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

Abstract

One sentence summary: A CGMMV-based vector enables gene function 21 studies in cucurbits, an extremely low efficiency species for genetic 22 42 hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and 43 cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene 44 could persist for over two months and the silencing effect of CGMMV-based vectors 45 could be passaged. These results demonstrate that CGMMV vector could serve as a 46 powerful and easy-to-use tool for characterizing gene function in cucurbits. 47 48 Keywords: Cucumber green mottle mosaic virus; viral vector; virus-induced gene 49 3 silencing; cucurbit plants 50 68 (VIGS) is a good alternative to gene transformation because of its simplicity, high 69 efficiency, and high throughput. 70 Gene silencing comprises transcriptional gene silencing (TGS) and 71 post-transcriptional gene silencing (PTGS). VIGS, a type of PTGS, is a natural 72 defense reaction that exists in a broad range of organisms. It confers resistance to 73 foreign nucleic acid invasion through PTGS at the RNA level. Because it can silence a 74 specific gene, leading to the loss of function of this gene, the potential of VIGS as a 75 tool to analyze gene function has been quickly recognized (Baulcombe, 1999). 76 4 In the past decades, a large number of viral vectors had been developed as 77 powerful tools for the functional verification of genes in plants (Ruiz et al., 1998; Liu 78 et al., 2002; Ding et al., 2006; Igarashi et al., 2009; Zhang et al., 2010; Sempere et al., 79 2011; Liu et al., 2016; Wang et al., 2016). To date, three different RNA viruses have 80 (CP). Only the 129 KDa and 186 KDa of replication-related proteins are translated 100 directly from the genomic RNA, whereas the 29-KDa MP and the 17.4-KDa CP are 101 translated from two subgenomic RNAs. There is an overlap between the MP and CP 102 ORFs (Ugaki et al., 1991). Viral vectors based on CGMMV for expressing foreign 103 genes have been constructed. Multiple cloning sites (MCS) were inserted adjacent to 104 the CP ORF, and the CP stop codon was altered to express the hepatitis B surface 105 antigen and a Dengue virus Epitope so that 20 and 44 foreign amino acids, 106 respectively, were expressed (Ooi et al., 2006; Teoh et al., 2009). Tobacco mosaic 107 virus (TMV), another member of the genus Tobamovirus, has been widely studied as a 108 5 model in this genus. TMV has successfully been developed as a VIGS vector by 109 including an additional duplicated copy of the CP subgenomic promoter (SGP) in the 110 viral genome (Kumagai et al., 1995).The CGMMV genome is similar to that of TMV, 111 and thus it was thought that methods similar to those used for TMV could be used to 112 create vectors based on CGMMV; unfortunately, results in this regard varied largely 113 129 Agricultural Sciences (Xinjiang, China), Tianjin Academy of Agricultural Sciences 130 (Tianjin, China) and Ningbo Academy of Agricultural Sciences (Ningbo, China), 131 respectively. All cucurbit seeds were soaked in ste...

A novel strategy for improving watermelon resistance to cucumber green mottle mosaic virus by exogenous boron application

“…Most of the DEGs identified in this study were found in the 45 DAP samples, suggesting that watermelon resistance to CGMMV infection in the later stage is more complicated. To validate this speculation, we selected 20 DEGs and analysed their roles in watermelon resistance to CGMMV infection using a CGMMV‐based VIGS vector reported previously (Liu et al, 2020). The results of pV190 VIGS assays for watermelon leaf were consistent with those for fruit except for DnaJ11 (Figures S2, 4 and 5).…”

“…Because watermelon is recalcitrant to genetic transformation, analyses of gene functions in this plant are hindered. To circumvent this obstacle, we decided to investigate the molecular mechanisms controlling CGMMV infection in watermelon fruits through reverse genetics using a CGMMV‐based virus‐induced gene silencing (VIGS) pV190 vector as described previously (Lange et al, 2013; Liu et al, 2020). Currently, two VIGS vectors (i.e., the CGMMV‐based pV190 vector and a tobacco rattle virus [TRV]‐based vector) have been reported to be effective in gene silencing in cucurbits (Fang et al, 2021; Liu et al, 2020).…”

“…To circumvent this obstacle, we decided to investigate the molecular mechanisms controlling CGMMV infection in watermelon fruits through reverse genetics using a CGMMV‐based virus‐induced gene silencing (VIGS) pV190 vector as described previously (Lange et al, 2013; Liu et al, 2020). Currently, two VIGS vectors (i.e., the CGMMV‐based pV190 vector and a tobacco rattle virus [TRV]‐based vector) have been reported to be effective in gene silencing in cucurbits (Fang et al, 2021; Liu et al, 2020). In this study, we found that the pV190 vector‐induced gene silencing in watermelon fruits lasted more than 54 days and thus is suitable for studies of CGMMV infection in watermelon fruits and possibly other cucurbit crops.…”

Efficient transformation and genome editing of watermelon assisted by genes that encode developmental regulators

“…Although successful and highly efficient transformation systems have been established in some species, transformation remains the bottleneck in the production of transgenic and genome-edited plants for watermelon as well as many other horticultural species. Transient expression methods, such as virus-induced gene silencing using virus vectors and protoplast-based transient expression assays, have recently been established in watermelon (Igarashi et al, 2009, Tian et al, 2017, Liu et al, 2020, Wang et al, 2021a, and these methods can be applied to study gene functions. However, stable transformation protocols are still the foundation for the genetic engineering of crop traits.…”

Biotechnological Application of Potyvirus-Based Vectors in Cucurbit Plants

“…Advances in transient transformation facilitates rapid and efficient understanding of gene function. One of this transient expression strategies is the so called virus-induced gene silencing (VIGS) (Liu et al, 2019).…”