Functional Characterization And Expression Patterns Of Rice Xylanase Inhibitors

Since their discovery in1997, three distinct type of xylanase inhibitors, with different structures and specificities were isolated from different plants. As the xylanase inhibitors can interact with xylanases from microbial, it has been hypothesized that xylanase inhibitors play an important role in plant defense. Here, we cloned a XIP-type xylanase inhibitor gene OsXIP-Ⅱ from rice and examined the activity of this protein. In order to facilitate the understanding of its contribution in rice growth, we have obtained OsXIP-Ⅱ overexpressed and suppressed transgenic rice plants, and also determined the resistance of transgenic plants overexpressing xylanase inhibitor protein to Magnaporthe oryzae. Furthermore, we used deep RNA sequencing combined with digital gene expression profile (DGE) analysis to rapidly identify and analyze the differentially expressed genes in xylanase inhibitor overexpressed transgenic rice plants. Moreover, to clarify the expression pattern of xylanase inhibitors, the pomotor::GUS vectors were constructed and the transgenic rice lines were obtained.Our results are as follows:(1) We cloned a XIP-type inhibitor gene OsXIP-Ⅱ from rice DNA, and expressed it in Escherichia coli. Over-expression and RNAi vectors were constructed and introduced respectively into rice, and hypothesized OsXIP-Ⅱ might be involved in environmental responses such as defense against phytopathogens.(2) To determin the resistance of transgenic plants overexpressing xylanase inhibitor to pathogen, we took RIXI as representative of rice XIP type xylanase inhibitors and RIXI-overexpressed and suppressed transgenic rice plants were obtained. In comparison with the wild-type plants, the RIXI-overexpressed transgenic plants had significantly increased levels of RIXI as well as defense related genes and showed resistance to Magnaporthe oryzae.(3) Deep RNA sequencing combined with digital gene expression profile (DGE) analysis was employed to rapidly identify and analyze the differentially expressed genes in xylanase inhibitor overexpressed transgenic rice plants. Geneontology (GO) analysis revealed that the differentially expressed genes were mainly involved in biological process and molecular function. The expression of several transcription factor families showed different expression patterns. Significantly enriched metabolic pathways, biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone signal transduction pathways were identified. And the plant-pathogen interaction pathway was the most significant pathway. The plant-pathogen interaction, plant hormone signal transduction and photosynthesis pathways were analyzed in detail.(4) Analysis of the expression patterns of XIP family genes in rice. The promotor sequences of RIXI, OsXIP and riceXIP were isolated and fused to GUS reporter gene to generate promotor::GUS constructs, and then introduced them into rice by Agrobacterium-mediated transformation. The GUS staining results showed that members of rice XIP family showed differential organ-specific expression in rice plants. RIXI primarily expressed in root, leaf, stem and vascular bundle, OsXIP primarily expressed in root and lemma vein, and riceXIP primarily expressed in root tip, lemma vein and stamen. We also found that RIXI was highly expressed in the leaf of young seedlings, while OsXIP and riceXIP were little expressed. In contrast, at the flowering stage, OsXIP was little expressed in leaf, and RIXI and riceXIP were not expressed. It suggested that members of rice XIP family occur in different fashions throughout rice development. The presence of signal peptides in XIP-type xylanase inhibitors suggested them to be located in the apoplastic space, and confocal laser scanning fluorescence microscopic observation of the GFP fluorescence from the XIP::GFP fusion protein provided evidence to support this.(5) Analysis of the response of XIP-type xylanase inhibitors to biotic and abiotic stresses. Two-week-old seedlings of the rice cultivar were treated with0.2mM MeJA,200mM sodium chloride (NaCl), Nilaparvata lugens, wounding, low temperature (8℃) and distilled water (Control), and the GUS activities in root and shoot were measured. The results shown that OsXIP and riceXIP expressions were were markedly induced by MeJA, NaCl, Nilaparvata lugens, wounding and low temperature; moreover, OsXIP expression were more significantly induced compared to riceXIP. RIXI expression was drastically induced by MeJA, Nilaparvata lugens, wounding and low temperature, with no significant induction by NaCl. The results showed that the response of XIP-type xylanase inhibitors to biotic and abiotic stresses were different, and the manner of induction by Nilaparvata lugens was very similar to that by wounding. These results suggest that the induction of XIP-family proteins expressions by Nilaparvata lugens may occur via a wounding-mediated pathway.Considering that the expression patterns and the manners of induction by the biotic and abiotic stresses of rice XIP-family proteins were different, we speculate that they have different functions in rice, i.e. RIXI may specialize in defense towards pathogens in young rice, whereas OsXIP and riceXIP may specialize in defense mechanism against pathogens in grown rice.