Yeast genes involved in growth inhibition by Pseudomonas syringae pv. syringae syringomycin family lipodepsipeptides

Abstract Saccharomyces cerevisiae genes encoding functions necessary for inhibition by the Pseudomonas syringae pv. syringae cyclic lipodepsipeptide, syringomycin-E, were identified by mutant analyses. Syringomycin-E-resistant mutants were isolated, shown to contain single recessive mutations, and divided into eight gene complementation groups. Representative strains from five groups were resistant to nystatin, and deficient in the plasma membrane lipid, ergosterol. All of the mutant strains were resistant to the related cyclic lipodepsipeptides, syringotoxin and syringostatin. The findings show that: 1) at least eight gene-encoded functions participate in the inhibitory response to syringomycin; 2) ergosterol is important for this response; 3) the three related lipodepsipeptides have similar modes of action.     

Syringomicin production is stimulated by cysteine in Pseudomonas syringae pv. syringae

Abstract The influence of cysteine and serine in the production of syringomycin by Pseudomonas syringae pv. syringae has been studied. Both amino acids increased toxin synthesis in wild-type strains, although cysteine has a higher stimulatory effect than serine. To corroborate the role of cysteine in the production of syringomycin, a Cys⁻ mutant of P. syringae pv. syringae was isolated by transpositional mutagenesis with Tn5; this Cys⁻ mutant did not produce syringomycin. Nevertheless, and after the addition of high concentrations of cysteine, the cys ∷Tn5 mutant recovered its ability to produce syringomycin. On the other hand, the addition of serine did not return the production of syringomycin to the sys ∷ Tn5 strain: all these data indicated that cysteine modulates the synthesis of syringomycin in P. syringae pv. syringae positively.     

First Report of Bacterial Leaf Spot of Parsley Caused by Pseudomonas syringae pv. apii in Turkey

Since March, 2011, typical leaf spot symptoms were observed on parsley in several fields inspected in Hatay and Adana provinces of Turkey. Incidence of the disease was 5–15% in the regions. Symptoms were characterized as angular to irregular, initially water soaked later brown to dark black spots. Spots often limited by veins which were visible from both adaxial and abaxial sides of leaves but were not present on stems. Fluorescent bacterial colonies were consistently isolated from typical leaf spots. Biochemical tests, fatty acid methyl ester (FAME) analysis, molecular, pathogenicity tests and sequence of 16S ribosomal DNA of bacterial isolates were performed to identify possible causal disease agent. The causal disease agent was identified as Pseudomonas syringae pv. apii based on symptoms, biochemical, molecular, pathogenicity tests and sequencing. To our knowledge, this is the first report of bacterial leaf spot on parsley caused by Pseudomonas syringae pv. apii in Turkey.     

植物病原细菌Pseudomonas syringae pv. tomato基因组中的信号肽分析

应用SignalP 3.0 对植物病原细菌Pseudomonas syringae pv. tomato DC3000菌株基因组中的全部5 615个ORFs进行了分析,确定其中679个ORFs所编码蛋白质的N-端有信号肽序列,其中已经命名并有注释的有107个ORFs。信号肽的长度以19 ~31 个氨基酸居多,其中最多的是23 个氨基酸的信号肽。具有信号肽的ORFs编码蛋白的长度大多为101~400 个氨基酸之间。同时,对组成信号肽的氨基酸种类作了系统的分析,发现组成信号肽的氨基酸中非极性氨基酸占48.54%,极性氨基酸占18.67%,带负电荷氨基酸占24.54%,带正电荷氨基酸仅占8.00%,出现最多的3种氨基酸依次为亮氨酸、丙氨酸和丝氨酸,最少的氨基酸是异亮氨酸,在切割位点-1端的氨基酸中83.211%均为丙氨酸,在切割位点后3位的氨基酸中最多的氨基酸也是丙氨酸。通过分析确定628个分泌类信号肽,36个信号肽具有RR-motif的保守区段,15个脂蛋白类信号肽,未发现Prepilin-like 信号肽和Bacteriocin and Pheromone信号肽。     

