植物病原细菌Ralstonia solanacearum GMI1000中分泌蛋白信号肽分析

利用SignalP 3.0、TMHMM 2.0、TargetP 1.01、LipoP 1.0和PSORTb蛋白分析软件并结合L值计算,对植物病原细菌Ralstonia solanacearum GMI1000菌株基因组中的全部3 440个ORFs进行了分析预测,确定其中186个ORFs所编码蛋白质的N-端有信号肽序列,且它们的氨基酸残基相对保守.其中134条具有分泌型信号肽,22条具有RR-motif型信号肽,30条具有信号肽酶Ⅱ型信号肽.对各类信号肽及其结构域的长度作了系统的分析.未发现Prepilin-like信号肽和细菌素和信息素信号肽.     

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.     

Tomato SlSAP3, a member of the stress-associated protein family,is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000

Tomato stress-associated proteins (SAPs) belong to A20/AN1 zinc finger protein family, some of which have been shown to play important roles in plant stress responses. However, little is known about the functions and underlying molecular mechanisms of SAPs in plant immune responses. In the present study, we reported the function of tomato SlSAP3 in immunity to Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlSAP3 attenuated while overexpression of SlSAP3 in transgenic tomato increased immunity to Pst DC3000, accompanied with reduced and increased Pst DC3000-induced expression of SA signalling and defence genes, respectively. Flg22-induced reactive oxygen species (ROS) burst and expression of PAMP-triggered immunity (PTI) marker genes SlPTI5 and SlLRR22 were strengthened in SlSAP3-OE plants but were weakened in SlSAP3-silenced plants. SlSAP3 interacted with two SlBOBs and the A20 domain in SlSAP3 is critical for the SlSAP3-SlBOB1 interaction. Silencing of SlBOB1 and co-silencing of all three SlBOB genes conferred increased resistance to Pst DC3000, accompanied with increased Pst DC3000-induced expression of SA signalling and defence genes. These data demonstrate that SlSAP3 acts as a positive regulator of immunity against Pst DC3000 in tomato through the SA signalling and that SlSAP3 may exert its function in immunity by interacting with other proteins such as SlBOBs, which act as negative regulators of immunity against Pst DC3000 in tomato.     

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.     

Host specificity,pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.     

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。     

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.     

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.     

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     

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