Virulence determinants of Pseudomonas syringae strains isolated from grasses in the context of a small type III effector repertoire.

BACKGROUND: Pseudomonas syringae is pathogenic to a large number of plant species. For host colonization and disease progression, strains of this bacterium utilize an array of type III-secreted effectors and other virulence factors, including small secreted molecules such as syringolin A, a peptide derivative that inhibits the eukaryotic proteasome. In strains colonizing dicotyledonous plants, the compound was demonstrated to suppress the salicylic-acid-dependent defense pathway. Here, we analyze virulence factors of three strains colonizing wheat (Triticum aestivum): P. syringae pathovar syringae (Psy) strains B64 and SM, as well as P. syringae BRIP34876. These strains have a relatively small repertoire of only seven to eleven type III secreted effectors (T3Es) and differ in their capacity to produce syringolin A. The aim of this study was to analyze the contribution of various known virulence factors in the context of a small T3E repertoire. RESULTS: We demonstrate that syringolin A production enhances disease symptom development upon direct infiltration of strains into wheat leaves. However, it is not universally required for colonization, as Psy SM, which lacks syringolin biosynthesis genes, reaches cell densities comparable to syringolin A producer P. syringae BRIP34876. Next, we show that despite the small set of T3E-encoding genes, the type III secretion system remains the key pathogenicity determinant in these strains, and that phenotypic effects of deleting T3E-coding genes become apparent only when multiple effectors are removed. CONCLUSIONS: Whereas production of syringolin A is not required for successful colonization of wheat leaves by P. syringae strains, its production results in increased lesion formation. Despite the small number of known T3Es encoded by the analyzed strains, the type III secretion system is essential for endophytic growth of these strains.

Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses.

The Pseudomonas syringae species complex has recently been named the number one plant pathogen, due to its economic and environmental impacts, as well as for its role in scientific research. The bacterium has been repeatedly reported to cause outbreaks on bean, cucumber, stone fruit, kiwi and olive tree, as well as on other crop and non-crop plants. It also serves as a model organism for research on the Type III secretion system (T3SS) and plant-pathogen interactions. While most of the current work on this pathogen is either carried out on one of three model strains found on dicot plants with completely sequenced genomes or on isolates obtained from recent outbreaks, not much is known about strains isolated from grasses (Poaceae). Here, we use comparative genomics in order to identify putative virulence-associated genes and other Poaceae-specific adaptations in several newly available genome sequences of strains isolated from grass species. All strains possess only a small number of known Type III effectors, therefore pointing to the importance of non-Type III secreted virulence factors. The implications of this finding are discussed.

Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome with syringolin A.

As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra.

Production of proteasome inhibitor syringolin A by the endophyte Rhizobium sp. strain AP16.

Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts.

Genome and Transcriptome Sequences of Pseudomonas syringae pv. syringae B301D-R.

Strains of the plant pathogen Pseudomonas syringae are commonly found in the phylosphere and are able to infect a number of agriculturally important crops. Here, we report a high-quality draft genome sequence of Pseudomonas syringae pv. syringae B301D-R, isolated from pears, which is a model strain for phytotoxin research in P. syringae.

The role of bacterial phytotoxins in inhibiting the eukaryotic proteasome.

The ubiquitin-26S proteasome degradation system (UPS) plays a pivotal role in almost all aspects of plant life, including defending against pathogens. Although the proteasome is important for plant immunity, it has been found to be also exploited by pathogens using effectors to increase their virulence. Recent work on the XopJ effector and syringolin A/syrbactins has highlighted host proteasome inhibition as a virulence strategy of pathogens. This review will focus on these recent developments.

Wheat gene TaS3 contributes to powdery mildew susceptibility.

KEY MESSAGE: Identification of TaS3 as a potential susceptibility gene encoding a protein homologous to ULP1 protease in wheat, which may regulate SUMO function facilitating powdery mildew attack. Some plant genes that are required for susceptibilities to certain pathogens are known as susceptibility genes or susceptibility factors, whose loss-of-function mutations can confer the plants resistances. To identify potential susceptibility genes to powdery mildew in wheat, differentially expressed genes in compatible and incompatible interactions between wheat and powdery mildew were examined by the cDNA chip assay. The genes exclusively expressed in the susceptible cultivar were interfered using biolistic transient transformation in wheat epidermal cells. The suppression of gene TaS3 (Triticum aestivum susceptibility) decreased the pathogen penetration by 19%, and its over-expression increased the disease susceptibility. The deduced protein from TaS3 belongs to the putative ubiquitin-like protease 1 peptidase domain family. Subcellular localization studies revealed that its protein was accumulated in the nucleus. Quantitative real-time polymerase chain reaction analysis revealed that TaS3 transcript was significantly induced in the compatible host. This suggests that TaS3 is a potential susceptible gene and its function may be related to regulate SUMO functions.

Arabidopsis YELLOW STRIPE-LIKE7 (YSL7) and YSL8 transporters mediate uptake of Pseudomonas virulence factor syringolin A into plant cells.

