After centrifugation at 100 g for 10 min at 4 °C, the supernatant was mixed with incubated at 4 °C for 2 h. After washing, the IP products were boiled at 100 °C for 10 min. Samples were separated by 10% Bis-Tris PAGE Gel (GenScript, Nanjing, China) and subjected to immunoblot analysis. The ubiquitin modifications were detected with the anti-Myc (1:10 000, Abmart, M20002) and anti-Ubi P4D1 (1:1000, Santa Cruz, sc-8017) antibodies. The immunoprecipitated OsWRKY proteins were detected with the anti-FLAG (1:10 000, GenScript, A00187) antibody .
Plants have intricate mechanisms that tailor their defence responses to pathogens. WRKY transcription factors play a pivotal role in plant immunity by regulating various defence signalling pathways. Many WRKY genes are transcriptionally activated upon pathogen attack, but how their functions are regulated after transcription remains elusive. Here, we show that OsWRKY7 functions as a crucial positive regulator of rice basal immunity against Xanthomonas oryzae pv. oryzae (Xoo). The activity of OsWRKY7 was regulated at both translational and post-translational levels. Two translational products of OsWRKY7 were generated by alternative initiation. The full-length OsWRKY7 protein is normally degraded by the ubiquiti... More
Plants have intricate mechanisms that tailor their defence responses to pathogens. WRKY transcription factors play a pivotal role in plant immunity by regulating various defence signalling pathways. Many WRKY genes are transcriptionally activated upon pathogen attack, but how their functions are regulated after transcription remains elusive. Here, we show that OsWRKY7 functions as a crucial positive regulator of rice basal immunity against Xanthomonas oryzae pv. oryzae (Xoo). The activity of OsWRKY7 was regulated at both translational and post-translational levels. Two translational products of OsWRKY7 were generated by alternative initiation. The full-length OsWRKY7 protein is normally degraded by the ubiquitin-proteasome system but was accumulated following elicitor or pathogen treatment, whereas the alternate product initiated from the downstream in-frame start codon was stable. Both the full and alternate OsWRKY7 proteins have transcriptional activities in yeast and rice cells, and overexpression of each form enhanced resistance to Xoo infection. Furthermore, disruption of the main AUG in rice increased the endogenous translation of the alternate stabilized form of OsWRKY7 and enhanced bacterial blight resistance. This study provides insights into the coordination of alternative translation and protein stability in the regulation of plant growth and basal defence mediated by the OsWRKY7 transcription factor, and also suggests a promising strategy to breed disease-resistant rice by translation initiation control.
关键词
OsWRKY7; Xoo; alternative translation; basal immunity; protein stability.