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.

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.

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 NaH¹³CO₃ revealed preferential ¹³C-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.

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. 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. 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.