽

Sombat Rukpratanporn ◽

Nuchnard Warin ◽

...

Keyword(s):

Mosaic Virus ◽

Immunosorbent Assay ◽

Mosaic Disease ◽

Cassava Mosaic Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Sri Lankan ◽

Stem Cuttings ◽

Field Samples ◽

Cassava Stem ◽

Cassava Mosaic Virus

Abstract Background Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). Methods Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. Results A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. Conclusions Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Download Full-text