Bacterial and plant natriuretic peptides improve plant defence responses against pathogens

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP‐like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP‐A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over‐expressing XacPNP, lines over‐expressing AtPNP‐A and AtPNP‐A‐deficient plants were generated. Plants over‐expressing XacPNP or AtPNP‐A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP‐deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP‐A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over‐expressing XacPNP or AtPNP‐A were more resistant to Pst infection than control plants, whereas PNP‐deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non‐host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.     

Comparative genomics reveals genes significantly associated with woody hosts in the plant pathogen Pseudomonas syringae

The diversification of lineages within Pseudomonas syringae has involved a number of adaptive shifts from herbaceous hosts onto various species of tree, resulting in the emergence of highly destructive diseases such as bacterial canker of kiwi and bleeding canker of horse chestnut. This diversification has involved a high level of gene gain and loss, and these processes are likely to play major roles in the adaptation of individual lineages onto their host plants. In order to better understand the evolution of P. syringae onto woody plants, we have generated de novo genome sequences for 26 strains from the P. syringae species complex that are pathogenic on a range of woody species, and have looked for statistically significant associations between gene presence and host type (i.e. woody or herbaceous) across a phylogeny of 64 strains. We have found evidence for a common set of genes associated with strains that are able to colonize woody plants, suggesting that divergent lineages have acquired similarities in genome composition that may form the genetic basis of their adaptation to woody hosts. We also describe in detail the gain, loss and rearrangement of specific loci that may be functionally important in facilitating this adaptive shift. Overall, our analyses allow for a greater understanding of how gene gain and loss may contribute to adaptation in P. syringae.     

Monitoring the rate of oxygen consumption in plant cell suspensions

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

High levels of cyclic‐di‐GMP in plant‐associated Pseudomonas correlate with evasion of plant immunity

The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour.     

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.     

Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity

The HrpZ1 gene product from phytopathogenic Pseudomonas syringae is secreted in a type-III secretion system-dependent manner during plant infection. The ability of HrpZ1 to form ion-conducting pores is proposed to contribute to bacterial effector delivery into host cells, or may facilitate the nutrition of bacteria in the apoplast. Furthermore, HrpZ1 is reminiscent of a pathogen-associated molecular pattern (PAMP) that triggers immunity-associated responses in a variety of plants. Here, we provide evidence that the ion pore formation and immune activation activities of HrpZ1 have different structure requirements. All HrpZ1 orthologous proteins tested possess pore formation activities, but some of these proteins fail to trigger plant defense-associated responses. In addition, a C-terminal fragment of HrpZ1 retains the ability to activate plant immunity, whereas ion pore formation requires intact HrpZ1. Random insertion mutagenesis of HrpZ1 further revealed the C terminus to be important for the PAMP activity of the protein. HrpZ1 binds to plant membranes with high affinity and specificity, suggesting that the activation of plant immunity-associated responses by HrpZ1 is receptor-mediated. Our data are consistent with dual roles of HrpZ1 as a virulence factor affecting host membrane integrity, and as a microbial pattern governing the activation of plant immunity during infection.     

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum . This locus named RCH2 (for recognition of C. higginsianum ) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 ( resistance to Ralstonia solanacearum 1 ) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2 , were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum . The screening identified an additional susceptible mutant ( rps4-21 ) that has a 5-bp deletion in the neighboring gene, RPS4-Ws , which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 ( Pst - avrRps4 ). The rps4-21 / rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4 . Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.     

冠毒素生物合成与调控的研究进展

冠毒素是80年代末发现的一种能引起多种植物弥散性黄萎病细菌毒素,它在结构和功能上与在胁迫反应中起作用的植物内源激素茉莉酸甲酯有显著的相似性。本文阐述了冠毒素在生物合成途径与遗传学研究及生物合成调控机制方面的研究进展。     

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.