Pathogen protein modularity enables elaborate mimicry of a host phosphatase.

Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.

Electronic Nose Differentiation between Quercus robur Acorns Infected by Pathogenic Oomycetes Phytophthora plurivora and Pythium intermedium.

Identification of the presence of pathogenic oomycetes in infected plant material proved possible using an electronic nose, giving hope for a tool to assist nurseries and quarantine services. Previously, species of Phytophthora plurivora and Pythium intermedium have been successfully distinguished in germinated acorns of English oak Quercus robur L. Chemical compound analyses performed by HS-SPME/GC-MS (Headspace Solid-Phase Microextraction/Gas Chromatography-Mass Spectrometry) revealed the presence of volatile antifungal molecules produced by oak seedlings belonging to terpenes and alkanes. Compounds characteristic only of Phytophthora plurivora or Pythium intermedium were also found. Methylcarveol occurred when germinated acorns were infected with Pythium, while neophytadiene (isomer 2 and 3) occurred only when infected with Phytophthora. Moreover, isopentanol was found in acorns infected with Phytophthora, while in control, isopentyl vinyl ether was not observed anywhere else. Among the numerous volatile compounds, isopentanol only occurred in acorns infected with Phytophthora and methylcarveol in acorns infected with Pythium.

The Fatty Acid Methyl Ester (FAME) profile of Phytophthora agathidicida and its potential use as diagnostic tool.

Phytophthora diseases cause devastation to crops and native ecosystems worldwide. In New Zealand, Phytophthora agathidicida is threatening the survival of kauri, an endemic, culturally and ecologically important tree species. The current method for detecting P. agathidicida is a soil bating assay that is time-consuming and requires high levels of expertise to assess, thus limiting the analytical sample throughput. Here, we characterized the fatty acid methyl ester (FAME) profile of P. agathidicida. We also compared it with the FAME profile of P. cinnamomi and assessed the efficacy of FAME analysis as a diagnostic tool for detecting the pathogen in soil samples. In FAME analysis, the total fatty acid content is isolated from a sample and converted to FAMEs for analysis, a process that takes less than a day. Unique fatty acid acyl chains can serve as biomarkers for specific organisms. We detected 12 fatty acids in P. agathidicida, two of which (20:4ω6 and 20:5ω3) show promise as potential Phytophthora specific biomarkers. Collectively, these findings advance our fundamental understanding of P. agathidicida biology and provide a promising technique to increase the rate of sample processing and the speed of pathogen detection for P. agathidicida in soil.

Phytophthora sojae effector Avr1d functions as an E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection.

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (ITC) assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor. Altogether, this study highlights a virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

Root Border Cells and Mucilage Secretions of Soybean, Glycine Max (Merr) L.: Characterization and Role in Interactions with the Oomycete Phytophthora Parasitica.

Root border cells (BCs) and their associated secretions form a protective structure termed the root extracellular trap (RET) that plays a major role in root interactions with soil borne microorganisms. In this study, we investigated the release and morphology of BCs of Glycine max using light and cryo-scanning electron microscopy (SEM). We also examined the occurrence of cell-wall glycomolecules in BCs and secreted mucilage using immunofluorescence microscopy in conjunction with anti-glycan antibodies. Our data show that root tips released three populations of BCs defined as spherical, intermediate and elongated cells. The mechanism of shedding seemed to be cell morphotype-specific. The data also show that mucilage contained pectin, cellulose, extracellular DNA, histones and two hemicellulosic polysaccharides, xyloglucan and heteromannan. The latter has never been reported previously in any plant root secretions. Both hemicellulosic polysaccharides formed a dense fibrillary network embedding BCs and holding them together within the mucilage. Finally, we investigated the effect of the RET on the interactions of root with the pathogenic oomycete Phytophthora parasitica early during infection. Our findings reveal that the RET prevented zoospores from colonizing root tips by blocking their entry into root tissues and inducing their lysis.

Effect of elicitors on holm oak somatic embryo development and efficacy inducing tolerance to Phytophthora cinnamomi.

