Virulence, accumulation of acetyl-coenzyme A, and pectate lyase synthesis are controlled by PhoP-PhoQ two-component regulatory system responding to organic acids of Erwinia chrysanthemi 3937

The PhoP-PhoQ two-components regulatory system is involved in the pathogenesis of animal, plant, and insect pathogenic bacteria in response to various environmental factors. To elucidate how this system contributes to the plant pathogenesis of Erwinia chrysanthemi 3937 (Ech 3937), marker-exchanged mutants of phoP and phoQ were constructed. Their role in the regulation of a major virulent factor, pectate lyase (Pel), in response to various organic acids was then tested. These mutants synthesized more Pel than did the wild type in the medium containing acetate or citrate as the sole source of carbon, but they synthesized less Pel than did the wild type in pyruvate or malate as the sole source of carbon. Synthesis of Pel did not differ in succinate, fumarate, or glycerol from the wild type. The phoP and phoQ mutants grown and resuspended in acetate or citrate also caused more maceration, and the wild type pretreated in pyruvate or malate caused more maceration than did the mutants. The level of intracellular acetyl-coenzyme A (acetyl-CoA) almost paralleled the synthesis of Pel in the wild type and in the mutants of the phoP and phoQ. These results suggested that acetyl-CoA may be involved in regulation of Pel synthesis through two-independent regulatory cascades via the PhoP-PhoQ system (in an opposite manner) in response to acetate/citrate and pyruvate/malate. However, ackA and pta genes, involved in the synthesis of acetyl-CoA in Escherichia coli, were not expressed as predicted on the basis of the level of acetyl-CoA. Thus there may be an additional regulation or pathway for the synthesis of acetyl-CoA in Ech 3937.     

d -Alanine-d -alanine ligase gene ( ddl ) of Erwinia chrysanthemi strain EC16 II: analysis of pectate lyase regulation using ddl ? mutant

 Erwinia chrysanthemi (Ech) triggers soft rot disease mainly by secreting pectate lyase (Pel), which is regulated in a complex manner by many regulatory genes. In a previous study, we used a gene dosage method to show that the ddl gene, which encodes d-alanine-D-alanine ligase, reduced Pel production and tissue maceration by Ech strain EC16n. In this study, the ddl ⁻ marker-exchanged mutant was shown to overcome the long growth lag caused by various salts in the growth medium and to increase Pel production over that by EC16n, especially in a medium containing magnesium salts. Thus, ddl seems to regulate Pel production in a negative manner. Because the profiles of a gel shift assay using the pelE promoter region as the target DNA with crude extracts of EC16n and ddl ⁻ mutant were distinguishable, Ddl is thought to affect the binding of other regulatory proteins. Expression of the ddl gene was induced in the medium containing a low-molecular-weight fraction of potato extract, but it was reduced in that containing both polygalacturonic acid (PGA) and the fraction. The repression of ddl expression by PGA should contribute in part to the in planta hyperinduction of Pel. Received: May 15, 2002 / Accepted: June 20, 2002     

水稻抗白叶枯病基因Xa47(t) a的敲除及抗性鉴定

为鉴定水稻Xa47(t)a基因对白叶枯病的抗性,以水稻种质L214为材料,通过构建针对Xa47(t)a基因的Xa47(t)a-Cas9敲除载体来获得敲除突变体,利用生物信息学技术对突变的Xa47(t)a基因进行突变类型分析,同时采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术分析突变体中Xa47(t)a及病程相关基因的表达情况,并于水稻孕穗期对突变体及其野生型植株接种11株白叶枯病菌Xanthomonas oryzae pv. oryzae菌株进行抗性鉴定。结果表明,在Xa47(t)a基因的第2外显子区域进行基因编辑后成功获得25株T₀代突变体株系;测序分析发现T₀代突变体株系中有13种不同的突变体类型,其中纯合突变体有3种类型,且突变位点均在靶标位点的11位碱基处缺失1～4个A碱基;氨基酸序列分析发现大部分突变体中Xa47a编码的蛋白翻译会提前终止,由原来的803个氨基酸变为144～166个氨基酸;qPCR分析结果表明突变体中Xa47(t)a及大部分病程相关基因的表达水平显著低于野生型株系;抗性鉴...     