大豆细菌性斑点病菌harpin编码基因的克隆与表达

摘要：【方法、目的】利用PCR方法从丁香假单胞菌大豆致病变种（Pseudomonas syringae pv. glycinea）Psg12菌株中克隆到1026bp的hrp基因。将其定向插入到表达载体pGEX-4T-1上,并转化宿主菌BL21,IPTG诱导表达后,SDS-PAGE显示其表达产物为分子量为61 kDa的融合蛋白质。【结果】该蛋白质在性质与功能上类似于已发现的harpins,即富含甘氨酸、不含半胱氨酸,热稳定以及对蛋白酶K敏感,能够在烟草上引起典型的过敏性反应,过敏性反应还可被真核生物代谢抑制     

丁香假单胞菌环式脂肽毒素的生理和分子生物学研究

综合评述了近10年来在丁香假单胞菌脂肽毒素生理和分子生物学研究上的发现。这些毒素依肽部AA数目可分两组。丁香假单胞霉素组(syringomycuns)已报告4个成员,肽部有9个AA;丁香假单胞肽毒素组有2个成员,肽部分别有22个和25个AA。肽部C端羧基与分子内羟基氨基酸残基(AA)的羟基酯化闭合成环,再由羟基脂肪酸酰化。两组毒素都诱导植物电解质渗漏、人和动物红血球溶解,其机制在于在细胞膜上形成二价阳离子可通过的寡体通道。对酵母菌的抑制作用受固醇的种类影响,以胆固醇的保护作用最强。丁香假单胞霉素的合成涉及一个多酶系统,有些负责肽合成,有些负责运输或调节,除受内源调节蛋白调节外,也受外源信号分子调节,尤其是受植物酚糖苷诱导。这些毒素具有抗真菌活性,对人和动物的一些病原霉菌有明显效果,在试验剂量无副作用,在医药上应用的前景良好。     

Effects of Pseudomonas syringae pv. morsprunorum Strain C28, Outer Membrane and LPS Preparations on Cherry Callus

Callus cultures were established from cherry (Prunus avium) cvs. Napoleon and Colt, respectively susceptible and resistant to race‐1 strains of Pseudomonas syringae pv. morsprunorum, by growth on Schenk–Hildebrandt medium. On Napoleon callus, necrosis began earlier and proceeded more rapidly when inoculated with the virulent race‐1 cherry isolate strain C28, than with mutants of diminished virulence derived from it, or with the virulent plum isolates D10 and D17. Colt tissue displayed poorer viability and showed susceptibility to strain C28 and the plum isolates. Callus from both sources was somewhat susceptible to the saprophytes P. aeruginosa NCIMB 8295 and P. fluorescens NCIMB 3756. Strain C28 grew on suspended Napoleon callus cells over a period of 3–4 days, causing leakage of UV‐absorbing compounds and K⁺, with a concomitant rise in extracellular pH. P. fluorescens NCIMB 3756 showed no growth on suspended callus for 6 days. EDTA‐extracted outer membrane (OM) from strain C28 caused leakage of UV‐absorbing material and K⁺, which was later reabsorbed, with little change in pH. The presence of OM suppressed the growth of a subsequent inoculum of strain C28, possibly due to complexation of the available Ca²⁺ and/or Mg²⁺ in the surrounding medium, by the component lipopolysaccharide (LPS). OM from the rough avirulent mutant strain C28‐2 induced leakage of K⁺, but not of UV‐absorbing material, and did not prevent the growth of subsequently inoculated strain C28. Smooth LPS from strain C28 did not cause leakage of K⁺ or of UV‐absorbing material and did not prevent growth of C28. The relevance of these findings is discussed in relation to disease.     

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.     

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.     

