Development of Infectious cDNA Clones of Citrus Yellow Vein Clearing Virus Using a Novel and Rapid Strategy

Abstract

Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch's postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus-host interactions.

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.…”

“…T-DNA cassettes do not require stable integration in host genomes, and transient expression is sufficient to achieve plant infection. This feature, together with Agrobacterium promiscuity and its extensive host range (Lacroix et al, 2006), made agro-inoculation a method successfully applied to dicot and monocot plants (Bhat et al, 2016;Grimsley et al, 1986Grimsley et al, , 1987Liou et al, 2014;Lu et al, 2012;Scofield and Nelson, 2009) as well as herbaceous and woody hosts, including citrus, grapevine or apple (Cui et al, 2018;Dawson and Folimonova, 2013;Kurth et al, 2012;Vel azquez et al, 2016;Zhang and Jelkmann, 2017).…”

RNA silencing machinery contributes to inability of BSBV to establish infection in Nicotiana benthamiana: evidence from characterization of agroinfectious clones of Beet soil-borne virus

“…Agroinoculation of virus is not always successful on N. benthamiana , as already described by other studies [49, 50]. Despite its susceptibility to many viruses [51], this plant species is not host for all of them thanks to, inter alia, its antiviral RNA silencing.…”

The Coat Protein of Citrus Yellow Vein Clearing Virus Interacts with Viral Movement Proteins and Serves as an RNA Silencing Suppressor

“…We tried to construct infectious clones of CYVCV, but they failed to infect N. benthamiana and other herbaceous plant species. Recently, another research group in China obtained CYVCV infectious clones that can infect Citrus sinensis (L) Osbeck seedlings through an Agrobacterium -mediated vacuum-infiltration strategy but that failed to infect N. benthamiana plants [65]. Additionally, CYVCV could be mechanically transmitted to several herbaceous plants with low efficiency.…”