Construction of full-length cDNA infectious clones of Chilli veinal mottle virus

Peng, Qiao; Yang, Daoyong; Yang, Ting; Cheng, Yongchao; Yang, Yufan; Xi, Dehui

doi:10.1016/j.virusres.2022.198948

Cited by 5 publications

(7 citation statements)

References 42 publications

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“…Multiple DNA fragments, with 20–30 bp homologous end flanking, could prime the scarless assembly in yeast using the endogenous repair machinery. Various studies have demonstrated the efficiency and capacity of HR-based cloning in the construction of infectious cDNA clones of plant plus-strand RNA viruses [ 7 , 8 , 9 , 24 , 25 , 26 , 27 ].…”

Section: Discussionmentioning

confidence: 99%

“…Using the yeast HR-based cloning method, Sun et al (2017) constructed an intronless PVY cDNA clone that could be obtained by rapid assembly and is highly infectious [ 4 ]. Similarly, highly infectious cDNA clones of the Sonchus yellow net virus , Soybean mosaic virus , and Chili veinal mottle virus were successfully constructed [ 4 , 8 , 9 ]. Hence, the yeast HR-based cloning method can be used to construct intronless plant virus cDNA clones.…”

Section: Discussionmentioning

confidence: 99%

“…Obtaining infectious clones of many plant viruses using traditional methods is difficult because of the large viral genomic RNA/DNA size, unexpected deletions, and toxicity to E. coli growth [ 5 , 6 , 7 ]. In contrast, homologous recombination (HR)-based cloning depends on yeast ( Saccharomyces cerevisiae ) and is a highly efficient and cost-effective method for the seamless assembly of DNA fragments, especially in potyvirus infectious clone construction [ 7 , 8 ]. Taking advantage of HR in yeast cells, which is referred to as gap-repair cloning, could achieve multiple DNA fragment assembly with 20–30 bp homologous ends [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

“…Owing to its high transformation efficiency, simplicity, and capacity for complex DNA assembly, HR in yeast has been used in high-throughput plasmid construction, genome synthesis, and genome engineering [ 4 ]. HR-based cloning in yeast has already been successfully applied to the construction of several plant RNA virus infectious clones for agrobacteria-based infiltration [ 8 , 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

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Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction

Feng

Guo

et al. 2023

IJMS

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Infectious cloning of plant viruses is a powerful tool for studying the reverse genetic manipulation of viral genes in virus–host plant interactions, contributing to a deeper understanding of the life history and pathogenesis of viruses. Yet, most of the infectious clones of RNA virus constructed in E. coli are unstable and toxic. Therefore, we modified the binary vector pCass4-Rz and constructed the ternary shuttle vector pCA4Y. The pCA4Y vector has a higher copy number in the E. coli than the conventional pCB301 vector, can obtain a high concentration of plasmid, and is economical and practical, so it is suitable for the construction of plant virus infectious clones in basic laboratories. The constructed vector can be directly extracted from yeast and transformed into Agrobacterium tumefaciens to avoid toxicity in E. coli. Taking advantage of the pCA4Y vector, we established a detailed large and multiple DNA HR-based cloning method in yeast using endogenous recombinase. We successfully constructed the Agrobacterium-based infectious cDNA clone of ReMV. This study provides a new choice for the construction of infectious viral clones.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction

Feng

Guo

et al. 2023

IJMS

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“…Chilli veinal mottle virus belongs to the genus Potyvirus of the family Potyviridae, which encodes one polyprotein and hydrolyzes into 11 mature viral proteins, including 6K2, NIb, HC-Pro, Vpg, etc. The membrane-bound protein 6K2 can generate the formation of endoplasmic reticulum (ER)-derived vesicles for intercellular movement of potyviral replication complexes (VRCs) and viral RNA replication [29][30][31], and it is critical for intercellular movement and systemic infection of potyviruses. NIb is a viral RNA-dependent RNA polymerase (RdRp).…”

Section: Introductionmentioning

confidence: 99%

ATG8f Interacts with Chilli Veinal Mottle Virus 6K2 Protein to Limit Virus Infection

Ji,

Zhou,

Yang

et al. 2023

Viruses

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Autophagy, as a conserved protein degradation pathway in plants, has also been reported to be intricately associated with antiviral defense mechanisms. However, the relationship between chilli veinal mottle virus (ChiVMV) and autophagy has not been investigated in the existing research. Here, we reveal that ChiVMV infection caused the accumulation of autophagosomes in infected Nicotiana benthamiana leaves and the upregulation of autophagy-related genes (ATGs). Moreover, the changes in gene expression were correlated with the development of symptoms. Treatment with autophagy inhibitors (3-MA or E-64D) could increase the infection sites and facilitate virus infection, whereas treatment with the autophagy activator (Rapamycin) limited virus infection. Then, ATG8f was identified to interact with ChiVMV 6K2 protein directly in vitro and in vivo. The silencing of ATG8f promoted virus infection, whereas the overexpression of ATG8f inhibited virus infection. Furthermore, the expression of 6K2-GFP in ATG8f- or ATG7-silenced plants was significantly higher than that in control plants. Rapamycin treatment reduced the accumulation of 6K2-GFP in plant cells, whereas treatment with the inhibitor of the ubiquitin pathway (MG132), 3-MA, or E-64D displayed little impact on the accumulation of 6K2-GFP. Thus, our results demonstrated that ATG8f interacts with the ChiVMV 6K2 protein, promoting the degradation of 6K2 through the autophagy pathway.

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MiR827 positively regulates the resistance to chilli veinal mottle virus by affecting the expression of FBPase in Nicotiana benthamiana

Tang,

Yang,

et al. 2024

Physiologia Plantarum

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MicroRNA(miRNA) is a class of non‐coding small RNA that plays an important role in plant growth, development, and response to environmental stresses. Unlike most miRNAs, which usually target homologous genes across a variety of species, miR827 targets different types of genes in different species. Research on miR827 mainly focuses on its role in regulating phosphate (Pi) homeostasis of plants, however, little is known about its function in plant response to virus infection. In the present study, miR827 was significantly upregulated in the recovery tissue of virus‐infected Nicotiana tabacum. Overexpression of miR827 could improve plants resistance to the infection of chilli veinal mottle virus (ChiVMV) in Nicotiana benthamiana, whereas interference of miR827 increased the susceptibility of the virus‐infected plants. Further experiments indicated that the antiviral defence regulated by miR827 was associated with the reactive oxygen species and salicylic acid signalling pathways. Then, fructose‐1,6‐bisphosphatase (FBPase) was identified to be a target of miR827, and virus infection could affect the expression of FBPase. Finally, transient expression of FBPase increased the susceptibility to ChiVMV‐GFP infection in N. benthamiana. By contrast, silencing of FBPase increased plant resistance. Taken together, our results demonstrate that miR827 plays a positive role in tobacco response to virus infection, thus providing new insights into understanding the role of miR827 in plant‐virus interaction.

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Construction of full-length cDNA infectious clones of Chilli veinal mottle virus

Cited by 5 publications

References 42 publications

Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction

Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction

ATG8f Interacts with Chilli Veinal Mottle Virus 6K2 Protein to Limit Virus Infection

MiR827 positively regulates the resistance to chilli veinal mottle virus by affecting the expression of FBPase in Nicotiana benthamiana

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