Regulation of Biosynthesis of Syringolin A, a Pseudomonas syringae Virulence Factor Targeting the Host Proteasome

Molecular & Cellular Proteomics

et al. 2014

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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. Molecular & Cellular

Section: Methodsmentioning

confidence: 99%

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

Svozil

Hirsch‐Hoffmann

Molecular & Cellular Proteomics

et al. 2014

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“…The SylA synthetase, a modular mixed non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS; see Box 1) is encoded by a gene cluster containing the five genes sylA, sylB, sylC, sylD, and sylE, the last three forming an operon [54,55] (Figure 2A). Sequence and architecture of these genes implied a SylA biosynthesis model also experimentally supported in most of its aspects [54][55][56][57][58] (Figure 2B).…”

Section: A T3e With Proteasome Inhibiting Activitymentioning

confidence: 99%

“…Proteasome inhibition is probably specific because cysteine proteases and trypsin were not inhibited. Although all three catalytic activities of the proteasome are inhibited in vitro, the β5 subunit with its ChTL activity is most sensitive, while the β1 subunit conferring CL activity is least sensitive to inhibition by Occurrence of SylA synthetase genes in P. syringae strainsThe SylA synthetase, a modular mixed non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS; see Box 1) is encoded by a gene cluster containing the five genes sylA, sylB, sylC, sylD, and sylE, the last three forming an operon [54,55] (Figure 2A). Sequence and architecture of these genes implied a SylA biosynthesis model also experimentally supported in most of its aspects [54][55][56][57][58] (Figure 2B).…”

mentioning

confidence: 99%

The role of bacterial phytotoxins in inhibiting the eukaryotic proteasome

2014

Trends in Microbiology

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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. AbstractThe ubiquitin-26S proteasome degradation system (UPS) plays a pivotal role in almost all aspects of plant life, including defending against pathogens. While the proteasome is important for plant immunity, it has been found to also be 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. The ubiquitin-26S proteasome degradation system (UPS) and plant immunityBesides the lysosome, the UPS is the main protein degradation system of eukaryotic cells that not only destructs misfolded proteins but is also involved in many cellular processes by degrading proteins regulating such processes [1,2]. Proteins destined for destruction by the proteasome become polyubiquitinated by a reaction cascade involving three enzymes designated E1, E2, and E3. Ubiquitin is activated in an ATP-dependent way by E1 and transferred to E2, from which it is normally directly transferred to a lysine residue of the target protein by E3, which, by selecting target proteins, confers specificity to the UPS [3].Polyubiquitination is achieved by multiple rounds in which further ubiquitin units are conjugated to an internal lysine residue of ubiquitin. Polyubiquitinated proteins are substrates for the proteasome, which is composed of two 19S regulatory particles (RP) capping a 20S core particle (CP). The RPs recognize polyubiquitinated proteins, which become deubiquitinated and unfolded in an ATP-dependent reaction, and regulate access of unfolded substrates to the proteolytic channel of the CP. The channel is formed by four stacked sevenmembered rings. The two identical outer rings consist of seven different α-subunits, whereas 3 the two identical inner rings consist of seven different β-subunits, three of which have proteolytic activities: the β1 subunit exhibits a caspase-like activity (CL), cleaving protein substrates after acidic residues, while the β2 and β5 subunits have trypsin-like (TL) and chymotrypsin-like (ChTL) activities cleaving after basic and hydrophobic residues, respectively. In all three catalytic subunits, the active site residue is the N-terminal threonineThe UPS is involved in the signaling pathways of nearly all plant hormones, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), hormones of particular importance for plant defense against pathogens [5]. SA plays a prominent role in defense reactio...

“…Amplification by RT-PCR of the intergenic region between PMI03_05100 and PMI03_05101 was not successful (Fig. 1C), indicating that these genes, unlike their homologs in syringolin A-producing P. syringae strains, were not transcribed as an operon (14). Similarly, the intergenic region between PMI03_05097 and PMI03_05099 was also not detected in the extracted RNA.…”

Section: Resultsmentioning

confidence: 98%

“…1B) (13). Whereas sylA encodes a LuxRtype transcriptional activator of the sylB gene and the sylCDE operon (14), sylE encodes a putative export facilitator involved in syringolin secretion. The sylC and sylD genes encode the NRPS/ PKS responsible for syringolin biosynthesis, whereas sylB encodes a desaturase thought to mediate the conversion of lysine to 3,4-dehydrolysine in the ring structure.…”

mentioning

confidence: 99%

Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16

Dudnik

Bigler

2014

Appl Environ Microbiol

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bSyringolin 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 betaand 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. Syringolin A was originally isolated from culture supernatants of the phytopathogenic gammaproteobacterium Pseudomonas syringae pv. syringae B301D-R based on its ability to elicit defense responses and pathogen resistance in rice plants (1, 2). It is a tripeptide derivative consisting an N-terminal valine and the two nonproteinogenic amino acids, 3,4-dehydrolysine and 5-methyl-4-amino-2-hexenoic acid; the first is N-acylated with an unusual ureido-valine moiety, and the latter two form a 12-member macrolactam ring (Fig. 1A). Syringolin A is the major variant of a family of related compounds in which one or both valine residues can be replaced by isoleucine and/or 3,4-dehydrolysine can be replaced by lysine (3). Syringolin A was shown to be a virulence factor in the interaction of P. syringae strain B728a with its host plant Phaseolus vulgaris (bean) where loss of syringolin A production resulted in a significantly diminished lesion number (4). The elucidation of the mode of action of syringolin A revealed that it irreversibly inhibited all three proteolytic activities (i.e., the caspase-, trypsin-, and chymotrypsin-like activities) of the eukaryotic proteasome by covalent ether bond formation with the active-site N-terminal threonine residues of the catalytic ␤1, ␤2, and ␤5 subunits of the 20S core proteasome (4). Proteasome inhibition suppresses the action of many plant hormones, including defense reactions mediated by the important defense hormones jasmonic acid (JA) and salicyl...