ABSTRACT
Viruses encode RNA silencing suppressors to counteract host RNA silencing-mediated defense responses. In this study, we demonstrate that VP53, VP37 and LCP encoded by RNA2 of broad bean wilt virus 2 (BBWV-2), a member of the genus Fabavirus, are strong suppressors of RNA silencing triggered by single-stranded sense RNA. They, however, had no effect on suppression of RNA silencing induced by double-stranded RNA. We provide evidence that these three suppressors can significantly limit the accumulation of small interfering RNAs (siRNAs) in tissues where the GFP gene has been silenced, and prevent the long distance spread of the induced silencing signal. Gel mobility shift assays showed that all three suppressors could bind ssRNA in a size-specific manner. Interestingly, VP37 and LCP, but not VP53, could reverse the silencing of a GFP gene in leaf tissue. Furthermore, these three proteins are capable of enhancing pathogenicity of potato virus X. Collectively, our findings indicate that viruses employ a more sophisticated strategy to overcome the host defense response mediated through suppression of RNA silencing during virus infection. As far as we are aware, this is the first report of RNA silencing suppressors encoded by a virus in the genus Fabavirus.
Subject(s)
Fabavirus/immunology , Fabavirus/physiology , Host-Pathogen Interactions , Nicotiana/immunology , Nicotiana/virology , RNA Interference , Viral Proteins/metabolism , Electrophoretic Mobility Shift Assay , Immune Evasion , Protein Binding , RNA, Small Interfering/metabolismABSTRACT
The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.
Subject(s)
Cucurbita/virology , Fabavirus/genetics , Fabavirus/isolation & purification , Genome, Viral , RNA, Viral/genetics , Sequence Analysis, DNA , Antibodies, Viral/immunology , Blotting, Western , China , Cluster Analysis , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Fabavirus/classification , Fabavirus/immunology , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino AcidABSTRACT
Broad bean wilt virus 1 (BBWV-1) causes damages in economically important plant crops such as pepper, bean, spinach, etc. Fast, cheap and reliable diagnostic tools are crucial to limit or control the disease. In this work, tissue blot immunoassay (TBIA), dot-blot (DB) and tissue-print (TP)-hybridization were developed for BBWV-1 diagnosis and evaluated for sensitivity, specificity and reliability in plants of several host species grown in the greenhouse or in the field, in comparison with ELISA and RT-PCR. RT-PCR followed by DB-hybridization provided the most sensitive and efficient diagnostic, but the virus was also detected in most samples by ELISA. Detection by TBIA or by TP-hybridization avoided sample processing, but they were less consistent and greatly depended on host species and tissue. DB-hybridization with probes corresponding to different genomic regions allowed universal detection of BBWV-1 and discrimination between genetically distant isolates.
Subject(s)
Fabavirus/genetics , Fabavirus/isolation & purification , Immunoassay/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Staining and Labeling/methods , Fabavirus/immunology , Plant Diseases/virology , Sensitivity and SpecificityABSTRACT
Six hybridoma cell lines secretion monoclonal antibodies(MAbs) against broad bean wilt virus(BBWV) were produced by fusing mouse myeloma cells(SP 2/0) with spleen cells from BAL B/c immunized by the BBWV particles. The hybridoma cell lines secreted MAbs stably after cultured in vitro for 3 months or stored in liquid nitrogen and then revived for several times. The titres of ascitic fluids of six MAbs ranged from 1:256,000 to 1:640,000 when measured by indirect ELISA. In agarose gel immunodiffusion test, it showed that the six MAbs represented the same isotype of murine antibodies, IgG1. Six MAbs could detect 4 tested BBWV isolates, but didn't crossreact with other 5 plant viruses. The result of Western blot showed that all the six MAbs can react with the 44.7 kD large coat protein subunit of BBWV. This is the first report of production of MAbs against BBWV.
