Harpin xoo在水稻中表达提高对白叶枯病不同小种抗性

用剪叶接种法测定10个转hrf1基因水稻株系对15个水稻白叶枯病菌小种的抗性.所有的转基因系对JxOV和PXO79小种的抗性与受体对照相比显著提高,RT-PCR结果显示,在转化hrf1基因的抗病水稻中,信号传导基因npr1表达明显增强,表明转hrf1基因水稻可能通过激发水稻中信号传导基因的表达,提高对白叶枯病菌不同小种的抗性.     

水稻白叶枯病菌harpin基因的克隆与表达

 用PCR方法从水稻黄单胞菌白叶枯致病变种(Xanthomonas oryzae pv.oryzae,Xoo) JxoIII菌株中克隆到编码诱导植物过敏性反应激发子的基因hrfA。同源性比较发现其推测产物与水稻白叶枯病菌菲律宾小种Pxo86的Hpa1有97.2%的序列一致性,比Hpa1少了一个-GGG-GG-氨基酸残基序列重复。基因经NdeI和BamHI双酶切后连到含T7启动子的高表达载体pET 30a (+)上构建重组质粒pHRF4,并转化宿主菌BL21(DE3)产生表达菌株BLHR4。经过IPTG诱导之后,BLHR4产生一分子量为15.6 kD的蛋白质。研究表明,该蛋白在性质与功能上类似于其它已发现的harpins,即能够在烟草上引起典型的过敏性反应,富含甘氨酸、热稳定以及对蛋白酶敏感。因此,我们把该蛋白定名为harpin_Xoo。harpin_Xoo还具有诱导植物抗病性的功能。     

Effect of Foliar Applied Plant Elicitors on Microbial and Nematode Populations in the Root Zone of Potato

Abstract

Systemic acquired resistance (SAR) is a process whereby a plant that successfully resists a pathogen becomes highly resistant to subsequent infection not only by the original pathogen but also by a wide variety of pathogens. Most SAR research has focused on resistance in leaves, so much less is known about the effectiveness of foliar applications of SAR compounds in the protection of plant roots and associated microorganisms in soil. This study was conducted to determine if foliar SAR‐inducing applications (BTH or harpin) negatively impact the potato root system beneficial rhizosphere microbial populations and activity or influence pathogenic nematode populations. Foliar applications of benzo (1,2,3) thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) and the microbial protein harpin applied in various combinations, timings, and rates showed no effects on microbial biomass, culturable bacteria, Pseudomonas populations, or N‐mineralization potentials over 2 years. No stimulatory or inhibitory effects on major bacterial populations were observed, indicating that SAR induction does not have a negative effect on general microbial populations or activities. BTH and harpin both reduced the numbers of lesion nematodes (Pratylenchus spp.) by potato harvest. BTH reduced root knot nematodes, Meloidogyne chitwoodi at the end of the season. In addition, BTH and high‐dose harpin (applied at the 4× rate) reduced the nematode infection index in comparison to the control. The SAR elicitors increased the population densities of nontarget free‐living nematodes in the soil compared to the control. Potato yields were not affected by plant elicitors but BTH and harpin both reduced the number of culled potatoes 26% compared to the control. Future studies are designed to determine if these plant elicitors have any direct effect on rhizosphere diversity or if plants with active defense pathways alter carbon flow and root exudates into the soil.     

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)化学诱变hrp基因突变体及相关性状的研究

 硫酸二乙酯(diethyl sulfate,DES)化学诱变水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)日本系统III号小种(JXOIII),获得16株hrp-突变体,其在感病寄主水稻IR26、汕优63上失去致病性和在非寄主烟草上失去过敏反应。所得的hrp-突变体并无黄色素缺失现象。16株hrp-突变体的淀粉酶、果胶酶、蛋白酶、纤维素酶和脂酶等胞外酶活性基本与野生型一致。JCM2092、JCM2211、JCM2252和JCM2457这4株hrp-突变体为III型泌出通道突变体,JCM0066等另12株hrp-突变体的超声波破碎液、离心上清液中无激发HR的信号物质存在。细胞组分分析表明,激发烟草HR的信号物质不是LPS而是蛋白类物质。来自JXOIII基因文库hrp基因克隆pUHRX245中的1.1kb SacI-KpnI片段,能够互补hrp-突变体JCM0066和JCM2482,使其恢复在烟草上激发HR的能力,但不能恢复其致病性。     

