Harpinpsta from Pseudomonas syringae pv. tabaci Is Defective and Deficient in Its Expression and HR-inducing Activity

To elucidate the role of harpins produced by Pseudomonas syringae, the corresponding hrpZ gene was isolated from P. s. pv. tabaci. The sequence information revealed that this gene carries a serious mutation with 326 bp lacking in the central region and potentially encodes only 140 N-terminal amino acids because of a frame shift. The investigation of biological properties using recombinant harpin indicated harpin_psta was incapable of inducing HR in both host and nonhost plants. Based on an immunoblot analysis to detect harpin from P. s. pathovars in hrp-inducing medium, the truncated harpin_psta was neither expressed nor secreted into the culture medium. These results suggest that harpin is not the sole determinant of the host-parasite specificity in P. s. pv. tabaci. Received 10 August 2000/ Accepted in revised form 21 December 2000     

利用编码harpin蛋白的基因遗传改良生防荧光假单胞菌2P24

荧光假单胞菌Pseudomonas fluorescens 2P24是根围促生细菌（PGPR）,具有Ⅲ型分泌系统（T3SS）。为了在2P24中表达植物过敏反应激发子harpin,赋予生防菌诱导抗病性能力,本文选择可在2P24中表达的来自Pseudomonas syringae pv.tomato DC3000的avrPto1基因启动子与水稻细菌性条斑病菌Xanthomonasoryzae pv.oryzicola harpin蛋白编码基因hpa1进行融合,实现了harpin蛋白在2P24的表达。重组菌株通过T3SS分泌harpin蛋白,可激发烟草产生过敏反应（HR）,激活HR途径的HIN1基因和HRS203J基因以及病程相关蛋白PR1a基因的转录表达。harpin重组菌株与2P24一样,对小麦赤霉病菌Fusarium graminearum和棉花枯萎病菌F.oxysporum f.sp.vasinfectum具有抑制作用。这为利用植物-病原物互作中激发植物产生抗病性的激发子来遗传改良生防微生物奠定了理论和实践基础。     

Erwinia amylovora hrpN mutants,blocked in harpin synthesis,express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units     

水稻细条斑病菌的化学诱变及hrp基因突变体的功能互补研究

硫酸二乙酯（ＤＥＳ）化学诱变水稻细条斑病菌（Ｘａｎｔｈｏｍｏｎａｓｏｒｙｚａｅｐｖ．ｏｒｙｚｉｃｏｌａ）ＲＳ１０５获得６株ｈｒｐ突变体,其在寄主水妥上失去致病性和在非寄主烟草上失去激发过敏反应（ｈｒｐｅｒｓｅｎｓｉｔｉｖｅ ｒｅｓｐｏｎｓｅ,ＨＲ）的能力。将水稻白叶枯病菌（Ｘ．ｏｒｙｚａｅｐｖ．ｏｒｙｚａｅ）日本系统Ⅲ号小种（ＪＯＸⅢ）基因文库和ＲＳ１０５基因文库互补ＲＳ１０５ｈｒｐ突变体Ｍ５５,     

Erwinia chrysanthemi分离株CSCL006 hrpN基因的克隆与高效表达

通过构建Erwiniachrysanthemi分离株CSCL006的DNA文库,克隆出hrpNCSCL006基因,测序结果显示该基因编码区长1020bp;推导的harpinCSCL006与ErwiniachrysanthemiEC16和3937编码的harpin蛋白同源性高,但与其它软腐菌的harpin蛋白同源性较低;在大肠杆菌中(Escherichiacoli)高效表达了hrpNCSCL006基因,重组harpinCSCL006蛋白分子量为34kD。以抗harpinEcc的抗体为探针,Westernblot证实该蛋白确为harpin;纯化的harpinCSCL006,能引起烟叶的过敏反应。     