Holm oak trees (Quercus ilex L.) mortality is increasing worryingly in the Mediterranean area in the last years. To a large degree this mortality is caused by the oomycete Phytophthora spp., which is responsible for forest decline and dieback in evergreen oak forest areas of the southwestern Iberian Peninsula. This study is based on the possibility of applying chemical elicitors or filtered oomycete extracts to holm oak somatic embryos (SE) in order to induce epigenetic memory, priming, that may increase tolerance to the pathogen in future infections. To this end, we first examined the effect of priming treatments on SE development and its oxidative stress state, to avoid elicitors that may cause damage to embryogenic tissues. Both, the sterile oomycete extracts and the chemical elicitor methyl jasmonate (MeJA) did not produce any detrimental effect on SE growth and development, unlike the elicitors benzothiadiazole (BTH) and p-aminobenzoic acid (PABA) that reduced the relative weight gain and resulted in necrotic and deformed SE when were applied at high concentrations (25 µM BTH or 50 µM PABA) in accordance with their high malondialdehyde content. No significant differences among elicitation treatments were found in dual culture bioassays, although those SEs elicited with 50 µM MeJA increased H2O2 production after challenged against active oomycete indicating the activation of stress response. Since this elicitation treatment did not produce any adverse effect in the embryogenic process we suggest that could be used in further priming experiments to produce holm oak plants adapted to biotic stress.

Proteomics Reveals the Mechanism Underlying the Inhibition of Phytophthora sojae by Propyl Gallate.

Phytophthora sojae is a serious soil-borne pathogen, and the major control measures undertaken include the induction of soybean-resistance genes, fungicides, and scientific and reasonable planting management. Owing to the safety and resistance of fungicides, it is of great importance to screen new control alternatives. In a preliminary study, we observed that propyl gallate (PG) exerts a considerable inhibitory effect on P. sojae and can effectively prevent and cure soybean diseases, although the underlying mechanism remains unclear. To explore the inhibitory mechanism of PG on P. sojae, we analyzed the differences in the protein profile of P. sojae before and after treatment with PG using tandem mass tag (TMT) proteomics. Proteomic analysis revealed that the number of differentially expressed proteins (DEPs) was 285, of which 75 were upregulated and 210 were downregulated, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways primarily comprised glycolysis, tricarboxylic acid cycle, fatty acid metabolism, secondary metabolite generation, and other pathways. Among the DEPs involved in PG inhibition of P. sojae are two closely related uncharacterized proteins encoded by PHYSODRAFT_522340 and PHYSODRAFT_344464, denoted PsFACL and PsCPT herein. The CRISPR/Cas9 knockout technique revealed that PsFACL and PsCPT were involved in the growth rate and pathogenicity. In addition, the results of gas chromatography-mass spectrometry (GC-MS) showed that there were differences in fatty acid levels between wild-type (WT) and CRISPR/Cas9 knockout transformants. Knocking out PsFACL and PsCPT resulted in the restriction of the synthesis and ß-oxidation of long-chain fatty acids, respectively. These suggest that PsFACL and PsCPT were also involved in the regulation of the fatty acid metabolism. Our results aid in understanding the mechanism underlying the inhibition of P. sojae growth by PG.

Structural analysis of Phytophthora suppressor of RNA silencing 2 (PSR2) reveals a conserved modular fold contributing to virulence.

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the "start" unit, various numbers of L-W-Y motifs as the "middle" units, and a degenerate L-W-Y as the "end" unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a "building block" to accelerate effector evolution in Phytophthora.

Characterisation of Stramenopile-specific mastigoneme proteins in Phytophthora parasitica.

Mastigonemes, tripartite tubular hairs on the anterior flagellum of Phytophthora zoospores, are instrumental for disease dissemination to new host plants. A previous study showed that PnMas2 was part of the tubular shaft of Phytophthora parasitica mastigonemes. In the current study, genes encoding two related proteins, PnMas1 and PnMas3, were identified in the genome of P. parasitica. PnMas1 interacts with PnMas2 and also occurs along the mastigoneme shaft. RNA-Seq analyses indicate that PnMas1 and PnMas2 genes have similar expression profiles both in vitro and in planta but that PnMas3 is expressed temporally prior to PnMas1 and PnMas2 during asexual development and plant infection. Immunocytochemistry and GFP-tagging document the occurrence of all three PnMas proteins within the specialised compartments of the ER during mastigoneme formation, but only PnMas1 and PnMas2 occur in mature mastigonemes on the flagellar surface. Anti-PnMas1 and anti-PnMas2 antibodies co-labelled two high-molecular-weight (~400 kDa) protein complexes in native gels but anti-PnMas3 antibodies labelled a 65 kDa protein complex. Liquid chromatography-mass spectrometry analysis identified PnMas1 and PnMas2 but not PnMas3 in flagellar extracts. These results suggest that PnMas3 associates with mastigonemes during their assembly within the ER but is not part of mature mastigonemes on the anterior flagellum. Phylogenetic analyses using homologues of Mas genes from the genomes of 28 species of Stramenopiles give evidence of three Mas sub-families, namely Mas1, Mas2 and Mas3. BLAST analyses showed that Mas genes only occur in flagellate species within the Stramenopile taxon.

