The stability of P1 of Turnip mosaic virus impacts the HC-Pro suppression of miRNA pathway

The P1/HC-Pro of potyvirus is the first discovered viral suppressor and HC-Pro needs to work with P1 for enhancing the suppression effect against post transcriptional gene silencing (PTGS) and inhibiting the microRNA (miRNA) pathway; however, the mechanism of P1/HC-Pro remains unclear. Furthermore, P1 is the most divergent protein in length and amino acid sequence among potyviruses. For studying the function of P1/HC-Pro, the antisera specific to P1, HC-N (1-267 a.a. of HC-Pro), HC-C (249-458 a.a. of HC-Pro) or CP were developed. In order to improve the quality of antiserum, fast protein liquid chromatography (FPLC) was used to purify immunoglobulin G (IgG) and to concentrate IgGs by protein centricon for enhancing the sensitivity. In this study, we demonstrated that the rapid turnover rate of P1 in vivo and the stability of P1 is crucial for miRNA pathway. Moreover, TuMV-infected plants and P1/HC-Pro transgenic Arabidopsis were treated with MG132 (26S proteasome inhibitor) to demonstrate that P1 is under an ubiquitin-independent degradation process. Interestingly, the P1 protein became stabilized and detectable while its N-terminus is fused with YFP (yellow fluorescent protein) or Flag tags and it caused a mild phenotype with serrated leaves in transgenic plants. Surprisingly, the P1dN600/HC-Pro transgenic Arabidopsis, which retained the conserved regions at the C-terminus of P1, became detectable and showed mild leaf serration in Arabidopsis. It suggests that the N-terminus of P1 regarding its stability is crucial for inhibiting miRNA pathway. We conclude that the stability of P1 affects the efficiency of miRNA suppression, and the N-terminus of P1 regulates the turnover rate. Study on the instability of P1will help clarify the role of P1/HC-Pro in miRNA pathway.