超敏蛋白对低温胁迫下茶树生理特性的影响

 超敏蛋白是一种诱导植物抗逆性的新型生物制剂。为了解超敏蛋白在低温胁迫过程中对茶树耐寒性的影响,以茶树品种‘迎霜’为试材,分别在常温和4 ℃下喷施超敏蛋白,研究了在低温胁迫下超敏蛋白对茶树叶片生理特性的影响。结果表明：常温下,超敏蛋白喷施的茶树叶片,在6 d内略微提高了渗透调节物质含量和抗氧化酶活性。低温胁迫下,茶树叶片中可溶性糖含量、可溶性蛋白含量、游离脯氨酸含量、丙二醛（MDA）含量、超氧化物歧化酶活性（SOD）、过氧化物酶活性（CAT）、过氧化氢酶（POD）活性均增加,叶绿素含量降低;喷施24 μg ? L^-1超敏蛋白后,低温下可在6 d内提高茶树叶片叶绿素、可溶性蛋白、可溶性糖、游离脯氨酸的含量,提高SOD、CAT、POD活性,从而缓解低温胁迫对茶树的伤害,增强茶树耐寒性。试验表明,喷施一定浓度的超敏蛋白可以有效提高茶树的耐寒性。     

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZ_Pst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.     

Effect of Harpin from Four Pathovars of Pseudomonas syringae on Pea Defense Responses

To investigate the role of the proteinaceous elicitor, harpin, on host and nonhost plants, we isolated the harpin-coding gene, hrpZ, from Pseudomonas syringae pvs. pisi, glycinea, tabaci and tomato. Effects of the recombinant harpin proteins on pea plants were analyzed and compared with the effects of the corresponding bacterial treatment. After inoculation of pea with pea pathogen P. syringae pv. pisi, the bacterial population increased and the accumulation of PAL-mRNA and pisatin was inhibited. The nonpathogenic pathovars, glycinea, tabaci and tomato induced both defense responses in pea. However, none of the harpins induced the hypersensitive reaction or accumulation of PAL-mRNA and pisatin in pea. Harpins from P. syringae pvs. glycinea, tomato and pisi did induce these defense responses in tobacco, however, suggesting that externally applied harpins either are not recognized or are nonfunctional in pea plants. Received 27 June 2000/ Accepted in revised form 21 February 2001     

异源Harpin对软腐欧氏杆菌与植物互作关系的影响

 本文通过三亲交配将带有梨火疫欧文氏杆菌功能完整的hrp基因簇的重组粘粒pCPP430转移到了胡萝卜软腐欧氏杆菌(Erwinia carotovora) Se9R中。从接合子中提纯的质粒与pCPP430的大小、酶切图谱相同;以该质粒为模板,用针对hrpN序列的引物经PCR扩增得到与hrpN大小相同的片段;用地高辛标记的hrpN作为探针进行Southern blotting分析,在接合子中的质粒上得到预期的杂交带。接合子可以在烟草叶片上稳定地引起过敏反应。另外,接合子可以在寄主马铃薯叶片上引起过敏反应样枯斑,表明异源harpin的表达及功能不受软腐菌的抑制。该接合子在体外产生的果胶酸裂解酶活性与受体菌Se9R无明显差别。低浓度的接合子在马铃薯块茎上的软腐症状明显低于受体菌Se9R。外源harpin表达可能不改变软腐欧氏杆菌的致病力、而是通过诱导马铃薯抗性达到减轻发病的作用。     

烟草野火病菌hrpZ基因的克隆及蛋白的原核表达

采用PCR方法从烟草野火病病原菌Psta218中扩增harpin编码基因hrpZ,将其克隆到原核表达载体pGEX-4T-1上,获得重组表达质粒pGEX-hrpZPsta。将重组质粒pGEX-hrpZPsta转化至大肠杆菌BL21(DE3)菌株,得到重组大肠杆菌BL21/pGEX-hrpZPsta。经IPTG诱导得到14.7 kD的蛋白质。该蛋白质与其他已发现的harpins一样,能够使烟草等植物产生过敏性坏死反应、富含甘氨酸、热稳定以及对蛋白酶K敏感。