Amyloidogenesis of type III-dependent harpins from plant pathogenic bacteria

Harpins are heat-stable, glycine-rich type III-secreted proteins produced by plant pathogenic bacteria, which cause a hypersensitive response (HR) when infiltrated into the intercellular space of tobacco leaves; however, the biochemical mechanisms by which harpins cause plant cell death remain unclear. In this study, we determined the biochemical characteristics of HpaG, the first harpin identified from a Xanthomonas species, under plant apoplast-like conditions using electron microscopy and circular dichroism spectroscopy. We found that His(6)-HpaG formed biologically active spherical oligomers, protofibrils, and beta-sheet-rich fibrils, whereas the null HR mutant His(6)-HpaG(L50P) did not. Biochemical analysis and HR assay of various forms of HpaG demonstrated that the transition from an alpha-helix to beta-sheet-rich fibrils is important for the biological activity of protein. The fibrillar form of His(6)-HpaG is an amyloid protein based on positive staining with Congo red to produce green birefringence under polarized light, increased protease resistance, and beta-sheet fibril structure. Other harpins, such as HrpN from Erwinia amylovora and HrpZ from Pseudomonas syringae pv. syringae, also formed curvilinear protofibrils or fibrils under plant apoplast-like conditions, suggesting that amyloidogenesis is a common feature of harpins. Missense and deletion mutagenesis of HpaG indicated that the rate of HpaG fibril formation is modulated by a motif present in the C terminus. The plant cytotoxicity of HpaG is unique among the amyloid-forming proteins that occur in several microorganisms. Structural and morphological analogies between HpaG and disease-related amyloidogenic proteins, such as Abeta protein, suggest possible common biochemical characteristics in the induction of plant and animal cell death.     

Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.     

Identification of a key functional region in harpins from <Emphasis Type="Italic">Xanthomonas</Emphasis> that suppresses protein aggregation and mediates harpin expression in <Emphasis Type="Italic">E. coli</Emphasis>

In the current study, we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E. coli. Our data suggested that the presence of two common features in harpins [Wei et al. (1992) Science 257:85-88], namely, high glycine content and lack of cysteine residues, were not sufficient for Xanthomonas to elicit hypersensitive response (HR) activity or heat stability. Additionally, bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids (QGISEKQLDQLL) was α-helical. Following site-directed mutagenesis deletion of this region, the three mutated harpin proteins, in cultures induced at 37°C, failed to elicit a HR in tobacco leaves. However, at 24°C, two mutated harpins retained the ability to elicit HR, albeit with lower expression levels than that noted with the wild-type. SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body. These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.     

Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally non-polar hrpZ deletion mutations, truncated HrpZ fragments, and hrmA mutations

Pseudomonas syringae pv. syringae , like many plant pathogenic bacteria, secretes a 'harpin' protein that can elicit the hypersensitive response (HR), a defensive cellular suicide, in non-host plants. The harpin-encoding hrpZ gene is located in an operon that also encodes Hrp secretion pathway components and is part of the functional cluster of hrp genes carried on cosmid pHIR11 that enables saprophytic bacteria like Escherichia coli and Pseudomonas fluorescens to elicit the HR in tobacco leaves. We have constructed functionally non-polar hrpZ deletion mutations, revealing that HrpZ is necessary for saprophytic bacteria carrying pHIR11 to elicit a typical HR, whereas it only enhances the elicitation activity of P. s. syringae . Partial deletion mutations revealed that the N-terminal 153 amino acids of HrpZ can enable E. coli MC4100-(pHIR11) to elicit a strong HR. hrpZ subclone products comprising the N-terminal 109 amino acids and C-terminal 216 amino acids, respectively, of the 341 amino acid protein were isolated and found to elicit the HR. P. fluorescens (pHIR11 hrmA  ::Tn phoA ) mutants do not elicit the HR, but cell fractionation and immunoblot analysis revealed that they produce and secrete wild-type levels of HrpZ. Therefore, elicitor activity resides in multiple regions of HrpZ, P. syringae produces elicitor(s) in addition to HrpZ, and HrpZ is essential but not sufficient for HR elicitation by saprophytic bacteria carrying pHIR11.     

The Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A₂₃₀ assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. ΔhrpZ::nptII or hrpW::ΩSp^r P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.     

