Isolation And Functional Characterization Of A CCCH-Type Zinc Finger Nucleic Acid-Binding Protein Gene C3H12in Rice

Rice is an important food for the world population. Bacterial blight is one of the main reason for the great loss in rice production and cause huge economic losses ever year, so researching and exploiting rice defence genes provides new sources for breeding rice varieties with good resistance and help to elucidate the mechanism of rice disease resistance.Zinc finger proteins harboring the motif with conserved three cysteine residues and one histidine are defined as CCCH-type zinc finger proteins. Evidence reveal that CCCH-type zinc finger proteins belong to a large family and contain at least67members in the rice genome, but their functions are poorly understood. Here we report that one of the rice CCCH-type zinc finger proteins, C3H12, containing five typical CX8-CX5-CX3-H (X represents any amino acid) zinc finger motifs, is involved in the rice-Xoo interaction.We confirmed that C3H12colocalized with a minor disease resistance quantitative trait locus (QTL) to Xoo and the enhanced resistance of randomly chosen plants in the QTL mapping population correlated with increased expression level of C3H12. We overexpressed C3H12in susceptible Japonica variety Mudanjiang8and suppressed C3H12in moderately susceptible Japonica variety Zhonghua11and moderately resistant indica variety Minghui63with T-DNA insertion and RNA interference respectively by Agrobacterium-mediated transformation. According to realtime PCR analysis, the phenotypes of C3H12-overexpressing and-knockout or-suppressing transgenic plants were cosegregated with the C3H12expression level. Overexpression of C3H12significantly enhanced resistance to Xoo compared to the wild type, and accompanied by the induced expression of jasmonic acid (JA)-signaling genes LOX AOS2Chtl PR5PR10and accumulation of JA after Xoo inoculation. In contrast, knockout or suppression of C3H12resulted in significantly reduced resistance to Xoo compared to the wild type, accompanied by the decreased levels of JA and expression of JA-signaling genes in rice. These results suggest that C3H12may positively and quantitatively regulates rice resistance to Xoo through JA-dependent patyway.We found that C3H12protein localized in the nucleus by subcellular localization experiment. Subsequently, we expressed and purified the recombinant C3H12protein with maltose binding protein (MBP) tag in vitro and found C3H12protein possessed the ability to bind nucleic acid in vitro nucleic acid binding assay, but without transactivation activity in yeast cells. These results suggest that C3H12protein may regulated gene expression in posttranscriptional level by binding to mRNA.In conclusion, C3H12, as a nucleic acid-binding protein, positively and quantitatively regulates rice resistance to Xoo and its function is likely associated with the JA-dependent pathway..