Expression of a metacaspase gene of Nicotiana benthamiana after inoculation with Colletotrichum destructivum or Pseudomonas syringae pv. tomato, and the effect of silencing the gene on the host response

Metacaspases are cysteine proteinases that have homology to caspases, which play a central role in signaling and executing programmed cell death in animals. A type II metacaspase cDNA, NbMCA1, was amplified from Nicotiana benthamiana infected with Colletotrichum destructivum. It showed a peak in expression at 72 h post-inoculation corresponding with the switch to necrotrophy by C. destructivum. Inoculation of N. benthamiana with an incompatible bacterium, Pseudomonas syringae pv. tomato, which should induce a non-host hypersensitive response (HR), did not result in an increase in NbMCA1 expression at the time of necrosis development at 20–24 h postinoculation. Virus-induced silencing of NbMCA1 resulted in three to four times more lesions due to C. destructivum compared with leaves inoculated with the PVX vector without the cloned metacaspase gene or inoculated with water only. However, virus-induced silencing of NbMCA1 did not affect the HR necrosis or population levels of P. syringae pv. tomato. Although this metacaspase gene does not appear to be involved in the programmed cell death of non-host HR resistance to P. syringae, it does affect the susceptibility of N. benthamiana to C. destructivum indicating a function in a basal defense response. Possible roles of NbMCA1could be in degrading virulence factors of the pathogen, processing pro-proteins involved in stress responses, eliminating damaged proteins created during stress, and/or degrading proteins to remobilize amino acids to fuel de novo synthesis of proteins involved in stress adaptations.     

Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance

Four glutathione S-transferase (GST) genes, NbGSTU1, NbGSTU2, NbGSTU3, and NbGSTF1, were amplified from cDNA of Nicotiana benthamiana leaves infected with Colletotrichum destructivum using primers based on conserved regions of N. tabacum GST sequences. Expression of NbGSTU1 and NbGSTU3 increased progressively during infection by either C. destructivum or Colletotrichum orbiculare, except for a slight decrease by NbGSTU1 late in the infection, whereas NbGSTU2 and NbGSTF1 expression remained relatively constant. Each of the four genes was cloned into a PVX vector for virus-induced gene silencing, and reduced expression of the four genes was detected by RT-PCR. A statistically significant increase in susceptibility of N. benthamiana to infection following gene silencing was found only for NbGSTU1-silenced plants, which had 130% more lesions and 67% more colonization by C. orbiculare compared with control plants. These results demonstrate that the different GST genes respond in different ways to fungal infection, and at least one plant GST gene has an important role in disease development.     

Early events in the pathogenicity of Pseudomonas syringae on Nicotiana benthamiana

Conserved microbial molecules known as PAMPs (pathogen-associated molecular patterns) elicit defence responses in plants through extracellular receptor proteins. One important PAMP is the flagellin protein derived from motile bacteria. We show here that the solanaceous species Nicotiana benthamiana perceives the flagellin proteins of both pathogenic and non-host species of Pseudomonas syringae. The response to flagellin required a gene closely related to that encoding the Arabidopsis thaliana flagellin receptor that we designated NbFls2. In addition, silencing of NbFls2 led to increased growth of compatible, non-host and non-pathogenic strains of P. syringae. Thus, flagellin perception restricts growth of P. syringae strains on N. benthamiana. Pathogenic bacteria secrete effector proteins into the plant cell to enhance virulence. We tested the ability of several unrelated effectors to suppress PAMP-mediated defences. The effector proteins AvrPto and AvrPtoB, but not AvrRps4, suppressed all responses tested including the hypersensitive response induced by non-host flagellins and the oomycete elicitor INF1. Strikingly, transient expression of avrPto or avrPtoB stimulated the growth of non-pathogenic Agrobacterium tumefaciensin planta, suggesting that multiplication of this species is also restricted by PAMP perception. Unexpectedly, AvrPtoB but not AvrPto required the defence-associated genes Rar1, Sgt1 and Eds1 for suppression. This observation separates the respective mechanisms of the two effectors, and suggests that AvrPtoB may target the defence machinery directly for its suppressive effect.     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.      

Self-protection of Pseudomonas syringae pv. "tabaci" from its toxin, tabtoxinine-beta-lactam

An extracellular toxin, tabtoxinine-beta-lactam (T beta L), is produced by Pseudomonas syringae pv. "tabaci." This toxin irreversibly inhibits its target, glutamine synthetase; yet P. syringae pv. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection of P. syringae pv. "tabaci," we compared the effects of T beta L on Tox+ (T beta L-producing, insensitive to T beta L) and Tox- (T beta L nonproducing, sensitive to T beta L) strains. The extent of protection afforded to the Tox- strain when induced to adenylylate glutamine synthetase was tested. We concluded that an additional protection mechanism was required. A detoxification activity was found in the Tox+ strain which opens the beta-lactam ring of T beta L to produce the inactive, open-chain form, tabtoxinine. Whole cells of the Tox+ strain incubated for 24 h with [14C]T beta L (0.276 mumol/3 X 10(10) cells) contained [14C]tabtoxinine (0.056 mumol), and the medium contained T beta L (0.226 mumol). Extracts of spheroplasts of the Tox+ stain also converted T beta L to tabtoxinine, whereas extracts of the Tox- strain did not alter T beta L. The conversion was time dependent and stoichiometric and was destroyed by boiling for 30 min or by the addition of 5 mM EDTA. Penicillin, a possible substrate and competitive inhibitor of this lactamase activity, inhibited the conversion of T beta L to tabtoxinine. Periplasmic fluid did not catalyze the conversion of T beta L.     

