Hydroperoxide bicyclase CYP50918A1 of Plasmodiophora brassicae (Rhizaria, SAR): Detection of novel enzyme of oxylipin biosynthesis.

The genome of the cabbage clubroot pathogen Plasmodiophora brassicae Woronin 1877 (Cercozoa, Rhizaria, SAR), possesses two expressed genes encoding the P450s that are phylogenetically related to the enzymes of oxylipin biosynthesis of the CYP74 clan. The cDNA of one of these genes (CYP50918A1) has been expressed in E. coli. The preferred substrate for the recombinant protein, the 13-hydroperoxide of α-linolenic acid (13-HPOT), was converted to the novel heterobicyclic oxylipins, plasmodiophorols A and B (1 and 2) at the ratio ca. 12:1. Compounds 1 and 2 were identified as the substituted 6-oxabicyclo[3.1.0]hexane and 2-oxabicyclo[2.2.1]heptane (respectively) using the MS and NMR spectroscopy, as well as the chemical treatments. The 18O labelling experiments revealed the incorporation of a single 18O atom from [18O2]13-HPOT into the epoxide and ether functions of products 1 and 2 (respectively), but not into their OH groups. In contrast, the 18O from [18O2]water was incorporated only into the hydroxyl functions. One more minor polar product, plasmodiophorol C (3), identified as the cyclopentanediol, was formed through the hydrolysis of compounds 1 and 2. Plasmodiophorols A-C are the congeners of egregiachlorides, hybridalactone, ecklonialactones and related bicyclic oxylipins detected before in some brown and red algae. The mechanism of 13-HPOT conversions to plasmodiophorols A and B involving the epoxyallylic cation intermediate is proposed. The hydroperoxide bicyclase CYP50918A1 is the first enzyme controlling this kind of fatty acid hydroperoxide conversion.

Genome-wide identification of genes encoding putative secreted E3 ubiquitin ligases and functional characterization of PbRING1 in the biotrophic protist Plasmodiophora brassicae.

The E3 ubiquitin ligases are key regulators of protein ubiquitination, which have been shown to be involved in a variety of cellular responses to both biotic and abiotic stresses in eukaryotes. However, the E3 ubiquitin ligase homologues in the soil-borne plant pathogen Plasmodiophora brassicae, the causal agent of clubroot disease of crucifer crops worldwide, remain largely unknown. In this study, we characterized secreted E3 ubiquitin ligases, a group of proteins known to be involved in virulence in many pathogens, in a plasmodiophorid P. brassicae. Genome-wide search in the P. brassicae genome retrieved 139 putative E3 ubiquitin ligases, comprising of 115 RING, 15 HECT, 1 HECT-like, and 8 U-box E3 ubiquitin ligases. Among these E3 ubiquitin ligases, 11 RING, 1 U-box, and 3 HECT were found to harbor signal peptide. Based on published RNA-seq data (Schwelm et al. in Sci Rep 5:11153, 2015), we found that these genes were differentially expressed in distinct life stages including germinating spores, maturing spores, and plasmodia. We characterized one potential secreted E3 ubiquitin ligase, PbRING1 (PBRA_000499). Yeast invertase assay showed that PbRING1 harbors a functional N-terminal signal peptide. PbRING1 also harbors a really interested new gene (RING) domain at its C terminus, which was found to display the E3 ligase activity in vitro. Collectively, this study provides a comprehensive insight into the reservoir of putative secreted E3 ligases in P. brassicae.

Arabidopsis thaliana expressing PbBSMT, a gene encoding a SABATH-type methyltransferase from the plant pathogenic protist Plasmodiophora brassicae, show leaf chlorosis and altered host susceptibility.

