Elucidating The Molecular Mechanism Of Fungal Elicitor MoHrip2-induced Disease Resistance In Rice

Magnaporthe oryzae hypersensitive responsive protein 2(Mo Hrip2) is an elicitor protein of the rice pathogen M. oryzae that could enhance disease resistance in rice seedlings against M. oryzae. To elucidate the mechanism of MoHrip2 in rice, seedlings were exposed to MoHrip2 and then the levels of several defense-related transcription factors(TFs), marker genes for the salicylic acid(SA) and jasmonic acid(JA) signaling pathways, and pathogenesis-related(PR) genes were assessed over time. The endogenous levels of SA and JA were also assessed over time. Differential expression among the total proteins extracted from rice leaves at 24 h was evaluated using differential-display 2-D gel electrophoresis and qRT-PCR after treatment with MoHrip2 or mock treatment. The qPCR data have revealed that all selected TFs were induced within 2 h after treatment(hpt) with highest expression at 4 hpt. Genes related to the SA and JA pathways(Os NPR1/OsNH1, OsPAL1, and OsLOX2) were also upregulated within 2 hpt compared with the controls, while OsEDS1 and OsAOS2 were downregulated. Similarly, PR genes(OsPR1a, Os PR2, OsPR3, OsPR4, Os PR5 and Os PR10) were also induced at various times(2–48 hpt). The highest expression was observed for glucanase(OsPR2) and chitinases(OsPR3 and OsPR4) earlier(2–4 hpt) after treatment; other PR genes(OsPR1a, OsPR5 and OsPR10) reached their highest expression at 24–48 hpt. Endogenous levels of SA and JA were upregulated in treated rice seedlings over the controls, further supporting the induction of defense-related genes. The 2D gel data have indicated that among ~1000 protein spots detected on each gel, 10 proteins were newly induced, 4 were upregulated, and 3 were downregulated in MoHrip2-treated samples compared with the mock controls. Seventeen differentially expressed proteins were detected using MS/MS analysis and categorized into six groups according to their putative function: defense-related transcriptional factors, signal transduction-related proteins, reactive oxygen species(ROS) production, programmed cell death(PCD), defense-related proteins, and photosynthesis and energy-related proteins. The q PCR results(relative expression level of genes) further supported the differential expression of proteins in Mo Hrip2-treated rice leaves. Moreover, four candidate-binding proteins of MoHrip2 were cached using poly His-Tag pulls down protein interaction kit(Thermo Scientific). The encoding genes were cloned for further confirming the interactions in vivo and in vitro. Bioinformatics analysis indicated that Mo Hrip2 might directly or indirectly interacts with peroxidases(ROS-related proteins).Conclusively, MoHrip2 triggers an early defense responses in rice via stress-related pathways, induced PR genes and triggered both SA and JA pathways, and the results provide a basis to commercially exploit these findings for genetic improving rice resistance and an evidence for elicitor-induced resistance at transcriptomics, proteomics and metabolomics level.