Function Identification Of A Novel Avirulent Gene And Cloning And Expression Of The Quorum Sensing Related Genes From Xanthomonas Oryzae PV.Oryzae

IS-PCR and rep-PCR were used to analyze the genetic diversity in a populationof 17 strains of Xanthomonas oryzae pv. oryzae collected from the west of Yunnanjaponica rice planted area and 7 strains from other areas. Genomic DNA from 24strains were amplified with 2 specific primers J3 and ERIC by PCR. The resultsrevealed 18 haplotypes, 14 bands by J3 primer and 13 bands by ERIC primer.Dendrograms were generated from the data sets obtained by using PHYLIP Version3.6 UPGMA analysis. 21 strains from China were grouped in 4 clusters, 3 strains fromthe Philippines was out of 4 clusters. 17 strains from the west of Yunnan are groupedin all 4 clusters, but 14 strains mainly grouped in 2 clusters. A novel avirulent gene was cloned from X. oryzae pv. oryzae strain PXO339, whichis the standard representative strain for race 9a in the Philippines, and is virulent toXa7 and also to Xa4,Xa10, Xa14, but avirulent to xa5. The full-length gene spans2118 bp and encodes a protein of 705 amino acids. Its molecular weight and IsolectricPoint is 74.7 kDa and 6.585, respectively. BLAST search in NCBI indicated that thegene belongs to avrBs3 gene family, and designated arp3 (AvrBs3-related protein,arp3). Alignment Arp3 with others AvrBs3–like proteins uncovered that theN-terminus of arp3 lacks of two segments, one is 144 bp, the other is 45 bp.Although the N-terminus character of arp3 is unique in avrBs3 gene family, it is socommonly in all Xoo strain genomic DNA. The central region of the arp3 containsonly 5.5 copies of 102 bp repeats, the smallest copy number of repeats found inavrBs3 gene family by now. Together with the repeats is heptad repeats,resemblingleucine zippers. Three functional nuclear localization signals (NLSs) and an acidicactivation domain (AAD) are also found in the C-terminal region. The functionalNLSs and AAD are necessary for the secretion of Avr protein into plant cell. Deletionof NLSs and AAD, Avr protein lost its virulent function. So here we assumed that theAvrBs3-like proteins are assembled proteins, which are suitable for the competitionbetween the pathogens and plants. The repeats in the middle region of Avr protein isthe basic functional domain. The N-terminus and C-terminus structures further helpAvr protein secretion from bacterium and into the plant cell, and express the normally III复旦大学博士学位论文 水稻白叶枯病菌无毒新基因的功能鉴定以及 Quorum Sensing 相关基因的克隆和表达function of Avr protein. Southern blotting data showed that the arp3 is present as a single-copy in genomicDNA of PXO339 and locus in plasmid clone. A prokaryotic expression systempGEX-4T-1-Arp3 was constructed. The arp3 could be expressed in vitro inEscherichia coli BL21 and a 128 kDa fusion protein was detected by Western analysis.Then the pGEX-4T-1-Arp3 expression vector was transformed into Xoo strain PXO99by electric transformation. The rice IRBB5, which contains xa5 resistant gene, wasinoculated with PXO99::pGEX-4T-1-Arp3 by clip inoculation when the rice seedlingsgrew at five leaves stages. The disease lesions of the inoculated leaves wereinvestigated after inoculation 11d. The results indicated that the disease in the leavesinoculated with PXO99::pGEX-4T-1-Arp3 is as serious as the leaves inoculated withPXO99only, which is susceptible to IRBB5. Arp3 may be a candidate avrxa5 gene andits function need to further identify. Two genes related to Quorum sensing were cloning from X. orzo pv. oryzae. One isXooR gene, which is a transcriptional factor and its full-length spans 765 bp. Theother is amino acid transporter (aat) gene, which neighbor to xooR, is a ATP-bindingprotein and the full-length spans 1377 bp. Southern blotting data showed that bothgenes are present as a single-copy in genomic DNA of PXO86. The prokaryoticexpression vector pET-24a-d (+) was used to construct the fusion protein expressionsystem. The vector wa...