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

丁香假单胞大豆致病变种两个III型效应因子的克隆及功能分析

为了研究Ⅲ型泌出效应因子在丁香假单胞大豆致病变种中的作用,利用反向PCR技术,首次从丁香假单胞大豆致病变种全基因组中克隆得到两个效应因子HopAB1和HopAF1基因的同源物,分别命名为HopAB1s和HopAF1s。生物信息学分析表明,HopAB1s基因全长是1 572 bp,编码523个氨基酸;HopAF1s基因全长是855 bp,编码284个氨基酸。即基因的登录号分别为JF826562和JF826563。保守功能区预测显示HopAB1s在N末端包含一个E3泛素连接酶功能区。将这2个基因克隆到PVX二元表达载体并转化农杆菌,利用农杆菌介导的瞬时侵染技术在本生烟中表达,发现2个效应因子均能抑制由鼠凋亡因子激发的细胞程序性死亡;将烟草疫霉接种在表达效应基因的区域,发现效应因子能促进烟草疫霉侵染烟草,因此本研究得到的两个效应因子是免疫抑制因子,为进一步研究该菌的致病机理奠定基础。     

Pseudomonas syringae pv. actinidiae detection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.     

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean ( Phaseolus vulgaris L.).
Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium , Erwinia and Pantoea .
Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria .
Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.     

Development of pea bacterial blight caused by Pseudomonas syringae pv. pisi in winter and spring cultivars of combining peas (Pisum sativum) with different sowing dates

Pea bacterial blight occurred by natural infection in a field trial on peas in 1995. Disease development in the winter cultivars Rafale, Frilene and Froidure was compared with that in the spring cultivars Baccara, Conquest and Bohatyr, each sown on six dates in October, November, December, mid-March, late March and April. Disease incidence had reached 100% plants affected in all treatments by mid-July. Disease severity was greater in winter-sown (October, November or December) than in spring-sown peas of each cultivar at each assessment. Significant (P < 0.05) differences in disease severity occurred between cultivars in the winter-sown plots in May and June and the spring cultivars were affected more severely than the winter cultivars. Comparison of areas under the disease progress curves for both disease incidence and severity also showed that the winter-sown peas were more affected by disease than spring-sown peas and that spring cultivars were more severely affected than winter cultivars. Yield was strongly correlated with disease severity. A linear regression model suggested that, for peas sown in October, November or December, a yield loss of 0.5 tha^-1 occurred for each 10% increase in canopy area affected by pea bacterial blight.     

Structure of the Lipopolysaccharide Side Chain of Pseudomonas syringae pv. tabaci Strain NCPPB 79 (=CFBP 1615), in Relation to O-serogroup

The lipopolysaccharide (LPS) side chain from Pseudomonas syringae pv. tabaci strain NCPPB 79 (=CFBP 1615) contained l ‐ and d ‐rhamnose, and GlcNAc. Using methylation analysis, periodate oxidation, Smith degradation and ¹H‐ and ¹³C‐nuclear magnetic resonance spectroscopy, the repeat unit was found to have the structure: This structure is correlated with a previously proposed serogrouping system. The involvement of LPS generally in plant disease is briefly discussed.     

Assessment of strains of Pseudomonas syringae pv. tomato from Tanzania for resistance to copper and streptomycin

Abstract

Fifty-six strains of Pseudomonas syringae pv. tomato (P.s. pv. tomato) were collected from tomato-producing areas in Tanzania and assessed for resistance to copper and antibiotics. The collection was done from three tomato-producing regions (Morogoro, Arusha and Iringa), representing three different ecological conditions in the country. After isolation and identification, the P. s. pv. tomato strains were grown on King's medium B (KB) amended with 20% copper sulphate (w/v). The strains were also assessed for resistance to antibiotics. Results indicated that there was widespread resistance of the P. s. pv. tomato strains to copper sulphate. The highest level of resistance was recorded from the Arusha region (Northern Tanzania), 83.3% of the P. s. pv. tomato strains from that region showed resistance to copper sulphate. This was followed by Iringa region (Southern Tanzania), from where strains of the pathogen were moderately resistant to copper sulphate, such that 54.0% of them were able to grow on the KB medium amended with 20% (w/v) of the copper compound.