表达Harpin蛋白质Hpa1功能片段的转基因小麦对赤霉病的抗性

来自水稻黄单胞菌的harpin蛋白质Hpa1有促进植物生长、诱导植物抗病性的功能,Hpa1序列10~42氨基酸片段（Hpa1_10-42）的活性比全长分子高1.3~7.5倍。为研究Hpa1_10-42在转基因小麦体内表达以后对赤霉病的影响和评价转基因小麦抗病水平与应用潜力,对6个小麦转基因系进行了测定。结果显示,Hpa1_10-42在转基因系T₃~T₅代呈现稳定表达,用禾谷镰刀菌一个分离物进行接种以后,转基因系发生赤霉病的程度较非转基因小麦显著降低,且转基因系T₃~T₅代赤霉病降低的趋势一致。另外,转基因系小麦病害减轻的程度,与在非转基因小麦上使用杀菌剂的效果相当,表明Hpa1_10-42转基因表达对小麦赤霉病有防治作用。     

产harpin的成团泛菌工程菌的构建

 梨火疫病菌(Erwinia amylovora)产生的胞外蛋白harpin是引发其非寄主植物过敏反应的因子。harpin蛋白还具有诱导植物抗性的功能。成团泛菌(Pantoea agglomerans)308R对链格胞属(Alternaria)的真菌具有拮抗活性,是一株抗利福霉素的自发突变株,对壮观霉素敏感。本文通过电击转化法将带有梨火疫病菌hrp基因簇的重组质粒pCPP430导入308R。所得转化子具有pCPP430的壮观霉素抗药性标记,在番茄上引起过敏反应;从转化子中提取到与pCPP430大小一样的质粒,其酶切物理图谱也与pCPP430完全相同;用地高辛标记的harpin的结构基因hrpN为探针对转化子中的质粒进行Southern blotting分析,结果在转化子中得到信号明显的杂交片段,而受体菌308R中未出现杂交信号;提取细菌的外壁的蛋白质进行PAGE分析,在大约44 kd的位置上308R(pCPP430)比308R明显多出1条带。这些结果表明成团泛菌能够表达梨火疫病菌的hrp基因簇,所产harpin蛋白也是有活性的。     

超敏蛋白对烟草黄瓜花叶病毒的诱导抗性及生长、品质的影响

为明确超敏蛋白对烟草黄瓜花叶病毒(Cucumber mosaic virus,CMV)的诱导抗性及其生长、品质的影响,采用半叶枯斑法和田间试验研究了10%超敏蛋白对枯斑三生烟和普通烟的诱导抗性及促生作用,并分析了对烤烟经济性状和内在质量的影响。结果表明,10%超敏蛋白在活体外对CMV无钝化作用,但预防、治疗效果显著,接种前48 h喷施预防效果达75.70%,接种后24 h喷施治疗效果达65.45%,均显著高于对照;10%超敏蛋白处理后烟草的株高、茎围、最大叶面积均高于其它处理,单位产量比20%吗啉胍·乙铜可湿性粉剂处理增产211.8 kg/hm2,增值3 519.2元/hm2;经10%超敏蛋白处理后的烤烟化学成分均处于优质烟的适宜质量分数范围内。表明超敏蛋白不仅有利于提高烟草抗性和促进烟株生长,且可以明显提高烟叶经济效益和烟叶内在质量。     

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.     

采后Harpin处理对损伤接种苹果梨黑斑病的影响

研究harpin处理对苹果梨采后黑斑病的影响,结果表明:离体试验harpin对黑斑病没有抑制作用。经损伤的苹果梨(Pyrus pyrifolia cv. Pingguoli),用15 mg/L、30 mg/L、60 mg/L的harpin溶液浸泡处理后接种链格孢(Alternaria alternata (Fr.) Keissl.),在低温条件下的贮藏结果表明:采后harpin浸泡处理可明显降低损伤接种苹果梨的黑斑病发病率,3种浓度中以30 mg/L的harpin溶液处理效果最好,而常温条件下对发病率无影响。在常温和低温条件下均可抑制病斑的扩展速度,3种浓度中以60 mg/L的harpin溶液处理效果最好。