Detection of Endogenous Phosphatidylinositol 4,5-bisphosphate in Phytophthora cinnamomi.

Plant diseases caused by Phytophthora species are serious threats to agriculture and the natural environment. Genome sequencing has revealed the lack of a gene for canonical phospholipase C (PLC), an enzyme that was hitherto thought to be ubiquitous in eukaryotes. PLC acts in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2 ), a membrane-bound phospholipid critical for signal initiation in many cellular processes. Previous studies have not provided evidence of endogenous PtdIns-4,5-P2 in Phytophthora and, in the absence of canonical PLC, argued for redundancy or loss in the PLC pathway in Phytophthora. Using liquid chromatography mass spectrometry, we have detected endogenous PtdIns-4,5-P2 in Phytophthora cinnamomi. This is the first identification of the phospholipid in the genus, and is significant because it indicates that the signal transduction pathway of the PLC product, inositol 1,4,5-trisphosphate (IP3 ), may have been retained in Phytophthora incorporating an as-yet unidentified homolog or analog of PLC.

Crystal structure of the RxLR effector PcRxLR12 from Phytophthora capsici.

RxLR genes are a prominent class of effectors in oomycetes, and almost half of these proteins contain a conserved sequence motif termed the WY domain, that may exist singly, or as divergent tandem repeats in different effectors. Here we describe the crystal structure of PcRxLR12 (63-488) from Phytophthora capsici at 3.0 Šresolution. The structure consists of five tandemly arrayed WY-domains linked to each other by short connecting helices. Superposition of the WY-2 domain on the other four domains of PcRxLR12, show that the first α-helix termed the K motif, and Loop 3 which connects α3 and α4 are the key regions of structural divergence between the WY domains. A similar pattern was observed when WY-2 was superposed on the 11 WY domains from other oomycete effectors. We also note that an added connecting helix between WY domains in some RXLR effectors, ensures that the WY domains are oriented in the same direction.

Analysis of the cocobiota and metabolites of Moniliophthora perniciosa-resistant Theobroma cacao beans during spontaneous fermentation in southern Brazil.

BACKGROUND: Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, yeasts, lactic acid, and acetic acid bacteria. The spontaneous fermentation of cocoa beans by Theobroma cacao TSH565 clonal variety, a highly productive hybrid resistant to Moniliophthora perniciosa and Phytophthora spp., was investigated. The natural cocobiota involved in the spontaneous fermentation of this hybrid in southern Brazil, was investigated by using both a culture-dependent microbiological analysis and a molecular analysis. The changes in the physicochemical characteristics and the kinetics of substrate utilization and metabolite production during fermentation were also evaluated. RESULTS: Yeasts (178) and bacteria (244) isolated during fermentation were identified by partial sequencing of the ITS and 16S rDNAs, respectively. After 144 h of fermentation, the indigenous yeast community was composed of Hanseniaspora spp., Saccharomyces spp., and Pichia spp. The bacterial population comprised Lactococcus spp., Staphylococcus spp., Acetobacter spp. and Lactobacilli strains. The kinetics of substrate transformation reflected the dynamic composition of the cocobiota. Substrates such as glucose, fructose, sucrose, and citric acid, present at the beginning of fermentation, were metabolized to produce ethanol, acetic acid, and lactic acid. CONCLUSION: The results described here provide new insights into microbial diversity in cocoa bean-pulp mass fermentation and the kinetics of metabolites synthesis, and pave the way for the selection of starter cultures to increase efficiency and consistency to obtain homogeneous and best quality cocoa products. © 2018 Society of Chemical Industry.

Antifungal and Cytotoxic ß-Resorcylic Acid Lactones from a Paecilomyces Species.

