Biological relevance of a stable biochemical interaction between the tombusvirus-encoded P19 and short interfering RNAs.
The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R-->W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR-->GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex approximately 21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for approximately 21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.
Omarov
Rustem
R
Department of Plant Pathology and Microbiology, Texas A&M University, 2132 TAMU, College Station, Texas 77843, USA.
Sparks
Kim
K
Smith
Lindsay
L
Zindovic
Jelena
J
Scholthof
Herman B
HB
D002850:Chromatography, Gel; D019281:Dimerization; D047468:Immunoprecipitation; D018551:Solanum lycopersicum / Q000821:virology; D010935:Plant Diseases / Q000821:virology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D017864:Tombusvirus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C493885:P19 protein, tomato bushy stunt virus; D034741:RNA, Small Interfering; D012367:RNA, Viral; D034743:RNA-Induced Silencing Complex; D014764:Viral Proteins