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

Abstract

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.

Molecular Characterization and Pathogenicity of an Infectious cDNA Clone of Youcai Mosaic Virus on Solanum nigrum

“…Then, the purified two fragments and the linearized pCA4Y vector were co-transformed into the yeast strain YPH500, and the positive yeast colonies were screened via colony PCR. Due to their existing 25 bp or longer overlapping sequences between these three fragments (frag1, frag2, and linearized vector), the two full-length YoMV cDNA sequence was easy to obtain using a yeast recombination system [31] (Figure 3D). Positive pCA4Y-YoMV-YZ plasmid was obtained and transformed into Agrobacterium EHA105 strain.…”

“…Purified Frag.1, Frag.2, and Stu I/BamH I-digested pCA4Y vector were mixed in vitro in a 2:2:1 molar ratio, and then transformed into yeast YPH500 strain as described previously [31]. Subsequently, a positive cDNA clone of full-length YoMV genomic RNA was obtained [31]. We provide a list of all pairs of primers used in the Supplementary Materials, Table S2.…”

“…The upper symptomatic leaves of N. benthamiana and S. nigrum were collected, then snap-frozen, and crushed in liquid nitrogen. Equal volume of 2 X SDS protein loading buffer was added to the powder as previously described [31]. Protein samples were subjected to electrophoresis in 12% SDS-polyacrylamide gel (SDS-PAGE) at voltage 120 V, then all proteins on the gel were transferred to a piece of nitrocellulose membrane (Cat.…”