Multiple copies of <Emphasis Type="Italic">AMT2</Emphasis> are prerequisite for the apple pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis> to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.     

D-Alanine-D-Alanine Ligase Gene (ddl) of Erwinia chrysanthemi Strain EC16 I. Isolation and Gene Dosage Effect on Pectate Lyase Synthesis

New regulatory gene for pectate lyase (Pel) production of Erwinia chrysanthemi strain EC16 was searched by observing the gene dosage effect of each cosmid library in Pel production. From this survey, a cosmid clone, p5A, had reduced in Pel production under both inducing and non-inducing conditions and caused less tissue maceration of potato tubers. The 2.64-kb HindIII fragment from p5A was found to be responsible for this phenotype and to contain one major ORF consisting of 1107 nucleotides, which had homology with ddlA (49.6%) and ddlB (49.6%) of Escherichia coli and with ddlA (44.6%) of Salmonella typhimurium. These genes had been shown to encode D-alanine-D-alanine ligase (Ddl), an enzyme which is involved in the synthesis of peptidoglycan. This ORF encodes 368 amino acid residues and its molecular weight is estimated to be 43 kDa. This ORF of EC16 could complement ddl deficient mutant of Escherichia coli. Received 15 May 2002/ Accepted in revised form 20 June 2002     

The in vivo sensitivity of ATPases to DDT,DDE, and physical immobilization in American cockroaches

American cockroaches injected with sublethal doses of DDT (0.75 μg/roach) at 5-day intervals showed a 40% reduction in oligomycin-sensitive Mg²⁺ATPase from muscle homogenates, and a 23% reduction of Na⁺-K⁺ATPase from nerve cords. Thus, the maximum effect measured occurred with the same enzyme and tissue as determined from in vitro studies. The metabolite, DDE, used at 15 μg per roach, gave no significant change in activity of the ATPase system following injection. In contrast, high single doses of DDT (7.5 μg/roach) and 100 μg DDE and dicofol per roach caused over 30% increase in oligomycin-sensitive Mg²⁺ATPase of muscle and a 10–15% increase in Na⁺-K⁺ATPase of nerve cords measured 24 and 48 hr later. While a similar response was observed for Mg²⁺ATPase activities in cockroaches that were immobilized, the increase in enzyme activities were much greater than that caused by the pesticides.     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

番木瓜环斑病毒西瓜株系弱毒突变体的筛选与应用

为防治番木瓜环斑病毒(Papaya ringspot virus,PRSV)在我国葫芦科作物上引起的病毒病害,以PRSV西瓜株系山东分离物(PRSV-SD)侵染性cDNA克隆为基础,采用定点突变方法将辅助成分-蛋白酶保守氨基酸137位和346位的天冬酰胺(N)和417位的缬氨酸(V)突变为丙氨酸(A),应用农杆菌浸润法接种西葫芦叶片并分析突变对PRSV-SD致病力的影响,筛选弱毒突变体,进而评价其交叉保护效果。结果表明,与野生型PRSV-SD相比,获得的3个突变体N137A、N346A和V417A,接种后在西葫芦植株上的症状明显减轻,衣壳蛋白在叶片中的积累水平分别为野生型PRSV-SD的24.0%、13.0%和4.0%,均为弱毒突变体。当保护间隔期为10 d时,弱毒突变体N137A具有完全的交叉保护效果,N346A可延迟发病15 d,而V417A无交叉保护效果。当间隔保护期为15 d时,弱毒突变体N137A和N346A的保护效率分别为100.0%和26.7%,而V417A无交叉保护效果。