Virus-induced gene silencing of pectin methylesterase protects Nicotiana benthamiana from lethal symptoms caused by Tobacco mosaic virus

2014 ◽  
Vol 141 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Giovanni Bubici ◽  
Anna Vittoria Carluccio ◽  
Fabrizio Cillo ◽  
Livia Stavolone
Keyword(s):  
Gene Silencing ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Pectin Methylesterase ◽  
Virus Induced Gene Silencing

scholarly journals A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

2019 ◽  
Author(s):  
Mei Liu ◽  
Zhiling Liang ◽  
Miguel A. Aranda ◽  
Ni Hong ◽  
Liming Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Nicotiana Benthamiana ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Economic Significance ◽  
Potential Alternative ◽  
Low Efficiency

AbstractCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiencies. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits. Here, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69-300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged. These results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function in cucurbits.One sentence summaryA CGMMV-based vector enables gene function studies in cucurbits, an extremely low efficiency species for genetic transformation.

scholarly journals Tobacco mosaic virus 126-kDa Protein Increases the Susceptibility of Nicotiana tabacum to Other Viruses and Its Dosage Affects Virus-Induced Gene Silencing

2008 ◽  
Vol 21 (12) ◽  
pp. 1539-1548 ◽  
Author(s):  
Phillip A. Harries ◽  
Karuppaiah Palanichelvam ◽  
Sumana Bhat ◽  
Richard S. Nelson
Keyword(s):  
Nicotiana Tabacum ◽  
Gene Silencing ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Brome Mosaic Virus ◽  
Virus Induced Gene Silencing ◽  
Suppressor Activity

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein–green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim

2013 ◽  
Vol 35 (5) ◽  
pp. 811-823 ◽  
Author(s):  
Hernan J. Villanueva-Alonzo ◽  
Rosa Y. Us-Camas ◽  
Luisa A. López-Ochoa ◽  
Dominique Robertson ◽  
Orlene Guerra-Peraza ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Capsicum Annuum ◽  
Nicotiana Benthamiana ◽  
Yucatan Peninsula ◽  
Virus Induced Gene Silencing ◽  
Yucatán Peninsula

scholarly journals Cucumber mosaic virus-induced gene silencing in banana

2019 ◽  
Vol 9 (1) ◽  
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

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

Plant Methods ◽  
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.

scholarly journals Identification and Verification of Genes Related to Pollen Development and Male Sterility Induced by High Temperature in Thermo-sensitive Genic Male Sterile Wheat Line YanZhan 4110S

2020 ◽  
Author(s):  
Xuetong Yang ◽  
Jiali Ye ◽  
Fuqiang Niu ◽  
Yi Feng ◽  
Xiyue Song
Keyword(s):  
Gene Silencing ◽  
Male Sterility ◽  
Mosaic Virus ◽  
Pollen Development ◽  
Bioinformatics Analysis ◽  
Virus Induced Gene Silencing ◽  
Wheat Line ◽  
Pollen Abortion ◽  
Hub Genes ◽  
Genic Male Sterility

Abstract Background: Environment-sensitive genic male sterility is of vital importance to hybrid vigor in crop production and breeding, therefore, it is meaningful to identify and study the function of the genes related to pollen development and male sterility, which still not fully understanding currently. In this study, Yanzhan 4110S, a new thermo-sensitive genic male sterility (TGMS) wheat line, and its near isogenic line Yanzhan 4110 were carried out cytological features observation, bioinformatics analysis to investgate the abortion state and identified the genes involved in pollen development which have fertility regulation function. Barely stripe mosaic virus-induced gene silencing was used to verify the genes function.Results: Cytological analysis showed pollen abortion event of Yanzhan 4110S occur at the later uninucleate stage (Lun) under higher temperature induction (day/night temperatures of 22 °C/20 °C), when the anthers were collected and assessed for transcriptomic profiling through high-throughput sequencing. We then in-depth analyzed the differentially expressed genes (DEGs) by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, the results showed that the occurrence of Yanzhan 4110S male-sterility most likely related to metabolic pathway, including phenylpropanoid biosynthesis in the biosynthesis of other secondary metabolites, starch and sucrose metabolism in carbohydrate metabolism, carbon fixation in photosynthetic organisms as well as carbon metabolism in energy metabolism. The weighted gene co-expression network analysis in the transcriptome profiles further identified some hub genes, where the key genes involved in those pathways were intersection between the unique DEGs of Yanzhan 4110S in anther and hub genes, totally 228 genes, which were highly related to pollen development including TaMut11 and TaSF3. Moreover, further verification through barely stripe mosaic virus-induced gene silencing elucidated that the silencing of TaMut11 and TaSF3 caused pollen abortion, finally resulting in the declination of fertility. So, the genes TaMut11 and TaSF3 are related to fertility conversion of Yanzhan 4110S.Conclusion: Through comparative transcriptome bioinformatics analysis, the genes TaMut11 and TaSF3 associated with pollen development and male sterility induced by high temperature were identified in Yanzhan 4110S, and verificated by barely stripe mosaic virus-induced gene silencing. These findings provided researching the abortive mechanism in environment-sensitive genic male sterility wheat.

scholarly journals Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

2020 ◽  
Vol 21 (4) ◽  
pp. 1414 ◽  
Author(s):  
Hui Li ◽  
Xiaobao Ying ◽  
Lina Shang ◽  
Bryce Redfern ◽  
Nicholas Kypraios ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Candidatus Liberibacter ◽  
Phloem Necrosis ◽  
Liberibacter Asiaticus ◽  
Green Fluorescent

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

scholarly journals Stability of Barley stripe mosaic virus–Induced Gene Silencing in Barley

2007 ◽  
Vol 20 (11) ◽  
pp. 1323-1331 ◽  
Author(s):  
Marianne Bruun-Rasmussen ◽  
Christian Toft Madsen ◽  
Stine Jessing ◽  
Merete Albrechtsen
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Barley Stripe Mosaic Virus ◽  
Mrna Levels ◽  
Virus Induced Gene Silencing ◽  
Qrt Pcr ◽  
Transient Nature ◽  
Polymerase Chain ◽  
Insert Length ◽  
Pds Gene

Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting photobleaching in infected barley plants was used as a reporter for silencing. In addition, downregulation of PDS mRNA was measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Using fragments of PDS ranging from 128 to 584 nucleotides in BSMV, we observed that insert length influenced stability but not efficiency of VIGS. Silencing was transient in most cases; however, the decrease in PDS mRNA levels measured by qRT-PCR began earlier and lasted longer than the photobleaching. Occasionally, silencing persisted and could be transmitted through seed as well as via mechanical inoculation, although large parts of the insert had been lost from the virus vector. The instability of the insert, observed consistently throughout our experiments, offers an explanation for the transient nature of silencing when using BSMV as a VIGS vector.

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