Syringolin A (SylA), a virulence factor secreted by certain strains of the plant pathogen Pseudomonas syringae pv. syringae, is an irreversible proteasome inhibitor imported by plant cells by an unknown transport process. Here, we report that functional expression in yeast of all 17 members of the Arabidopsis oligopeptide transporter family revealed that OLIGOPEPTIDE TRANSPORTER1 (OPT1), OPT2, YELLOW STRIPE-LIKE3 (YSL3), YSL7, and YSL8 rendered yeast cells sensitive to growth inhibition by SylA to different degrees, strongly indicating that these proteins mediated SylA uptake into yeast cells. The greatest SylA sensitivity was conferred by YSL7 and YSL8 expression. An Arabidopsis ysl7 mutant exhibited strongly reduced SylA sensitivity in a root growth inhibition assay and in leaves of ysl7 and ysl8 mutants, SylA-mediated quenching of salicylic-acid-triggered PATHOGENESIS-RELATED GENE1 transcript accumulation was greatly reduced compared with the wild type. These results suggest that YSL7 and YSL8 are major SylA uptake transporters in Arabidopsis. Expression of a YSL homolog of bean, the host of the SylA-producing P. syringae pv. syringae B728a, in yeast also conferred strong SylA sensitivity. Thus, YSL transporters, which are thought to be involved in metal homeostasis, have been hijacked by bacterial pathogens for SylA uptake into host cells.

High-Quality Draft Genome Sequence of Pseudomonas syringae pv. Syringae Strain SM, Isolated from Wheat.

Pseudomonas syringae is one of the most widespread plant pathogens that can cause significant damage to crop plantations. Here, we announce a noncontiguous finished genome sequence of Pseudomonas syringae pv. syringae strain SM, isolated from hexaploid wheat. The genome sequence revealed the smallest described complement of type III effectors.

Manipulation of host proteasomes as a virulence mechanism of plant pathogens.

The ubiquitin-26S proteasome degradation system (UPS) in plants is involved in the signal transduction of many cellular processes, including host immune responses triggered by pathogen attack. Attacking pathogens produce effectors that are translocated into host cells, where they interfere with the host's defense signaling in very specific ways. Perhaps not surprising in view of the broad involvement of the host proteasome in plant immunity, certain bacterial effectors exploit or require the host UPS for their action, as currently best studied in Pseudomonas syringae. Intriguingly, some P. syringae strains also secrete the virulence factor syringolin A, which irreversibly inhibits the proteasome by a novel mechanism. Here, the role of the UPS in plant defense and its exploitation by effectors are summarized, and the biology, taxonomic distribution, and emerging implications for virulence strategies of syringolin A and similar compounds are discussed.

Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat.

The Gram-negative gammaproteobacterium Pseudomonas syringae is one of the most wide-spread plant pathogens and has been repeatedly reported to cause significant damage to crop plantations. Research on this pathogen is very intensive, but most of it is done on isolates that are pathogenic to Arabidopsis, tomato, and bean. Here, we announce a high-quality draft genome sequence of Pseudomonas syringae pv. syringae B64 which is the first published genome of a P. syringae strain isolated from wheat up to date. The genome sequence will assist in gaining insights into basic virulence mechanisms of this pathogen which has a relatively small complement of type III effectors.

Heterologous expression of a Photorhabdus luminescens syrbactin-like gene cluster results in production of the potent proteasome inhibitor glidobactin A.

Syrbactins are cyclic peptide derivatives which are known to inhibit the eukaryotic proteasome by irreversible covalent binding to its catalytic sites. The only two members of this family characterized to date, syringolin A and glidobactin A, are secreted by certain strains of Pseudomonas syringae pv. syringae and strain K481-B101 from the order Burkholderiales, respectively. Syrbactins are the products of mixed non-ribosomal peptide/polyketide synthases encoded by gene clusters with a characteristic architecture. Similar, but not identical gene clusters are present in several other bacterial genomes, including that of Photorhabdus luminescens subsp. laumondii TT01, which is therefore hypothesized to be able to produce a syrbactin-type proteasome inhibitor. Here we report the cloning of the putative syrbactins synthetase encoding gene cluster of Ph. luminescens into a cosmid vector and its heterologous expression in Pseudomonas putida. Analysis of culture supernatants of transformed Ps. putida by HPLC and mass spectrometry revealed the presence of glidobactin A, indicating that the syrbactins-like gene cluster of Ph. luminescens encodes a glidobactin A synthetase and that this organism has the capacity to synthesize glidobactin A.

One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor.

Natural products represent valuable lead structures for drug discovery. However, for most bioactive compounds no cellular target is yet identified and many substances predicted from genome analysis are inaccessible due to their life stage-dependent biosynthesis, which is not reflected in common isolation procedures. In response to these issues, an NMR-based and target-directed protease assay for inhibitor detection of the proteasome was developed. The methodology is suitable for one-shot identification of inhibitors in conglomerates and crude culture broths. The technique was applied for analysis of the different life stages of the bacterium Photorhabdus luminescens, which resulted in the isolation and characterization of cepafungin I (CepI), the strongest proteasome inhibitor described to date. Its biosynthesis is strictly regulated and solely induced by the specific environmental conditions determined by our methodology. The transferability of the developed technique to other drug targets may disclose an abundance of novel compounds applicable for drug development.