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.     

Calcium efflux as a component of the hypersensitive response of Nicotiana benthamiana to Pseudomonas syringae

Using a model plant Nicotiana benthamiana, we have demonstrated that initial calcium uptake in response to the HR (hypersensitive response)-causing pathogen Pseudomonas syringae pv syringae 61 is followed by net calcium efflux initiated at about 12 h after the bacterial challenge and sustained for at least 48 h. Our data suggest that calcium not only acts as an important second messenger in the activation of resistance responses but may also be a downstream mediator of later cell death acceleration and completion of the defense reaction. Accordingly, we propose that the existing model of HR should be amended to include a PM Ca(2+) ATP pump as an important component of the HR to pathogens in plants.     

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.     

GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528

A two-component system comprising GacS and GacA affects a large number of traits in many Gram-negative bacteria. However, the signals to which GacS responds, the regulation mechanism for GacA expression, and the genes GacA controls are not yet clear. In this study, several phenotypic tests and tobacco-leaf pathogenicity assays were conducted using a gacA deletion mutant strain (BL473) of Pseudomonas syringae pv. tabaci 11528. To determine the regulation mechanism for gacA gene expression and to identify GacA-regulated genes, we conducted quantitative RT-PCR and electrophoretic mobility shift assay (EMSA) experiments. The results indicated that virulence traits related to the pathogenesis of P. syringae pv. tabaci 11528 are regulated coordinately by GacA and iron availability. They also revealed that several systems coordinately regulate gacA gene expression in response to iron concentration and bacterial cell density and that GacA and iron together control the expression of several virulence genes. EMSA results provided genetic and molecular evidence for direct control of virulence genes by GacA.     

Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae.

One of the chromosomal regions of Pseudomonas syringae pv. syringae encoding pathogenicity factors had been mapped into a 3.9-kilobase-pair fragment in previous studies. Promoter probe analysis indicated the existence of a promoter near one end of the fragment. DNA sequencing of this fragment revealed the existence of a consensus promoter sequence in the region of the promoter activity and two open reading frames (ORFs) downstream. These ORFs, ORF1 and ORF2, encoded putative polypeptides of 40 and 83 kilodaltons, respectively. All ORF1::Tn5 as well as ORF2::Tn5 mutant strains were nonpathogenic on susceptible host bean plants and were unable to elicit hypersensitive reactions on nonhost tobacco plants. The deduced amino acid sequence of the 83-kilodalton polypeptide contained features characteristic of known integral membrane proteins. Fusion of the lacZ gene to ORF2 led to the expression of a hybrid protein inducible in Escherichia coli. The functions of the putative proteins encoded by ORF1 and ORF2 are unknown at present.     

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.     

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Deltaorf1 and Deltaorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Deltaorf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Deltaorf1, Deltaorf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis.     

Cloning and sequencing of a molluscan endo-beta-1,4-glucanase gene from the blue mussel, Mytilus edulis.

Using polymerase chain reaction, cloning and sequencing techniques, a complementary DNA encoding a low molecular mass cellulase (endo-1,4-beta-D-glucanase, EC 3.2.1.4) has been identified in the digestive gland of the marine mussel, Mytilus edulis. It contains a 5' untranslated region, a 633-nucleotide ORF encoding a 211 amino-acid protein, including a 17 amino-acid signal peptide and a complete 3' untranslated region. At the C-terminal end of the purified mature protein, a 13 amino-acid peptide is lacking in comparison to the protein sequence deduced from the ORF. This peptide is probably removed as a consequence of post-translational amidation of the C-terminal glutamine. The endoglucanase genes have been isolated and sequenced from both Swedish and French mussels. The coding parts of these two sequences are identical. Both genes contain two introns, the positions of which are conserved. However the length of the introns are different due to base substitutions, insertions or deletions showing the existence of interspecies length polymorphism. The percentage of similarity for the introns of the two gene sequences is 96.9%. This is the first time a molluscan cellulase is characterized at DNA level. Amino acid sequence-based classification has revealed that the enzyme belongs to the glycosyl hydrolase family 45 [B. Henrissat (Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France), personal communication]. There is no cellulose binding domain associated with the sequence.     

Differential effects of flagellins from Pseudomonas syringae pv. tabaci,tomato and glycinea on plant defense response

To investigate the factor that determines incompatible interactions between Pseudomonas syringae pv. tabaci and non-host plants, an elicitor of hypersensitive reaction (HR) was partially purified from the supernatant of a nutrient-poor medium of bacterial culture by DEAE column chromatography. The major protein in the elicitor-active fractions was identified as a flagellin which is a component of flagellar filaments. The flagellins purified from P. syringae pv. tomato and glycinea, incompatible pathogens of tobacco plants, induced fragmentation of chromosomal DNA and oxidative burst accompanied by programmed cell death in tobacco (Nicotiana tabacum) Bright Yellow (BY-2) cells, but the flagellin from pv. tabaci, a compatible pathogen, did not. However, the amino acid sequences of flagellins deduced from fliC genes showed a high homology among these P. syringae pathovars. In particular, the amino acid sequences of pv. tabaci and glycinea are completely identical. However, both recombinant flagellins produced in Escherichia coli possess HR-inducing activity in BY-2 cells. These results indicate that the post-translational modification of flagellins has an important role for HR-inducing ability in tobacco cells. Furthermore, we discuss the cause of a different elicitor activity among flagellins on tobacco cells and the role of flagellins in the determining specificity.