Hibiscus chlorotic ringspot virus coat protein inhibits trans-acting small interfering RNA biogenesis in Arabidopsis

1 Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, 117545 Singapore 2 Functional Genomics Laboratory, Institute of Molecular and Cell Biology, 138673 Singapore 3 Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA 4 Adjunct Investigator, Temasek Life Sciences Laboratory, 1 Research Link, 117604 Singapore Correspondence Sek-Man Wong dbswsm{at}nus.edu.sg Many plant and animal viruses have evolved suppressor proteins to block host RNA silencing at various stages of the RNA silencing pathways. Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is capable of suppressing the transiently expressed sense-RNA-induced post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana . Here, constitutively expressed HCRSV CP from transgenic Arabidopsis was found to be able to rescue expression of the silenced GUS transgene. The HCRSV CP-transgenic Arabidopsis (line CP6) displayed several developmental abnormalities: elongated, downwardly curled leaves and a lack of coordination between stamen and carpel, resulting in reduced seed set. These abnormalities are similar to those observed in mutations of the genes of Arabidopsis RNA-dependent polymerase 6 ( rdr6 ), suppressor of gene silencing 3 ( sgs3 ), ZIPPY ( zip ) and dicer-like 4 ( dcl4 ). The accumulation of microRNA (miRNA) miR173 remained stable; however, the downstream trans -acting small interfering RNA (ta-siRNA) siR255 was greatly reduced. Real-time PCR analysis showed that expression of the ta-siRNA-targeted At4g29770, At5g18040, PPR and ARF3 genes increased significantly, especially in the inflorescences. Genetic crossing of CP6 with an amplicon-silenced line (containing a potato virus X–green fluorescent protein transgene under the control of the 35S cauliflower mosaic virus promoter) suggested that HCRSV CP probably interfered with gene silencing at a step after RDR6. The reduced accumulation of ta-siRNA might result from the interference of HCRSV CP with Dicer-like protein(s), responsible for the generation of dsRNA in ta-siRNA biogenesis. A list of the genes tested and the primers used in this study is available with the online version of this paper.