Cloning Of A New High Bacterial Blight Resistance Related Gene And Research On Internal Resistance Mechanism In Rice

Bacterial blight caused by Xanthomonas oryzae pv.oryzae?Xoo?is a devastating rice disease,which can cause a large lose of rice production.Using of resistance cultivars is an economical and environment-friendly means to protect rice from Xoo.This needs constantly explore new Xoo resistance genes and study these genes' function.In this study we isolated and cloned the resistant target gene in induced lesion mimic mutant hpil3 with high resistant and broad spectrum to Xoo,took proteomic experiment,screened interacting genes.And we also researched on RCA in the role of reistant to Xoo in Oryza meyeriana.The main research findings are as follows:We isolated and clone the mutant target gene using by the map-based cloning strategy based on next-generation genome resequencing.The target gene is located on 20-22 Mb region in chromosome 4.During this region seven candidate genes were detacted,and among them one more possible gene was found through sequencing verification of mutant location and bioinformatics analysis.The transgenic function complementary test showed that wild type gene can make hpil3 recover to its wild type and meanwhile lose Xoo resistance,which confirms that the gene is hpil3 target gene.Real-time fluorescence quantitative PCR?RT-q PCR?showed that the expression level of PR1 a,PR1b,PR5 and PR10 was significantly higher in mutant than that in its wild type Dalixiang?DLX?,while PR9 was slightly higher.HPIL3 expression level in hpil3 was significantly higher than wild type.This may be due to the loss of function caused by a mutation that led to increased expression of the gene in the plant.The expression level of HPIL3 in mutant is induced by Xoo.After inocculated with Xoo HPIL3 expression quality increased rapidly.Analysis suggests that the gene was correlated with rice resistance to Xoo.291 differently expressed proteins?expression ratio ?1.5,t-test?0.05?between hpil3 and DLX were identified by two-dimensional difference gel electrophoresis?2D-DIGE?combining mass spectrometry analysis.74 proteins were up-regulated and 217 proteins were down-regulated.The identified proteins are involved in metabolism modify of amino acid and protein,photosynthesis,nucleotide metabolism,defense,energy metabolism,sugar metabolism and redox,etc.Further data analysis showed that the main metabolic pathways in hpil3 including photosynthesis,glycolysis/glycolysis,pentose phosphate pathway,and Calvin cycle.Most of these metabolic pathways are negative correlated with the phenotype of the mutant.Some disease-related proteins such as PR10 and 14-3-3 protein were up-regulated while AAA type of ATPase which negative regulation of cell apoptosis was down-regulated.Meanwhile antioxidant enzymes including superoxide dismutase and peroxidase were up-regulated.These results showed that defense response happened in hpil3,meanwhile various related metabolic pathways are also affected.ROS scavenging system also plays an active role in order to meinain cell homostasis.We screened four HPIL3 interacting genes by using Yeast two hybrid.And the four fulllength genes were tested by Yeast two hybrid.Among the four candidate interacting genes,the plastocyanin and 23 k Da pepitide of PS?are chloroplast proteins.Two others are a YL1 nucleoprotein containing C-terminal domain and a stress response protein.It was speculated that the protein coding by mutant gene can not interact with related proteins and this mediate the active of downstream signaling pathway on disease resistance.Large amout of H₂O₂ induced by Xoo innoculation can lead to the association of RCA and thylakoid in Oryza meyeriana.In this study we found the inoculation of Xoo can induce H₂O₂ burst in Oryza meyeriana.And the period when Xoo infection triggered the “H₂O₂ burst” coincided precisely with the movement of RCA from the stroma to the thylakoid membrane,suggesting a causal link.H2 DCFDA staining experiment showed that H₂O₂ was generated in the chloroplast of O.meyeriana.In vitro experiments showed that H₂O₂ can induce the transfer of RCA and this transfer occurred only in O.meyeriana but not in the susceptible cultivated rice.Mixed these two component from the two rice materials,it didn't happen.Taken together,we isolated the target gene of hpil3 using the strategy combining the map-based cloning and the next-generation genome sequencing and confirmed this target gene through functional complementation.The results showed that HPIL3 negatively regulated the programme cell death and the lesion mimic phenotype,that is,the mutation and loss of function of HPIL3 caused the phenotype of lesion mimic in hpil3.RT-q PCR analysis showed that 4 PR genes significantly up-regulated.The proteomic-based analysis showed that HPIL3 was involved in the mutant phenotype of lesion mimic through the regulation on multiple functions in different pathways.In addition,four candidate interactive genes with HPIL3 were detected by yeast two hybrids.The association of RCA with the thylakoid membrane in O.meyeriana may helps protect the thylakoid membrane against oxidative damage from H₂O₂.Therefore,in addition to its universal function of regulating photosynthesis,RCA appears to play a novel role in the resistance of O.meyeriana to Xoo.The present work provides an important gene resource and theoretical basis for the further research on Xoo resistance response in rice as well as resistance breeding.