Study On The Rice Secreted Proteins And Their Involvement In Rice Resistance To Bacterial Blight

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most destructive bacterial disease world wide. The wild rice Oryza meyeriana shows high resistance to Xoo. Here, We investigate the functions of rice secreted proteins from the wild rice meyeriana and the cultivated rice nipponbare in Xoo-disease resistance. And the main results are summarized as follow:1. Semi-quantitative PCR and quantitative real time PCR were used to investigate the mRNA expression of Xoo-responsive proteins from meyeriana suspension culture. Eight Xoo-responsive proteins showed the similar changes in mRNA expression and protein abundance. Additionally, the enzymatic activity of peroxidase were up-regulated in both meyeriana and nipponbare after inoculation with Xoo, while the base enzyme activity in meyeriana was over three times higher than in nipponbare rice.2. Two signal proteins (OmCS1, OmSCP8) and two pathogensis-related proteins (OmPOP11, OmR40c1) were chosen as candidate proteins for subsequent functional analysis. OmCS1 contains a Chondroitin Sulfate domain. Over-expression of gene OmCS1 in rice significantly enhanced the rice tolerance to Xoo. OsPAL and OsNPRl involved in SA synthesis and accumulation were both up-regulated in OmCSl transgenic rice, which suggests that OmCSl may enhance rice tolerance to Xoo through SA-dependent pathway. OmPOP11 is a member of the Prolyl Oligopeptidase (POP) family. Over expression of gene OsPOPll in rice can also enhance the tolerance to Xoo.OmSCP8 contains a serine carboxypeptidase domain, similar with SCP domain of OsBRS1 in Brassinosteroids signal pathway. Over expression of OmSCPS in rice can enhance the tolerance to Xoo. OsPAL1 was up-regulated in OmSCP8 transgenic plant.3. OmR40c1 is a member of Ricin_B family. It was shown as a plasma membrane-localized protein by confocal microscopy and can agglutinate with rabbit blood red cells. PRs and JA/SA-synthesis-related-genes in OmR40cl and 5’UTR-OmR40cl transgenic plants were both down-regulated. Over expression of OmR40cl or 5’UTR-OmR40cl in rice didn’t enhance the rice tolerance to Xoo. However, the seed germination rate, plant height and cellular concentration of Na+ and K+ of the OmR40cl transgenic lines were all higher than that of the wild type rice when challenged by salt stress, which suggests OmR40cl may enhance rice salt tolerance.4. We performed a secretome protomics to investigate the secreted proteins from susceptible cultured cells challenged by Xoo.32 protein spots corresponding to 11 unique proteins were identified by MS:seven from rice and four from Xoo. The seven Xoo-responsive rice proteins are involved in cell defense, Redox and cell wall modification. Three Xoo proteins (Xoo0842, Xoo3479 and Xoo3654) were cell membrane-localized proteins. Over expression of Xoo3654 in P10 slightly reduced the pathogenicity of Xoo, which suggests Xoo3654 is likely a negative regulator of Xoo virulence.5. The chitinase OsCHIT16 was a Xoo-responsive protein from nipponbare suspension culture. The ORF of OsCHIT16 was shorter than that in OmCHIT16. Both OmCHIT16 and OsCHIT16 were cell membrane localized proteins. Over expression of 0sCHIT16 and OmCHIT16 in rice both enhanced rice tolerance to Xoo. A number of PRs genes and JA/SA-synthesis-related-genes in the transgenic rice plants were both down-regulated, which suggests CHIT16 enhance the tolerance to Xoo through the JA or SA independent pathway, and the difference in DNA sequences between them may not affect the gene function of CHIT16 in cultivated rice and wild rice.In summary, We perfomed a secretome proteomics to investigate the secreted proteins from susceptible rice suspension cultured cells challenged by Xoo, and found a protein Xoo3654 is likely a negative regulator of Xoo virulence. We showed that overexpression of OmCS1, OsPOP11, OmSCP8, OsCHIT16 and OsCHIT16 can all enhance rice tolerance to Xoo, but further study are required to understand the molecular mechanims of the enhanced Xoo resistance in the transgenics rice lines. Together, those results not only help us better understand the interaction of resistannt and susceptible rice with Xoo, but also serve as a reference for studying the interaction between other plants and their pathogens.