Regulation of biosynthesis of syringolin A, a Pseudomonas syringae virulence factor targeting the host proteasome.

Many strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae synthesize the virulence factor syringolin A, which irreversibly inactivates the eukaryotic proteasome. Syringolin A, a peptide derivative, is synthesized by a mixed nonribosomal peptide/polyketide synthetase encoded by five clustered genes, sylA to sylE. Biosynthesis of syringolin A, previously shown to be dependent on the GacS/GacA two-component system, occurs in planta and in vitro but only under still culture conditions in a defined medium. Here, we show that the sylC, sylD, and sylE genes of P. syringae pv. syringae B301D-R form an operon transcribed by promoter sequences located between the sylCDE operon and the sylB gene residing on opposite strands. Assays of overlapping sylB and sylCDE promoter deletions translationally fused to the lacZ gene defined promoter sequences required for gene activity both in vitro and in planta. Activation of both promoters depended on the sylA gene encoding a helix-turn-helix (HTH) LuxR-type transcription factor which was shown to directly bind to the promoters. Activity of the sylA gene, in turn, required a functional salA gene, which also encodes an HTH LuxR-type transcription factor. Furthermore, evidence is presented that acyl-homoserine lactone-mediated quorum-sensing regulation is not involved in syringolin A biosynthesis but that oxygen concentration appears to play a role.

Activity enhancement of the synthetic syrbactin proteasome inhibitor hybrid and biological evaluation in tumor cells.

Syrbactins belong to a recently emergent class of bacterial natural product inhibitors that irreversibly inhibit the proteasome of eukaryotes by a novel mechanism. The total syntheses of the syrbactin molecules syringolin A, syringolin B, and glidobactin A have been achieved, which allowed the preparation of syrbactin-inspired derivatives, such as the syringolin A-glidobactin A hybrid molecule (SylA-GlbA). To determine the potency of SylA-GlbA, we employed both in vitro and cell culture-based proteasome assays that measure the subcatalytic chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities. We further studied the inhibitory effects of SylA-GlbA on tumor cell growth using a panel of multiple myeloma, neuroblastoma, and ovarian cancer cell lines and showed that SylA-GlbA strongly blocks the activity of NF-κB. To gain more insights into the structure-activity relationship, we cocrystallized SylA-GlbA in complex with the proteasome and determined the X-ray structure. The electron density map displays covalent binding of the Thr1O(γ) atoms of all active sites to the macrolactam ring of the ligand via ether bond formation, thus providing insights into the structure-activity relationship for the improved affinity of SylA-GlbA for the CT-L activity compared to those of the natural compounds SylA and GlbA. Our study revealed that the novel synthetic syrbactin compound represents one of the most potent proteasome inhibitors analyzed to date and therefore exhibits promising properties for improved drug development as an anticancer therapeutic.

Pseudomonas syringae virulence factor syringolin A counteracts stomatal immunity by proteasome inhibition.

The peptide derivative syringolin A, a product of a mixed nonribosomal peptide and polyketide synthetase, is secreted by certain strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Syringolin A was shown to be a virulence factor for P. syringae pv. syringae B728a because disease symptoms on its host Phaseolus vulgaris (bean) were greatly reduced upon inoculation with syringolin A-negative mutants. Syringolin A's mode of action was recently shown to be irreversible proteasome inhibition. Here, we report that syringolin A-producing bacteria are able to open stomata and, thus, counteract stomatal innate immunity in bean and Arabidopsis. Syringolin A-negative mutants, which induce stomatal closure, can be complemented by exogenous addition of not only syringolin A but also MG132, a well-characterized and structurally unrelated proteasome inhibitor. This demonstrates that proteasome activity is crucial for guard cell function. In Arabidopsis, stomatal immunity was salicylic acid (SA)-dependent and required NPR1, a key regulator of the SA-dependent defense pathway whose proteasome-dependent turnover has been reported to be essential for its function. Thus, elimination of NPR1 turnover through proteasome inhibition by syringolin A is an attractive hypothesis to explain the observed inhibition of stomatal immunity by syringolin A.

Syrbactin class proteasome inhibitor-induced apoptosis and autophagy occurs in association with p53 accumulation and Akt/PKB activation in neuroblastoma.

Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV-3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC(50): 0.004microM) and dexamethasone-resistant MM1.RL cells (IC(50): 0.005microM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC(50): 0.015microM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53 and led to apoptotic cell death in a time-dependent manner. GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser(473) and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate.

BACKGROUND: Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for. RESULTS: We have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH(13)CO(3) revealed preferential (13)C-labeling at the ureido carbonyl position. CONCLUSION: The results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.

Syringolin A selectively labels the 20 S proteasome in murine EL4 and wild-type and bortezomib-adapted leukaemic cell lines.

The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition.

Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a K(i)' of 8.65 +/- 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics.