Cucumber mosaic virus 2b protein inhibits RNA silencing pathways in green alga Chlamydomonas reinhardtii

Abstract

The functions of RNA silencing are repression of endogenous gene expression and antiviral defense in plants and animals. Cucumber mosaic virus 2b (CMV2b) is a suppressor of RNA silencing in higher plants. In the present study, we evaluated the RNA silencing suppressor activity of CMV2b in Chlamydomonas reinhardtii. Before transformation, we modified CMV2b codons to increase the GC content for optimal expression in C. reinhardtii. Inhibition of Maa7 silencing was detected in CMV2b-expressing Maa7-IR44 strains, indicating that CMV2b suppressed siRNA pathways in C. reinhardtii as in higher plants. In addition, mRNA expression targeted for cleavage by miRNA was significantly higher in CMV2b-expressing strains, but increased accumulation of miRNA was not detected. These results indicate that the suppression of miRNA pathways is mediated by CMV2b in C. reinhardtii. Interestingly, expression of both Argonaute 1 (AGO1) and Dicer-like 1 (DCL1), regulated by a bidirectional promoter, was reduced in CMV2b-expressing strains, suggesting that CMV2b may affect transcription factors involved in RNA silencing pathways. Furthermore, reduction of AGO2 and AGO3 expression was detected in CMV2b-expressing strains. Taken together, our results demonstrate that CMV2b may suppress both siRNA and miRNA pathways, and also impair AGOs and DCL1 expression in C. reinhardtii.