Out of seven strains of P. s. pv. tomato from Morogoro region (Central Tanzania) included in the study, five (71.5%) were resistant to copper sulphate. The only strain of P. s. pv. tomato from the Dodoma region (Central Tanzania, but with a different ecological condition from the Morogoro region) included in the study was unable to grow on the medium containing 20% copper sulphate. None of the P. s. pv. tomato strains in the four regions included in the study were resistant to streptomycin sulphate. These results suggest that in the Arusha and Iringa regions of Tanzania, there might be possibilities of excessive use of copper compounds in tomato production, such that strains of P. s. pv. tomato strains in the areas have become resistant to the compounds.     

Cloning of genes by mRNA differential display induced during the hypersensitive reaction of soybean after inoculation with Pseudomonas syringae pv. glycinea

Soybean (Glycine max[L.] Merr.) cell suspension cultures (cv. Williams 82) inoculated with the pathogenic bacteria Pseudomonas syringae pv. glycinea respond with a hypersensitive reaction (HR) when the bacteria express the avirulence gene avrA. A mRNA differential display was established for this system to allow the identification of genes induced during the HR. Six PCR-fragments (DD1–DD6) from the differential display analysis were identified, which are induced during the HR. Database searches revealed that the fragment DD1 encodes chalcone isomerase and DD2 was identified as ubiquitin. The fragment DD3 shares significant homology to the signalling molecule 14-3-3. The partial DD4 product is homologous to the enhancer of rudimentary from Drosophila and an uncharacterized homologue of it from Arabidopsis. The fragment DD5 is similar to glucose-6-phosphate dehydrogenase which provides NADPH to the cell. The PCR-product DD6 seems to be a new leucine-rich-repeat disease resistance gene from soybean, which is significantly induced during the HR. All of the identified genes are clearly induced during a HR in infected plants of the same cultivar, indicating that results from the cell culture model system can be transferred to intact plants. These studies show that complex mRNA differential display is a powerful tool to identify new induced gene in plant-pathogen interactions.     

Structure of the Lipopolysaccharide Side-chain of Pseudomonas syringae pv. syringae Strain S29

Using ¹H‐ and ¹³C‐nuclear magnetic resonance spectroscopy, the repeat unit of the lipopolysaccharide side‐chain from Pseudomonas syringae pv. syringae strain S29 was shown to have the following structure: This structure is identical with that of the side‐chain of Pseudomonas syringae pv. mori CFPB 1656. a     

In vitro Antibiosis by Pseudomonas syringae Pss22d,Acting Against the Bacterial Blight Pathogen of Soybean Plants,Does Not Influence In planta Biocontrol

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species‐specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3‐methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co‐inoculated with either Pss22d wild‐type, a syringopeptin/syringomycin‐negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg‐negative mutant (Pss22d.1) into wounds of pin‐pricked leaves of greenhouse‐grown soybean plants, respectively. In all three cases, the wild‐type Pss22d and its toxin‐deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d’s antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra‐species antagonism in planta.     

Phytotoxin production by Pseudomonas syringae pv. syringae: Syringopeptin production by syr mutants defective in biosynthesis or secretion of syringomycin

Abstract Syringomycin and syringopeptin are lipodepsipeptide phytotoxins produced by Pseudomonas syringae pv. syringae . Four syr genes were identified previously and hypothesized to be involved in the regulation ( syrA ), biosynthesis ( syrB and syrC ), or export ( syrD ) of syringomycin. This study determines the influence of syr mutations on the composition of phytotoxic metabolites produced by P. syringae pv. syringae strain B301D-R. Levels of syringomycin and syringopeptin produced in liquid cultures were estimated by reverse phase HPLC analyses and differential antimicrobial assays. Significant quantities of syringopeptin were produced by both syrB and syrC mutants despite their inability to produce syringomycin. Only trace quantities of both lipodepsipeptides were produced by syrA and syrD mutants of P. syringae pv. syringae . These results indicate that syringomycin and syringopeptin are synthesized by separate pathways, but may share common mechanisms for secretion and regulation.