The plant pathogenic protist Plasmodiophora brassicae causes clubroot disease of Brassicaceae. This biotrophic organism can down-regulate plant defence responses. The previously characterised P. brassicae PbBSMT methyltransferase has substrate specificity for salicylic, benzoic and anthranilic acids. We therefore propose a role for the methylation of SA in attenuating plant defence response in infected roots as a novel strategy for intracellular parasitism. We overexpressed PbBSMT under the control of an inducible promoter in Arabidopsis thaliana and performed physiological, molecular and phytopathological analyses with the transgenic plants under control and induced conditions in comparison to the wild type. Upon induction, transcription of PbBSMT was associated with: (1) strong leaf phenotypes from anthocyanin accumulation and chlorosis followed by browning; (2) increased plant susceptibility after infection with P. brassicae that was manifested as more yellow leaves and reduced growth of upper plant parts; and (3) induced transgenic plants were not able to support large galls and had a brownish appearance of some clubs. Microarray data indicated that chlorophyll loss was accompanied by reduced transcription of genes involved in photosynthesis, while genes encoding glucose metabolism, mitochondrial functions and cell wall synthesis were up-regulated. Our results indicate a role for PbBSMT in attenuation of host defence responses in the roots by metabolising a plant defence signal.

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.

Isolation, cloning and large scale expression of glutathione-S-transferase (GST) protein of Polymyxa betae.

The plasmodiophoromycete Polymyxa betae, an obligate parasite of sugar-beet roots, is a natural vector of Beet necrotic yellow vein virus (BNYVV). To develop protein based diagnosis for any pathogenic agents including P. betae, a specific immunogenic protein has to be prepared. The glutathione-S-transferase (GST) is expressed in all the morphologically different stages of the pathogen's life cycle, and then it is a good candidate as an immunogenic agent for developing of specific antibodies and diagnostic purposes. The present study describes isolation, cloning and large scale expression and purification of P. betae GST protein. For this aim, total RNA was initially isolated from infected plants and corresponding cDNA was constructed by using reverse transcriptase and oligo-dT primer as well as mRNA as a template. The gene encoding GST was isolated and PCR-amplified from the synthesized cDNA by using specific primers. The amplified fragments were preliminary cloned into pTZ57R/T cloning vector. Intact clone containing right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. Next, GST encoding region having right sequence was recovered and sub-cloned into pET28a bacterial expression vector. Large scale expression of recombinant protein was performed in BL21-de3 strain of E. coli and purification was carried out under native situation through Immobolized metal ion affinity chromatography (IMAC) in column containing Ni-NTA agarose beads. Successful expression and purification steps were confirmed by SDS-PAGE followed by western blotting analysis. These results confirmed the high purity and integrity of GST protein which was around 21 kDa. Generally, the total yield of the purified protein in the culture medium was estimated at around 3.5 mg/mL. After purification, a major part of the purified proteins was precipitated identified as excess GST. To improve the solubility, the final concentration of purified protein was reduced to 0.5 mg/mL.

Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination.

Clubroot, caused by Plasmodiophora brassicae, is one of the most serious diseases of cultivated cruciferous crops in the world. However, the basis for pathogenicity in P. brassicae is not well understood. In this study, a serine protease gene (PRO1) was cloned from P. brassicae and its molecular characteristics were investigated. Southern analysis and specific polymerase chain reaction (PCR) amplification indicated that PRO1 is a single-copy gene present in a broad range of P. brassicae pathotypes. Northern analysis revealed that the expression of PRO1 was induced during plant infection, and that the quantity of transcript fluctuated according to the stage of pathogenesis. Amino acid sequence analysis suggested that the encoded protein (Pro1) belongs to the S28 family of proteases, with a predicted signal peptide and a theoretical molecular mass of 49.4 kDa. The open reading frame (ORF) of PRO1 was transferred into Pichia pastoris and Pro1 was heterologously produced. Pro1 showed proteolytic activity on skimmed milk and N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, and the activity could be inhibited by serine protease inhibitors and the chelating agent ethylenediaminetetraacetic acid. The optimal temperature of Pro1 was 25 degrees C, and it exhibited high activity at pH 6.0-6.4. These values coincide with the temperature and pH conditions favourable for P. brassicae resting spore germination in the field. When Pro1 was used to treat canola root exudates, it enhanced the stimulating effect of the root exudates on P. brassicae resting spore germination, indicating that Pro1 may play a role during clubroot pathogenesis by stimulating resting spore germination through its proteolytic activity.