Two amino acids near the N ‐terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity

Summary Cucumber mosaic virus ( CMV ) 2b suppresses RNA silencing primarily through the binding of double‐stranded RNA ( dsRNA ) of varying sizes. However, the biologically active form of 2b remains elusive. Here, we demonstrate that the single and double alanine substitution mutants in the N ‐terminal 15th leucine and 18th methionine of CMV 2b exhibit drastically attenuated virulence in wild‐type plants, but are efficiently rescued in mutant plants defective in RNA ‐dependent RNA polymerase 6 ( RDR6 ) and D icer‐like 4 ( DCL4 ). Moreover, the transgenic plants of 2b, but not 2blm ( L15A/M18A ), rescue the high infectivity of CMV ‐Δ2b through the suppression of antiviral silencing. L15A , M18A or both weaken 2b suppressor activity on local and systemic transgene silencing. In contrast with the high affinity of 2b to short and long ds RNAs , 2blm is significantly compromised in 21‐bp duplex small interfering RNA (siRNA) binding ability, but maintains a strong affinity for long dsRNAs . In cross‐linking assays, 2b can form dimers, tetramers and oligomers after treatment with glutaraldehyde, whereas 2blm only forms dimers, rather than tetramers and oligomers, in vitro . Together, these findings suggest that L15 and M 18 of CMV 2b are required for high affinity to ds‐ siRNAs and oligomerization activity, which are essential for the suppression activity of 2b on antiviral silencing.