Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector

Abstract

The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.

Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly

“…Furthermore, the repeatability is not reliable [ 38 ]. For the small-genome viruses, PCR along with ligation or fusion is useful for the infectious clone construction [ 39 , 40 ]. Miciak et al described a high-copy plasmid-based method that permits individual adenoviral genes to be easily manipulated in isolation and then recombined with other mutations or transgenes [ 41 ].…”

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…More recently, co‐transformation of yeast cells with 25 different overlapping fragments allowed correct assembly of a 590‐kb molecule (Gibson et al ., ). This finding highlights the extreme potential of in vivo yeast assembly, which has been used for target mutagenesis of a polerovirus clone (Liang et al ., ), and later to assemble binary infectious clones of members of genera Trichovirus , Potyvirus and Mandarivirus (Cui et al ., ; Sun et al ., ; Youssef et al ., ). Bacteria provide high transformation efficiency, plasmid yields and rapid growth rates, but homologous recombination efficiencies reported in E. coli are orders of magnitude lower than those of yeast.…”

“…These have been linked to toxicity of unwanted viral expression products in bacterial hosts, and several approaches were therefore designed to tackle instability issues. Although inconvenient for routine cloning procedures, assembly chassis that better tolerate toxic, difficult constructs, such as yeast and Agrobacterium, have been used for binary clone assembly (Sun et al, 2017;Tuo et al, 2017;Youssef et al, 2011). Better choices for repeated plasmid manipulations are E. coli strains suitable for cloning unstable DNA, that is, Stbl2 and Stbl4 (ThermoFisher), and SURE2 (Agilent Technologies, Santa Clara, CA).…”

P3N-PIPO Interacts with P3 via the Shared N-Terminal Domain To Recruit Viral Replication Vesicles for Cell-to-Cell Movement

“…The TuMV mutants, e.g., TuMV-GFP/ΔP3N-PIPO, TuMV-6K2mCh/ΔP3N-PIPO, TuMV-6K2mCh/ΔP3N/ ΔPIPO, and TuMV-6K2mCh/ΔP3C, were constructed by yeast homologous recombination using the yeast-Escherichia coli-Agrobacterium shuttle vector pCB301-2-HDV as described previously (55). The polymerase slippage site was mutated to 5=-GAGAAGA-3= in TuMV-GFP/ΔP3N-PIPO and TuMV-6K2mCh/ ΔP3N-PIPO, and the region encoding aa 5 to 152 of the P3N domain in TuMV-6K2mCh/ΔP3N/ΔPIPO was deleted using TuMV-6K2mCh/ΔP3N-PIPO as the template.…”

“…Confocal microscopy. Confocal microscopy analysis was performed as described previously (36,55). A 0.5-by 0.5-cm leaf patch was excised from the infiltrated leaf area and was monitored with a Leica TCS SP8 confocal laser scanning microscope (Leica, Germany) or Nikon A1 HD25 confocal microscope (Nikon, Japan) at the desired time point.…”