Post-translational modification of flagellin determines the specificity of HR induction

Flagellin, a constituent of the flagellar filament, is a potent elicitor of hypersensitive cell death in plant cells. Flagellins of Pseudomonas syringae pvs. glycinea and tomato induce hypersensitive cell death in their non-host tobacco plants, whereas those of P. syringae pv. tabaci do not remarkably induce it in its host tobacco plants. However, the deduced amino acid sequences of flagellins from pvs. tabaci and glycinea are identical, indicating that post-translational modification of flagellins plays an important role in determining hypersensitive reaction (HR)-inducibility. To investigate genetically the role of modification of flagellin in HR-induction, biological and phytopathological phenotypes of a flagella-defective Delta fliC mutant and Delta fliC mutants complemented by the introduction of the flagellin gene (fliC) from different pathovars of P. syringae were investigated. The Delta fliC mutant of pv. tabaci lost flagella, motility, the ability to induce HR cell death in non-host tomato cells and virulence toward host tobacco plants, whereas all pv. tabaci complemented by the introduction of the fliC gene of pvs. tabaci, glycinea or tomato recovered all the abilities that the Delta fliC mutant had lost. These results indicate that post-translational modification of flagellins is strongly correlated with the ability to cause HR cell death.     

The Δ<Emphasis Type="Italic">fliD</Emphasis> mutant of <Emphasis Type="Italic"> Pseudomonas syringae </Emphasis>pv.<Emphasis Type="Italic"> tabaci</Emphasis>, which secretes flagellin monomers,induces a strong hypersensitive reaction (HR) in non-host tomato cells

To investigate the role of flagella and monomer flagellin in the interaction between Pseudomonas syringae pv. tabaci and plants, non-polar fliC and fliD mutants were produced. The ORFs for fliC and fliD are deleted in the DeltafliC and DeltafliD mutants, respectively. Both mutants lost all flagella and were non-motile. The DeltafliC mutant did not produce flagellin, whereas the DeltafliD mutant, which lacks the HAP2 protein, secreted large amounts of monomer flagellin into the culture medium. Inoculation of non-host tomato leaves with wild-type P. syringae pv. tabaci or the DeltafliD mutant induced a hypersensitive reaction (HR), whereas the DeltafliC mutant propagated and caused characteristic symptom-like changes. In tomato cells in suspension culture, wild-type P. syringae pv. tabaci induced slight, visible HR-like changes. The DeltafliC mutant did not induce HR, but the DeltafliD mutant induced a remarkably strong HR. Expression of the hsr203J gene was rapidly and strongly induced by inoculation with the DeltafliD mutant, compared to inoculation with wild-type P. syringae pv. tabaci. Furthermore, introduction of the fliC gene into the DeltafliC mutant restored motility and HR-inducing ability in tomato. These results, together with our previous study, suggest that the flagellin monomer of pv. tabaci acts as a strong elicitor to induce HR-associated cell death in non-host tomato cells.     

Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato

Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell. Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive. Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions. Pseudomonas syringae pv. tomato T1 causes an HR-like cell death on Nicotiana benthamiana. This rapid cell death was delayed significantly in plants inoculated with P. syringae pv. tomato expressing avrPto. In addition, P. syringae pv. tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines. Transient expression of avrPto in both N. benthamiana and tomato prf3 plants also was able to suppress nonhost HR. Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens. AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions. Experiments with avrPto mutants revealed several residues important for the suppression effects. AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N. benthamiana. These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion.     

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.     

The Active Oxygen Response of Cell Suspensions to Incompatible Bacteria Is Not Sufficient to Cause Hypersensitive Cell Death

The inoculation of tobacco (Nicotiana tabacum L.) suspension cells with bacterial pathogens that elicit the hypersensitive response (HR) in leaves has been shown to elicit production of active oxygen. This response occurs in two phases, the second of which occurs 1 to 3 h after bacterial addition and is unique to HR-causing interactions. The relationship between the phase II active oxygen response and the HR was characterized using Pseudomonas syringae pv syringae and P. fluorescens (pHIR11), which contains a cosmid clone of the hrp/hrm region from P. syringae pv syringae. TnphoA mutations in complementation groups II through XIII of the hrp cluster blocked the phase II active oxygen response, whereas mutations in the group I hrmA locus did not affect phase II. Despite the normal active oxygen response, bacteria with mutations in the hrmA region did not cause the HR in intact tobacco leaves nor did they induce hypersensitive cell death in cell suspensions. The data indicate that the bacteria do not require the hrmA region to elicit active oxygen production, but a full and intact hrp/hrm region is required to elicit hypersensitive cell death. Therefore, the phase II active oxygen response does not directly cause hypersensitive cell death nor is the response itself sufficient to trigger the HR.     

HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions

Pseudomonas syringae pv. tomato DC3000 causes bacterial speck disease in tomato, and it elicits the hypersensitive response (HR) in non-host plants such as Nicotiana tabacum and Nicotiana benthamiana. The compatible and incompatible interactions of DC3000 with tomato and Nicotiana spp., respectively, result in plant cell death, but the HR cell death occurs more rapidly and is associated with effective plant defense. Both interactions require the Hrp (HR and pathogenicity) type III secretion system (TTSS), which injects Hop (Hrp outer protein) effectors into plant cells. Here, we demonstrate that HopPtoN is translocated into tomato cells via the Hrp TTSS. A hopPtoN mutant produced eightfold more necrotic 'speck' lesions on tomato leaves than did DC3000, but the mutant and the wild-type strain grew to the same level in infected leaves. In non-host N. tabacum leaves, the hopPtoN mutant produced more cell death, whereas a DC3000 strain overexpressing HopPtoN produced less cell death and associated electrolyte leakage in comparison with wild-type DC3000. Transient expression of HopPtoN via infection with a PVX viral vector enabled tomato and N. benthamiana plants to tolerate, with reduced disease lesions, challenge infections with DC3000 and P. syringae pv. tabaci 11528, respectively. HopPtoN showed cysteine protease activity in vitro, and hopPtoN mutants altered in the predicted cysteine protease catalytic triad (C172S, H283A and D299A) lost HR suppression activity. These observations reveal that HopPtoN is a TTSS effector that can suppress plant cell death events in both compatible and incompatible interactions.     

Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.     

Role of a xyloglucan-specific endo-beta-1,4-glucanase inhibitor in the interactions of Nicotiana benthamiana with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

A xyloglucan-specific endo-β-1,4-glucanase inhibitor cDNA, NbXEGIP1 , was amplified from diseased leaves of Nicotiana benthamiana . The sequence was similar to the tomato xyloglucan-specific endo-β-1,4-glucanase inhibitor (XEGIP) and tobacco nectarin IV genes that have been described as binding and inactivating fungal Family 12 xyloglucan-specific endo-β-1,4-glucanases. Expression of NbXEGIP1 was not detected in healthy leaves, but the gene was induced during the later stages of infection by the fungi Colletotrichum destructivum and C. orbiculare . Induction of NbXEGIP1 also occurred during disease development by the bacterium Pseudomonas syringae pv. tabaci and during the hypersensitive response produced by P. syringae pv. tabaci expressing avrPto . A portion of NbXEGIP1 was cloned into a tobacco rattle virus vector for virus-induced gene silencing in N. benthamiana . Silencing NbXEGIP1 did not affect the interactions with either Colletotrichum species but did significantly reduce population levels of P. syringae pv. tabaci in the compatible interaction and P. syringae pv. tabaci expressing avrPto in the incompatible interaction. In the susceptible response to P. syringae pv. tabaci , silencing of NbXEGIP1 also resulted in visibly wilted leaves several hours prior to necrosis, which was not observed in control plants. This was related to a significantly higher level of electrolyte leakage and higher expression of a defensin gene from infected NbXEGIP1 -silenced leaves compared with control leaves. Silencing appeared to be specific as it did not affect expression of a related gene, NbXEGIP2 . NbXEGIP1 may act as an inhibitor of a bacterial enzyme that degrades the xyloglucan–cellulose plant cell-wall network, and degradation of the cell wall results in host membrane disruption and signalling of defence responses.     

Contributions of the effector gene hopQ1-1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

To study the role of type III-secreted effectors in the host adaptation of the tobacco ( Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci , a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean ( Phaseolus vulgaris ) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1-1 _Pph1448A were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1-1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1-1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine-tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non-host species.     

