Interactions Among TTSS Effectors Of Xanthomonas Oryzae Pv.oryzicola And Rice Genes Involved In The Interaction With TTSS Effectors

Xanthomonas oryzae pv.oryzicola (Xooc) is the critical agent causing leaf streak disease in rice. The pathogen possesses hrp genes as other gram-negative plant pathogenic bacteria. The hrp genes confer the pathogenic bacteria to the ability to trigger hypersensitive response (HR) on nonhost plants and to be pathogenic on susceptible host plants. The clustered hrp genes also encode conserved proteins composing of a type III secretion system that delivers pathogenicity effectors into host cells.Harpin is a term of proteins of gram-negative plant pathogenic bacteria which triggersHR on nonhost plants. Protein harpin_Xooc encoded by the hpa1 gene in the hrp cluster of Xooc triggers a HR in tobacco. Reverse-transcriptional polymerase chain reaction(RT-PCR) demonstrated that PR-1a,hin1 and hsr203J genes in tobacco, and NPR1, OsPR-1a, OsPR-1b, and PAL genes in rice were activated transcriptionally after the spray with harpin_Xooc. This indicated that a systemic acquired resistance(SAR) was induced through the salicylic acid signaling pathway marked with activation of PR-1a,hin1 and hsr203J genes in tobacco and NPR1, OsPR-1a, and PAL genes in rice respectively. The extracted protein was industrially developed into soluble granule, commercially termed Yilite, containing 1% of harpin_Xooc. Application of harpin_Xooc in rice showed that harpin_Xooc, substituted chemical fungicides in the field, induced SAR in rice with control efficiencies equivalent to those by application of Tncyclazole and Jingangmycin, respectively, against rice blast (Magnaporthe grisea), rice sheath blight (Rhizoctonia solani) and rice false smut (Claviceps orysae-sativae ). The rice yield was increased over 6% compared with that in the plots sprayed chemical fungicides. The factor for the yield increase was mainly due to the grain weight increased. This is the first report that hpa1 gene product induced systemic acquired resistance in plants and controlled rice diseases efficiently in the field.A yeast-two hybrid system was used to investigate interactions among twenty-seven genes in hrp cluster of Xooc. The result demonstrated that there were positioned interactions among products of nine hrc genes, hrpE and hrpF assembling a typeâ…¢secretion system (TTSS). HrcN interacted with all components of the TTSS but HrpF. HrcC interacted with HrcR and HrcJ. HrcR interacted with HrcT, HrcV and HrcS. The interactions among other TTSS effectors in the hrp duster indicated that HrpB5, HrpB4, HrpB2, HpaF and Hpa4 were likely chaperones for secretion of TTSS effector via the TTSS. The interactions of other TTSS effectors, AvrXa3, Avr/pth28, Avr/pth13, AvrBs2, PopC and Pga, with twenty-seven hrp genes of Xooc showed that these six TTSS effectors interacted with HrpB5, HrpB4 and HrpB2. The result also revealed that different AvrBs3 family proteins interacted with different TTSS effectors when secreted via TTSS in schedule. The results all the above provided scientific clues for understanding molecular mechanisms of TTSS effectors of Xooc with rice.Constructing a cDNA library is the basis for cloning eukaryote genes. In this study, RNA and mRNA were extracted from rice leaves which were injected with 10⁸ cells of Xooc after 10 hours. The rice cDNA library was constructed using SMART^TM technology in a plasmid pGADT-7-sfiAB. Polymerase chain reaction demonstrated that OsPR-1a, OsPR-1b, and PAL genes, related in pathogenesis in rice, were transcipitionally expressed when using the rice eDNA library. The SfiI enzyme digestion of randomly selected clones from the rice cDNA library indicated that the inserts of the cDNA library in plasmid pGADT-7-SfiAB were mainly between 500bp-2000bp. The average was about 1000 bp. The above data indicated that the quality of the rice cDNA library was good for the following usage.In order to clone a rice gene which interacted with TTSS effectors of Xooc, twenty-seven genes in the hrp cluster of Xooc were constructed in a vector pGBKT-7 as the baits for screening the rice cDNA library using the yeast-two hybrid system. Totally 60 positives clones were got which would grow on a SD/-Ade/-Leu/-Trp/-His medium. The bait genes and the prey genes were amplified from the positive yeast clones using T7 sequencing primer, 3' AD Sequencing Primer and 3' DNA-BD Sequencing Primer in pGADT-7 and pGBKT-7 vectors. Big inserted prey fragment and its corresponding bait gene were isolated and sequenced. The sequencing results indicated that HpaB in the hrp duster of Xooc interacted with a polyubiquitin (Pubq) in rice. The Pubq gene has a 690bp open-reading frame, encoding a 229 amino acid protein. The PUBQ protein is similar to RUBQ1 and RUBQ2 at the C-termini. This is the first confirmation that the TTSS effector HpaB interacted with PUBQ in rice. The interactions provide a clue for further understanding the roles of HpaB and PUBQ in the interactions of Xooc-rice system.