'Candidatus Phytoplasma mali' Genome Encodes a Protein that Functions as an E3 Ubiquitin Ligase and Could Inhibit Plant Basal Defense.

Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein-protein interactions between the causal agent of apple proliferation 'Candidatus Phytoplasma mali' and its host plant. Here, we report that the 'Ca. P. mali' strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been reported before. However, this is, to our knowledge, the first time that a phytoplasma effector protein with E3 ligase activity has been reported.

The crystal structure of the malic enzyme from Candidatus Phytoplasma reveals the minimal structural determinants for a malic enzyme.

Phytoplasmas are wall-less phytopathogenic bacteria that produce devastating effects in a wide variety of plants. Reductive evolution has shaped their genome, with the loss of many genes, limiting their metabolic capacities. Owing to the high concentration of C4 compounds in plants, and the presence of malic enzyme (ME) in all phytoplasma genomes so far sequenced, the oxidative decarboxylation of L-malate might represent an adaptation to generate energy. Aster yellows witches'-broom (Candidatus Phytoplasma) ME (AYWB-ME) is one of the smallest of all characterized MEs, yet retains full enzymatic activity. Here, the crystal structure of AYWB-ME is reported, revealing a unique fold that differs from those of `canonical' MEs. AYWB-ME is organized as a dimeric species formed by intertwining of the N-terminal domains of the protomers. As a consequence of such structural differences, key catalytic residues such as Tyr36 are positioned in the active site of each protomer but are provided by the other protomer of the dimer. A Tyr36Ala mutation abolishes the catalytic activity, indicating the key importance of this residue in the catalytic process but not in the dimeric assembly. Phylogenetic analyses suggest that larger MEs (large-subunit or chimeric MEs) might have evolved from this type of smaller scaffold by gaining small sequence cassettes or an entire functional domain. The Candidatus Phytoplasma AYWB-ME structure showcases a novel minimal structure design comprising a fully functional active site, making this enzyme an attractive starting point for rational genetic design.

Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens.

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas.

The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.

Analysis of expressed genes of the bacterium 'Candidatus phytoplasma Mali' highlights key features of virulence and metabolism.

'Candidatus Phytoplasma mali' is a phytopathogenic bacterium of the family Acholeplasmataceae assigned to the class Mollicutes. This causative agent of the apple proliferation colonizes in Malus domestica the sieve tubes of the plant phloem resulting in a range of symptoms such as witches'--broom formation, reduced vigor and affecting size and quality of the crop. The disease is responsible for strong economical losses in Europe. Although the genome sequence of the pathogen is available, there is only limited information on expression of selected genes and metabolic key features that have not been examined on the transcriptomic or proteomic level so far. This situation is similar to many other phytoplasmas. In the work presented here, RNA-Seq and mass spectrometry shotgun techniques were applied on tissue samples from Nicotiana occidentalis infected by 'Ca. P. mali' strain AT providing insights into transcriptome and proteome of the pathogen. Data analysis highlights expression of 208 genes including 14 proteins located in the terminal inverted repeats of the linear chromosome. Beside a high portion of house keeping genes, the recently discussed chaperone GroES/GroEL is expressed. Furthermore, gene expression involved in formation of a type IVB and of the Sec-dependent secretion system was identified as well as the highly expressed putative pathogenicity-related SAP11-like effector protein. Metabolism of phytoplasmas depends on the uptake of spermidine/putescine, amino acids, co-factors, carbohydrates and in particular malate/citrate. The expression of these transporters was confirmed and the analysis of the carbohydrate cycle supports the suggested alternative energy-providing pathway for phytoplasmas releasing acetate and providing ATP. The phylogenetic analyses of malate dehydrogenase and acetate kinase in phytoplasmas show a closer relatedness to the Firmicutes in comparison to Mycoplasma species indicating an early divergence of the Acholeplasmataceae from the Mollicutes.

[Cloning, expression and characterization of tRNA-isopentenyltransferase genes (tRNA-ipt) from paulownia witches'-broom phytoplasma].