Eight new ß-resorcylic acid lactones (RALs), including the hypothemycin-type compounds paecilomycins N-P (1-3) and the radicicol-type metabolites dechloropochonin I (4), monocillins VI (5) and VII (6), 4'-hydroxymonocillin IV (7), and 4'-methoxymonocillin IV (8), along with nine known RALs (9-17), were isolated from the cultures of Paecilomyces sp. SC0924. Compounds 1 and 2 feature a novel 6/11/5 ring system, and 3 is the first 5'-keto RAL. The structures of 1-8 were elucidated on the basis of extensive spectroscopic analysis, X-ray diffraction analysis, and theoretical calculations of ECD spectra. Compounds 3, 5, and 6 exhibit cytotoxicity against MCF-7, A549, and HeLa cells, and compounds 5 and 7 display antifungal activity against Peronophythora litchii.

Probing the Functions of Carbohydrate Binding Modules in the CBEL Protein from the Oomycete Phytophthora parasitica.

Oomycetes are microorganisms that are distantly related to true fungi and many members of this phylum are major plant pathogens. Oomycetes express proteins that are able to interact with plant cell wall polysaccharides, such as cellulose. This interaction is thought to be mediated by carbohydrate-binding modules that are classified into CBM family 1 in the CAZy database. In this study, the two CBMs (1-1 and 1-2) that form part of the cell wall glycoprotein, CBEL, from Phytophthora parasitica have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained indicate that CBEL's CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a fungal family 1 CBM, has produced two variants. The first one lacks its native CBM, whereas the second contains the CBEL CBM1-1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on wheat straw. However, intriguingly the addition of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB variant lacking a CBM1. This suggests that the potentiating effect of CBM1-1 might not require the formation of a covalent linkage to TvXynB.

Bioinformatics Analysis Reveals Abundant Short Alpha-Helices as a Common Structural Feature of Oomycete RxLR Effector Proteins.

RxLR effectors represent one of the largest and most diverse effector families in oomycete plant pathogens. These effectors have attracted enormous attention since they can be delivered inside the plant cell and manipulates host immunity. With the exceptions of a signal peptide and the following RxLR-dEER and C-terminal W/Y/L motifs identified from the sequences themselves, nearly no functional domains have been found. Recently, protein structures of several RxLRs were revealed to comprise alpha-helical bundle repeats. However, approximately half of all RxLRs lack obvious W/Y/L motifs, which are associated with helical structures. In this study, secondary structure prediction of the putative RxLR proteins was performed. We found that the C-terminus of the majority of these RxLR proteins, irrespective of the presence of W/Y/L motifs, contains abundant short alpha-helices. Since a large-scale experimental determination of protein structures has been difficult to date, results of the current study extend our understanding on the oomycete RxLR effectors in protein secondary structures from individual members to the entire family. Moreover, we identified less alpha-helix-rich proteins from secretomes of several oomycete and fungal organisms in which RxLRs have not been identified, providing additional evidence that these organisms are unlikely to harbor RxLR-like proteins. Therefore, these results provide additional information that will aid further studies on the evolution and functional mechanisms of RxLR effectors.

Phenotypic analysis of Phytophthora parasitica by using high throughput phenotypic microarray.

OBJECTIVE: We studied the phenotypic characterization of Phytophthora parasitica Dastur var. nicotianae. METHODS: Phenotypic characterization of the pathogen was studied to provide information for disease management program by using BIOLOG phenotype MicroArray (PM ). Using PM plates 1 to 10, 950 different phenotypic characterizations were tested. RESULTS: P. parasitica was able to metabolize 74% of tested carbon sources, 96% of nitrogen sources, 100% of sulfur sources, and 98% of phosphorus sources. Most informative utilization patterns for carbon sources of P. parasitica were organic acids and carbohydrates, and for nitrogen were various amino acids. The pathogen presented 285 different nitrogen pathways. It had wide range adaptabilities in osmolytes with up to 1% sodium chloride, up to 3% potassium chloride, up to 5% sodium sulfate, up to 20% ethylene glycol, up to 2% sodium formate, up to 5% urea, and up to 2% sodium lactate. It also exhibited active metabolism under pH values between 3.5 and 10, with optimal pH of around 7.0. The pathogen showed both decarboxylase and deaminase activities in the presence of various amino acids. CONCLUSION: These phenotypic characterizations of P. parasitica provided the theoretical basis for the next study of the pathogen in physiology and metabolism, and provided potential new way for tobacco black shank management.