Characterization of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis

Residues in annexin XI-A essential for binding of calcyclin (S100A6) were examined by site-directed mutagenesis. GST fusion proteins with the calcyclin binding site of annexin XI-A, GST-AXI 34-62 and GST-AXI 49-77 bound to calcyclin-Sepharose Ca2+-dependently. The mutants GST-AXI L52E, M55E, A56E and M59E lost the binding ability, whereas GST-AXI A57E retained the ability. These results demonstrate that the hydrophobic residues L52, M55, A56 and M59 on one side surface of the alpha-helix are critical for the binding. Assays with GST fusion proteins and synthesized peptides corresponding to the calcyclin binding site indicated that other regions around the calcyclin binding site are important to stabilize the conformation.     

Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region

Escherichia coli pyruvate oxidase is a membrane-associated flavoprotein dehydrogenase which is greatly activated by lipids and detergents. The carboxyl-terminal region of the protein has been shown to play a critical role in the interaction with lipids. We report mutations generated by chemical and oligonucleotide-mediated site-directed mutagenesis of the poxB gene which result in enzymes defective in lipid activation. Nine mutants were isolated which encode enzymes with point mutations in the carboxyl-terminal segment of the protein. Two mutant lesions introduced termination codons giving enzymes lacking the last nine or three amino acids of the protein which were unable to interact with detergents in vitro and were unable to function in vivo. Of the missense mutants isolated, two were most informative. One was the substitution of Glu-564 with proline in the PoxB16 oxidase. This residue lies in the center of a putative lipid-binding amphipathic alpha-helix (Arg-558 to Thr-568) located close to the carboxyl terminus. Strains producing the PoxB16 oxidase were devoid of oxidase activity in vivo, the enzymes could not be activated by Triton X-100, and were activated poorly by phospholipids in vitro. These results indicated that the PoxB16 oxidase lacked normal lipid-binding ability. Another mutant oxidase (PoxB15) in which proline was substituted for Asp-560 at the beginning of the amphipathic alpha-helix had normal oxidase activity. These findings indicate that the amphipathic alpha-helix structure plays an essential role in the activation and lipid-binding properties of the enzyme. The second informative missense mutation was the substitution of the carboxyl-terminal arginine with glycine. This enzyme showed normal activation in vitro by phospholipids and some detergents, and somewhat reduced activity in vivo. This mutant enzyme appeared to dissociate from detergent vesicles more readily than does the normal enzyme. A model for the membrane interaction of the carboxyl terminus based on the properties of these mutant proteins is presented.     

Identification of an elicitor active site within the three-dimensional structure of the tobacco mosaic tobamovirus coat protein.

The coat protein (CP) of tobacco mosaic tobamovirus (TMV) elicits the hypersensitive response (HR) conferred by the N' gene from Nicotiana sylvestris. This study presents evidence demonstrating a critical role for a specific CP structural site in eliciting this HR. Based on the known structure of the TMV CP, specific substitutions were created within the CP of the elicitor strain P20L to identify structural areas essential for host recognition. Of 32 substitutions made, 14 conferred either a temperature-sensitive (loss of the HR at 29 degrees C) or a knockout (loss of the HR at 25 degrees C) HR phenotype in N.sylvestris. These essential residues were noncontiguous in position; however, within the three-dimensional CP structure, all resided primarily along the right face of the molecule's helical bundle. Substitutions that did not affect the HR phenotype either were located outside of this area or were conservative in change. In addition, placing two temperature-sensitive substitutions within the same CP resulted in lowering temperature sensitivity from 29 to 27 degrees C. This additive effect suggests that residues essential for HR elicitation contribute independently to host recognition. This feature is characteristic of recognition surfaces. The presence of a specific elicitor active site within the three-dimensional structure of the TMV CP is consistent with binding of a host-encoded receptor and demonstrates the importance of CP structure in HR specificity.     

Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors

Harpins are a subset of type III secretion system (T3SS) substrates found in all phytopathogenic bacteria that utilize a T3SS. Pseudomonas syringae pv. tomato DC3000 was previously reported to produce two harpins, HrpZ1 and HrpW1. DC3000 was shown here to deploy two additional proteins, HopAK1 and HopP1, which have the harpin-like properties of lacking cysteine, eliciting the hypersensitive response (HR) when partially purified and infiltrated into tobacco leaves, and possessing a two-domain structure similar to that of the HrpW1 class of harpins. Unlike the single-domain harpin HrpZ1, the two-domain harpins have C-terminal enzyme-like domains: pectate lyase for HopAK1 and lytic transglycosylase for HopP1. Genetic techniques to recycle antibiotic markers were applied to DC3000 to generate a quadruple harpin gene polymutant. The polymutant was moderately reduced in the elicitation of the HR and translocation of the T3SS effector AvrPto1 fused to a Cya translocation reporter, but the mutant was unaffected in the secretion of AvrPto1-Cya. The DC3000 hrpK1 gene encodes a putative translocator in the HrpF/NopX family and was deleted in combination with the four harpin genes. The hrpK1 quadruple harpin gene polymutant was strongly reduced in HR elicitation, virulence, and translocation of AvrPto1-Cya into plant cells but not in the secretion of representative T3SS substrates in culture. HrpK1, HrpZ1, HrpW1, and HopAK1, but not HopP1, were independently capable of restoring some HR elicitation to the hrpK1 quadruple harpin gene polymutant, which suggests that a consortium of semiredundant translocators from three protein classes cooperate to form the P. syringae T3SS translocon.     

Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein.

The baculovirus GP64 envelope fusion protein (GP64 EFP) is the major envelope glycoprotein of the budded virion and has been shown to mediate acid-triggered membrane fusion both in virions and when expressed alone in transfected cells. Using site-directed mutagenesis and functional assays for oligomerization, transport, and membrane fusion, we localized two functional domains of GP64 EFP. To identify a fusion domain in the GP64 EFP of the Orgyia pseudotsugata multiple nuclear polyhedrosis virus (OpMNPV), we examined two hydrophobic regions in the GP64 EFP ectodomain. Hydrophobic region I (amino acids 223 to 228) is a cluster of 6 hydrophobic amino acids exhibiting the highest local hydrophobicity in the ectodomain. Hydrophobic region II (amino acids 330 to 338) lies within a conserved region of GP64 EFP that contains a heptad repeat of leucine residues and is predicted to form an amphipathic alpha-helix. In region I, nonconservative amino acid substitutions at Leu-226 and Leu-227 (at the center of the hydrophobic cluster) completely abolished fusion activity but did not prevent GP64 EFP oligomerization or surface localization. To confirm the role of region I in membrane fusion activity, we used a synthetic 21-amino-acid peptide to generate polyclonal antibodies against region I and demonstrated that antipeptide antibodies were capable of both neutralizing membrane fusion activity and reducing infectivity of the virus. In hydrophobic region II, mutations were designed to disrupt several structural characteristics: a heptad repeat of leucine, a predicted alpha-helix, or the local hydrophobicity along one face of the helix. Single alanine substitutions for heptad leucines did not prevent oligomerization, transport, or fusion activity. However, multiple alanine substitutions or proline (helix-destabilizing) substitutions disrupted both oligomerization and transport of GP64 EFP. In addition, a deletion that removed region II and the predicted alpha-helix was defective for oligomerization, whereas a larger deletion that retained region II and the predicted helix was oligomerized. These results indicate that region II is required for oligomerization and transport and suggest that the predicted helical structure of this region may be important for this function. Thus, by using mutagenesis, functional assays, and antibody inhibition, two functional domains were localized within the baculovirus GP64 EFP: a fusion domain located at amino acids 223 to 228 and an oligomerization domain located at amino acids 327 to 335 within a predicted amphipathic alpha-helix.     

Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.     

Identification of an essential component of the elicitation active site of the EIX protein elicitor

Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense.     

Elicitation of the hypersensitive response by Pseudomonas syringae

The hypersensitive response (HR), a rapid local necrosis accompanied by the production of antimicrobial compounds, is a manifestation of the primary defense mechanisms through which plants prevent colonization by potentially pathogenic microorganisms. The pathogen factors that trigger HR-associated defense mechanisms have yet to be established. Application of transposon mutagenesis and molecular cloning techniques have provided new insights into the bacterial elicitation of the HR. This review focuses on the interaction of Pseudomonas syringae with non-host plants as a model system for the bacterial induction of the HR and discusses (a) the genetics of Pseudomonas syringae pathogenicity and HR elicitation, (b) the environmental conditions controlling expression of Pseudomonas syringae pathogenicity/HR determinants, (c) the nature of the HR elicitor and (d) early plant responses to the pathogen.     

The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants.

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.