MdBT2 suppresses ApNMV genomic RNA accumulation by promoting 1a ubiquitination and degradation
Given that 1a plays critical roles in viral genomic RNA replication according to its closely related model virus BMV, we thus hypothesized that MdBT2-mediated 1a degradation might inhibit ApNMV genomic RNA replication in apple. To verify our hypothesis, we transformed the infectious clone of ApNMV into apple ‘GL3’ leaves of WT,MdBT2-OE , and MdBT2-anti via vacuum method. Five days post-transformation, the leaves were collected and prepared for extraction of total proteins and RNAs. We first tested the protein level of 1a using anti-1a specific antibody, and found that it was lower inMdBT2-OE leaves, but was higher in MdBT2-anti leaves compared to that of WT (Fig. 6A), suggesting MdBT2 promoted the protein degradation of 1a during virus infection. We next verify the ubiquitination of 1a mediated by MdBT2, and found that the amount of poly-ubiquitinated 1a was higher in MdBT2-OE , but lower inMdBT2-anti leaves compared to that of WT when detected with anti-1a antibody (Fig. 6B, upper image). Also, more ubiquitinated proteins were present in MdBT2-OE leaves, but less inMdBT2-anti leaves compared to that in WT when detected with anti-Ubi antibody (Fig. 6B, lower image), suggesting MdBT2 promoted ApNMV 1a protein ubiquitination and degradation during virus infection.
We then determined the role of MdBT2 in ApNMV viral RNA replication via Northern blot by using a DIG-labeled probe that targeting the CP-coding seqence of ApNMV. The results showed that accumulated viral RNA levels in MdBT2-OE leaves were lower, but they were higher inMdBT2-anti leaves compared to that in WT leaves (Fig. 6C), suggesting MdBT2 played a negative role in ApNMV replication in apple. These data indicated that MdBT2 might repress the ApNMV genomic RNA replication by promoting the ubiquitination and degradation of 1a protein.