Mechanisms Involved in Symptom Modulation by Turnip Crinkle Virus and its Associated Subviral RNAs

Abstract

Turnip crinkle virus (TCV) (family Tombusviridae, genus Carmovirus) is a positive- strand RNA virus. SatC, a satellite RNA associated with TCV, intensifies symptoms of TCV on all symptomatic hosts. Arabidopsis protoplast assays indicated that TCV virion levels are substantially reduced by the presence of satC or when two amino acids are inserted at the N-terminus of the coat protein (CP), resulting in similarly enhanced symptoms. Since the TCV CP is an RNA silencing suppressor, increased levels of the resultant free CP could augment silencing suppression resulting in enhanced colonization of the plant. Cloning and sequencing of virus-derived small RNAs (vsRNAs) accumulating in Arabidopsis plants infected with TCV with or without satC showed that the majority of

vsRNAs are ~21-nt, purine-rich sequences. One TCV vsRNA species, TvsRNA5, is complementary to 3' UTR sequences in transcripts of 12 Arabidopsis genes. Transcript levels of 3 of these genes were reduced 2.4- to 4-fold by TCV infection, but restored to normal levels when infected with TCV containing a deletion in the TvsRNA5 sequence. This deletion did not affect levels of virus, but resulted in symptom attenuation in infected plants. These results suggest that at least some vsRNAs are specifically altering expression of host genes leading to phenotypic changes in host plants. In addition, a technique has been established for detection of viral RNAs in whole plants, which makes use of the binding of the CP of MS2 bacteriophage (CP MS2 ) to a 19 base hairpin (hp). Protoplast co-transfection of TCV containing the hairpin and a fusion protein construct consisting of CP MS2, GFP and a nuclear localization signal (NLS) relocated GFP from the nucleus to the cytoplasm, indicating the presence of virus. TCV movement was also tracked by observing cytoplasmic GFP fluorescence in infected transgenic plants expressing the fusion protein. This technique should be amenable for detection of any virus with a transformable host.