OBJECTIVE: To identify the tRNA-ipt gene of phytoplasmas and analyze the relationship between tRNA-ipt and synthesis of cytokinin as well as pathogenesis in phytoplasmas. METHODS: The paulownia witches'-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E. coli and specific antibody was prepared. Then the growth curve and cytokinin contents of E. coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA respectively. RESULTS: The length of tRNA-ipt genes from PaWB as well as mulberry dwarf, periwinkle virescence and Chinaberry witches'-broom phytoplasmas were 876 bp. All these genes encoded the proteins consisting of 291 amino acids. They contained and indentical motif (GPTASGKT) at N-terminal region related to the ATP or GTP binding sites. Four phytoplasma tRNA-IPTs shared the 99.1-99.5%, amino acid sequence indentity with each other, shared 95.4-99.3% sequence identity with the same group phytoplasmas, whereas the less than 70% identity with 16SrX apple proliferation and 16SrXII Australia grapevine yellows phytoplasmas. The expression of the tRNA-IPT protein and localization in the phloem in phytoplasma-infected paulownia were confirmed by Western blotting and immunofluorescence detection. The determination of growth curve suggested that the growth rate increase of E. coli was affected by the transformation of exogenous tRNA-ipt gene,which might be associated with the cytokinin accumulation. CONCLUSION: This protein was assumed to be involved in the cytokinin synthesis in phytoplasmas as well as other bacteria, which may play an important role in pathogenic processes of phytoplasmas and symptom development.

The AAA+ ATPases and HflB/FtsH proteases of 'Candidatus Phytoplasma mali': phylogenetic diversity, membrane topology, and relationship to strain virulence.

Previous examination revealed a correlation of phytopathogenic data of 'Candidatus Phytoplasma mali' strains and the DNA sequence variability of a type ATP00464 hflB gene fragment. To further investigate such a relationship, all distinct genes previously annotated as hflB in the genome of 'Ca. P. mali' strain AT were fully sequenced and analyzed from a number of representative mild, moderate, and severe strains. The re-annotation indicated that the sequences encode six AAA+ ATPases and six HflB proteases. Each of the nine distinct deduced AAA+ proteins that were examined formed a coherent phylogenetic cluster. However, within these groups, sequences of three ATPases and three proteases from mild and severe strains clustered distantly, according to their virulence. This grouping was supported by an association with virulence-related amino acid substitutions. Another finding was that full-length genes from ATPase AP11 could only be identified in mild and moderate strains. Prediction of the membrane topology indicated that the long ATPase- and protease-carrying C-terminal tails of approximately half of the AAA+ proteins are extracellular, putatively facing the environment of the sieve tubes. Thus, they may be involved in pathogen-host interactions and may compromise phloem function, a major effect of phytoplasma infection. All full-length genes examined appear transcriptionally active and all deduced peptides show the key positions indicative for protein function.

Functional characterization and gene expression profiling of superoxide dismutase from plant pathogenic phytoplasma.

The rapid production of huge amounts of reactive oxygen species (ROS) is one of the responses of animal and plant cells induced under stress conditions, such as pathogenic bacterial infection. To protect against the cytotoxic ROS, it is important for pathogenic bacteria to inactivate ROS by employing their antioxidant enzymes like superoxide dismutase (SOD). Here, we cloned and characterized the sodA gene from the plant pathogenic bacterium, 'Candidatus Phytoplasma asteris' OY-W strain. This is the first description of gene expression and antioxidant enzymatic activity of SOD from a phytoplasma. We also demonstrated the sodA gene product (OY-SOD) functions as Mn-type SOD. Since other Mollicutes bacteria such as mycoplasmas do not possess sodA probably due to reductive evolution, it is intriguing that phytoplasmas possess sodA despite their lack of many metabolic genes, suggesting that OY-SOD may play an important role in the phytoplasma colonization of plants and insects. Moreover, Western blot analysis and real-time PCR revealed that OY-SOD is expressed when the phytoplasma is grown in both plant and insect hosts, suggesting it is functioning in both hosts. Possible role of SOD in protection against damage by host-derived ROS is discussed.

Automated RFLP pattern comparison and similarity coefficient calculation for rapid delineation of new and distinct phytoplasma 16Sr subgroup lineages.

Phytoplasmas are cell wall-less bacteria that cause numerous diseases in several hundred plant species. During adaptation to transkingdom parasitism in diverse plant and insect hosts, phytoplasma evolution has given rise to widely divergent lineages. Since phytoplasmas cannot be cultured in a cell-free medium, measurable phenotypic characters suitable for conventional microbial classification are mostly inaccessible. Currently, phytoplasma differentiation and classification are mainly dependent on restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences. Extending our recent efforts in the exploitation of computer-simulated 16S rRNA gene RFLP analysis and virtual gel plotting for rapid classification of phytoplasmas, we have developed a Perl program for automated RFLP pattern comparison and similarity coefficient calculation. This program streamlines virtual RFLP pattern analysis and has led to the establishment of a criterion for phytoplasma 16Sr subgroup classification and to the delineation of new and distinct subgroup lineages in the clover proliferation phytoplasma group (16SrVI).