Infection of tobacco with different Pseudomonas syringae pathovars leads to distinct morphotypes of programmed cell death

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) infiltrated with either of two pathovars of Pseudomonas syringae- an avirulent strain of P. syringae pv. tabaci (Pst) or the non-host pathogen P. syringae pv. maculicola M2 (Psm) - developed a hypersensitive response (HR). There were considerable differences in HR phenotype, timing and sequence of cell dismantling between the two pathosystems. Following Psm infiltration, the first macroscopic signs were visible at 4.5 h post-infiltration (hpi). Simultaneously, increased plasma membrane permeability was observed, suggesting that the loss of cell membrane integrity initiates the macroscopic HR evoked by Psm. In contrast, after Pst treatment there was a distinct time lapse between the first signs of tissue collapse (9 hpi) and the occurrence of plasma membrane discontinuity (12 hpi). Ultrastructural studies of cells undergoing the HR triggered by Psm and Pst revealed distinct patterns of alterations in morphology of organelles. Moreover, while different forms of nuclear degeneration were observed in leaf zones infiltrated with Pst, we failed to detect any abnormalities in the nuclei of Psm-treated tissue. In addition, application of synthetic caspase inhibitors (Ac-DEVD-CHO, Ac-YVAD-CMK) abolished HR induced by Pst, but not Psm. Our observations suggest that different cell death mechanisms are executed in response to Psm and Pst. Interestingly, pre-inoculation with Pst, but not with Psm, induced a long-distance acquired resistance (LDAR) response, even though locally a typical set of defense responses, including acquired resistance, was activated locally in response to Psm. The failure of Psm to induce LDAR may be due to the rapid degeneration of bundle sheath cells resulting from Psm infection.     

Arabidopsis mutants compromised for the control of cellular damage during pathogenesis and aging

Mutants of Arabidopsis thaliana which exhibit accelerated cell death in response to pathogens were isolated and characterized to gain insight into how symptom severity and disease resistance are modulated. This paper describes mutants that fall into one of two complementation groups that were identified. A novel feature of these mutants is that they are unable to control the rate and extent of cell death after exposure to a variety of stimuli that induce senescence responses. Thus, accelerated cell death ( acd1 ) mutants show rapid, spreading necrotic responses to both virulent and avirulent Pseudomonas syringae pv. maculicola or pv. tomato pathogens and to ethylene. In addition, they develop necrotic lesions as they age and are sensitive to mechanical stress in a developmentally controlled manner. The acd1 mutants are also susceptible to opportunistic pathogens and show decreased growth inhibition of a heterologous pathogen of bean. The signal for lesion formation is not necessarily due to pathogens or wounding since plants grown aseptically also develop necrotic lesions. The lesions formed under a variety of conditions resemble those produced during a pathogen-induced rapid cell death response (the hypersensitive response, HR). Analysis of these acd1 mutants may help to explain the molecular basis of the HR and the relationship between this response and the normal process of senescence.     

Amino acid sequence of bacterial microbe-associated molecular pattern flg22 is required for virulence

Flagellin proteins derived from Pseudomonas syringae pv. tabaci 6605 and flg22Pa (QRLSTGSRINSAKDDAAGLQIA), one of the microbe-associated molecular patterns (MAMP) in bacterial flagellin, induce cell death and growth inhibition in Arabidopsis thaliana. To examine the importance of aspartic acid (D) at position 43 from the N-terminus of a flagellin in its elicitor activity, D43 was replaced with valine (V) and alanine (A) in P. syringae pv. tabaci flagellin and flg22Pta. The abilities of flagellins from P. syringae pv. tabaci D43V and D43A to induce cell death and growth inhibition were reduced, whereas the abilities of flg22PtaD43V and flg22PtaD43A were abolished. These results indicate that D43 is important for elicitor activity in P. syringae pv. tabaci. When tobacco plants were inoculated with each bacterium by the spray method, both P. syringae pv. tabaci D43V and D43A mutants had remarkably reduced ability to cause disease symptoms. Both mutants had reduced or no swimming and swarming motilities and adhesion ability. In P. syringae pv. tabaci D43V, little flagellin protein was detected and few flagella were observed by electron microscopy. These results indicate that mutant flagella are unstable and that flagellar motility is impaired. Thus, the amino acid residue required for MAMP activity is important for the intrinsic flagellar function.     

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.