2b proteins of highly virulent cucumber mosaic virus strains are associated with suppression of DCL4 activity.

Abstract

RNA silencing constitutes a fundamental antiviral defense mechanism in plants. Although its role in virus resistance has been extensively studied, the relationship between the dicing activity of Dicer-an essential enzyme in RNA silencing-and antiviral defense remains unclear. To address this knowledge gap, we previously established a simple in vitro dicing assay that enables the direct assessment of the activities of two Dicers, DCL3 and DCL4. We hypothesized that this assay could reveal how viral infection modulates DCL3 and DCL4 activities. Here, we report a negative correlation between NbDCL4 activity and symptom severity using the Nicotiana benthamiana-cucumber mosaic virus (CMV) system. We demonstrate that NbDCL4 activity is specifically suppressed in plants infected with highly virulent subgroup I (sg I) strains of CMV. In vitro dicing assays and small RNA sequencing analyses further reveal that NbDCL4 activity toward long double-stranded RNA (dsRNA) remains unaffected, whereas its activity toward shorter dsRNAs (~50 nt) is markedly inhibited in CMV(sg I)-infected plants. Subsequent analyses using a 2b-deficient disarmed mutant and a less virulent subgroup II strain of CMV indicate that this inhibition is mediated by the sg I 2b protein, a viral suppressor of RNA silencing. Taken together, these findings provide mechanistic insights into the molecular basis of CMV virulence, suggesting that the severe symptoms induced by highly virulent CMV strains are, at least in part, attributable to 2b-mediated inhibition of DCL4 activity.