Project

Discipline

proteasome inhibition; syringolin; plant-pathogen interactions; stomatal immunity; photorhabdus

Lead
Lay summary
) strains that inhibits the proteasome in host cells by a novel mechanism. Pss(syringae  pv. Pseudomonas syringae Bacterial plant pathogens are able to manipulate physiology and defense mechanisms of their hosts by means of so-called effectors, which often are proteins encoded by the pathogen that are injected into the host cells by a special transport system, the type III secretion system. In addition to type III effector proteins, phytopathogenic bacteria are known to synthesize a variety of small molecules acting as toxins or virulence factors. In previous projects, we have identified such a virulence factor, named syringolin A. Syringolin A is a small peptide derivative secreted by certain The experiments proposed in this research project aim at the further elucidation of the biology of syringolin A and similar compounds (collectively dubbed syrbactins). To reach its target, syringolin A must enter plant cells. We will try to elucidate how syringolin A is taken up by plant cells and to identify the uptake transport system that is postulated to exist. These experiments will involve functional expression of candidate Arabidopsis peptide/oligopeptide transporter cDNAs, or if necessary, an Arabidopsis cDNA library, in yeast. Other experiments will aim at the identification of the exact mechanisms of how proteasome inhibition benefits the pathogen. is postulated to produce. The proposed research has the potential to enlarge the syrbactin class of proteasome inhibitors and to predict occurrence, structure and function of syrbactins from genome and metagenome sequence data with high confidence. Because proteasome inhibitors are a promising class of anti-cancer agents, the results of the proposed research may also have medical implication.Photorhabdus luminescens . In the current project, we will  isolate and structurally and biochemically characterize the putative syringolin A analogon which the insect pathogen Photorhabdus asymbioticaand Burkholderia oklahomensis , an insect pathogen, as well as the human pathogens Photorhabdus luminescensa dangerous human pathogen and causal agent of melioidosis, Burkholderia pseudomallei, . We previously have isolated and characterized the gene clusters encoding the syringolin A and glidobactin A synthetases which turned out to exhibit a characteristic gene architecture. Similar gene clusters were found in five taxa among the more than 1500 completely sequenced bacterial genomes, including BurkholderialesSyringolin A defines a novel class of proteasome inhibitors, dubbed syrbactins, that includes glidobactin A, an antifugal and anti-tumor agent produced by a bacterium belonging to the

Direct link to Lay Summary

Last update: 21.02.2013

Number	Title	Start	Funding scheme

Bacterial plant pathogens have evolved sophisticated means such as type III effectors to subvert physiology and defenses of their hosts. In addition to type III effector proteins, phytopathogenic bacteria can synthesize a variety of small molecules acting as toxins or virulence factors. We have previously isolated syringolin A, a small peptide derivative secreted by certain Pseudomonas syringae pv. syringae (Pss) strains, and shown that it is a virulence factor in the interaction of Pss B728a with its host Phaseolus vulgaris (bean) which inhibits the eukaryotic proteasome by a novel mechanism. The experiments proposed in this research application aim at the further elucidation of the biology of syringolin A and similar compounds (collectively dubbed syrbactins).Recent results showed that proteasome inhibition by syringolin A counteracts stomatal immunity in bean and Arabidopsis. In Arabidopsis, stomatal closure triggered by syringolin A-deficient Pss B728a mutants required the salicylic acid (SA)-mediated defense pathway and was dependent on NPR1, the key transcriptional regulator of the SA pathway. The fact that NPR1 requires turnover by the proteasome for proper function provides a likely explanation for syringolin A’s inhibitory effect on stomatal immunity and defense gene transcription. Surprisingly, stomatal closure was independent of ICS1, the isochorismate synthase responsible for most of the SA needed for pathogen defense responses. We propose follow-up experiments to clarify how the SA needed for stomatal immunity is synthesized, taking advantage of Arabidopsis mutants (ics2, pal1, pal2, pal3, pal4, and combinations) affected in genes proposed to play a role in SA synthesis in plants.To reach its proteasome target, syringolin A must be taken up by plant cells, presumably by active transport because of its hydrophilic nature. It is still an unresolved question how uptake works. Experiments are proposed to identify the syringolin A transporters(s) by functional expression of candidate Arabidopsis peptide/oligopeptide transporter cDNAs, or if necessary, an Arabidopsis cDNA library, in yeast.The fact that proteasome inhibition by syringolin A will likely also affect activity of effectors requiring the host proteasome for their action, syringolin A biosynthesis may be expected to be tightly regulated. This is corroborated by the fact that syringolin A production in vitro only occurs in still cultures in a medium mimicking in planta conditions. In experiments with reporter gene constructs, we have identified the promoters driving the syringolin A synthetase structural genes of Pss B301D-R as well as the LuxR-type transcription factor SylA both in vitro and in planta. Activity of the sylA gene depends on the salA gene, itself encoding a LuxR-type transcription factor. All these promoters are essentially inactive under aerobic in vitro conditions but can be activated under microoxic conditions. The experiments proposed should elucidate the complex regulation and shed light on the involvement of the Pss homolog of the E. coli FNR oxygen sensor protein in syringolin A biosynthesis in vitro and in planta.Finally, we propose to isolate and structurally and biochemically characterize the putative syringolin A analogon which the insect pathogen Photorhabdus luminescens (Plu) is postulated to produce. Cloning of the syringolin A and glidobactin A synthetase genes has allowed the identification of homologous genes with slightly different architectures from a number of human and animal pathogens, including Plu. The proposed research has the potential to enlarge the syringolin A class of proteasome inhibitors (dubbed syrbactins) and to predict occurrence, structure and function of syrbactins from genome and metagenome sequence data with high confidence.