Use of the vital stain FUN-1 indicates viability of Phytophthora capsici propagules and can be used to predict maximum zoospore production.

The fluorescent vital dye FUN®-1 (2-chloro-4-[2,3-dihydro-3-methyl-{benzo-1,3-thiazol-2-yl}-methylidene]-1-phenylquinolinium iodide) was evaluated as a tool to assess Phytophthora capsici sporangia and zoospore metabolic activity and viability. Under aerobic conditions, mycelia, sporangia and zoospores cultured on agar medium and stained with FUN-1 exhibited red fluorescent cylindrical intravacuolar structures (CIVS) that were clearly visible at 100× magnification. Encysted zoospores did not exhibit CIVS after exposure to FUN-1 dye. Over 7 d there was a significant reduction in the percent of sporangia containing CIVS, which corresponded with a significant increase in zoospore formation and release. The decline in the percentage of metabolically active sporangia and increase in the number of zoospores fit both a linear and log regression model. The FUN-1 dye was suitable for distinguishing between live and dead sporangia and effective in monitoring the change in metabolic activity of sporangia over time. It will be useful in determining parameters, including P. capsici culture age, that maximize production of zoospores in vitro.

A high-temperature tolerant species in clade 9 of the genus Phytophthora: P. hydrogena sp. nov.

A previously unknown Phytophthora species was isolated from irrigation water in Virginia, USA. This novel species produces abundant noncaducous and nonpapillate sporangia in soil water extract solution. It sometimes produces chlamydospores and hyphal swellings in aged cultures and in Petri's solution. This species has optimum vegetative growth at 30 C and grows well at 35 C. The lowest and highest temperatures for growth are 5 and 40 C. All isolates examined in this study are compatibility type A1 and produce mostly plerotic oospores when paired with an A2 mating-type tester of P. cinnamomi. Sequence analyses of the rDNA internal transcribed spacer (ITS) regions and the mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene placed this species in clade 9 of the genus Phytophthora. These characteristics support the description of this taxon as a new species for which we propose the name P. hydrogena sp. nov. Further phylogenetic and physiological investigations of clade 9 species revealed a high-temperature tolerant cluster including P. hydrogena, P. aquimorbida, P. hydropathica, P. irrigata, P. chrysanthemi, P. insolita, P. polonica and P. parsiana. These species all grow well at 35 C. The monophyly of the species in this heat-tolerant cluster except P. insolita and P. polonica is highly supported by the maximum-likelihood analyses of the ITS and cox 1 sequences.

Structure reveals regulatory mechanisms of a MaoC-like hydratase from Phytophthora capsici involved in biosynthesis of polyhydroxyalkanoates (PHAs).

BACKGROUND: Polyhydroxyalkanoates (PHAs) have attracted increasing attention as "green plastic" due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved in linking the ß-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R)-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic. METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R)-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC. CONCLUSIONS/SIGNIFICANCE: The data in our study reveal the regulatory mechanism of an (R)-hydratase, providing information on enzyme engineering to produce low cost PHAs.

Escherichia coli-based expression system for the heterologous expression and purification of the elicitin ß-cinnamomin from Phytophthora cinnamomi.

Elicitins are sterol carrier proteins from the Oomycete genera Phytophthora and Phytium and elicit a hypersensitive response in many economically important plants, in some cases causing a systemic acquired resistance. Their recombinant expression in bacteria is complicated by the presence of three disulfide bonds in the elicitin structure. In consequence, elicitins have so far only been produced in soluble form by isolation from native Phytophthora or Phytium strains or by recombinant expression in the yeast Pichia pastoris. Here, for the first time, we report the soluble expression of the elicitin ß-cinnamomin from Phytophthora cinnamomi in Escherichia coli by secretion of the protein into the periplasm. ß-Cinnamomin yields have been significantly improved after careful selection of the optimum secretion signal sequence. In total, 17.6 mg ß-cinnamomin per liter cell culture have been obtained in shake flasks with the secretion signal sequence of the maltose-binding protein MalE from E. coli. Furthermore, by making use of a C-terminal His-tag, ß-cinnamomin purification has been significantly simplified with only one step of immobilized metal ion affinity chromatography yielding protein of high purity (>90%). The established protocol has further been successfully applied to the soluble expression of another elicitin.