Current View on Phytoplasma Genomes and Encoded Metabolism

Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced ‘Candidatus Phytoplasma' genomes that include those of ‘Ca. P. asteris' strains OY-M and AY-WB, ‘Ca. P. australiense,' and ‘Ca. P. mali'. These genomes are characterized by chromosome condensation resulting in sizes below 900 kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. ‘Ca. P. mali' is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP

The linear chromosome of the plant-pathogenic mycoplasma 'Candidatus Phytoplasma mali'

BACKGROUND: Phytoplasmas are insect-transmitted, uncultivable bacterial plant pathogens that cause diseases in hundreds of economically important plants. They represent a monophyletic group within the class Mollicutes (trivial name mycoplasmas) and are characterized by a small genome with a low GC content, and the lack of a firm cell wall. All mycoplasmas, including strains of 'Candidatus (Ca.) Phytoplasma asteris' and 'Ca. P. australiense', examined so far have circular chromosomes, as is the case for almost all walled bacteria. RESULTS: Our work has shown that 'Ca. Phytoplasma mali', the causative agent of apple proliferation disease, has a linear chromosome. Linear chromosomes were also identified in the closely related provisional species 'Ca. P. pyri' and 'Ca. P. prunorum'. The chromosome of 'Ca. P. mali' strain AT is 601,943 bp in size and has a GC content of 21.4%. The chromosome is further characterized by large terminal inverted repeats and covalently closed hairpin ends. Analysis of the protein-coding genes revealed that glycolysis, the major energy-yielding pathway supposed for 'Ca. P. asteris', is incomplete in 'Ca. P. mali'. Due to the apparent lack of other metabolic pathways present in mycoplasmas, it is proposed that maltose and malate are utilized as carbon and energy sources. However, complete ATP-yielding pathways were not identified. 'Ca. P. mali' also differs from 'Ca. P. asteris' by a smaller genome, a lower GC content, a lower number of paralogous genes, fewer insertions of potential mobile DNA elements, and a strongly reduced number of ABC transporters for amino acids. In contrast, 'Ca. P. mali' has an extended set of genes for homologous recombination, excision repair and SOS response than 'Ca. P. asteris'. CONCLUSION: The small linear chromosome with large terminal inverted repeats and covalently closed hairpin ends, the extremely low GC content and the limited metabolic capabilities reflect unique features of 'Ca. P. mali', not only within phytoplasmas, but all mycoplasmas. It is expected that the genome information obtained here will contribute to a better understanding of the reduced metabolism of phytoplasmas, their fastidious nutrition requirements that prevented axenic cultivation, and the mechanisms involved in pathogenicity

HfIB gene-based phytopathogenic classification of 'Candidatus Phytoplasma mali' strains and evidence that strain composition determines virulence in multiply infected apple trees

Analysis of pathological and molecular data of ‘Candidatus Phytoplasma mali’ accessions from 27 apple trees differing considerably in symptomatology was used to molecularly characterize and classify strains of the infecting apple proliferation phytoplasma. Single-strand conformation polymorphism and sequence analysis of a variable fragment of ATP00464-type hflB gene revealed that these sources consisted of single-strain and multiple-strain accessions that occurred in similar numbers. The latter group was composed of two to five distinct strains. Analysis of cloned sequences of mild and severe single-strain accessions resulted in two groups of reads that clustered, according to their virulence, distantly in the phylogram. Based on this data, the clustering patterns of multiple-strain accession sequences indicated that nearly all of them were composed of mild and severe strains. The distinct clustering of sequences representing mild and severe strains was associated with a range of molecular markers at the nucleotide and amino acid level. Data indicate that the virulence of multiple-strain accessions is determined by the ratio of the occurring mild and severe strains in that mild accessions were characterized by the predominance of sequences representing mild strains and vice versa. There is evidence that shifts in the population and other events may occur that drastically alter virulence of multiple-strain accessions. </jats:p

Molecular differentiation of severe and mild strains of &apos;Candidatus Phytoplasma mali&apos; and evidence that their interaction in multiply infected trees determines disease severity

Abstract Previous work has shown that multiple infections of apple trees by distinctly different strains of &apos;Candidatus Phytoplasma mali&apos; are widespread. In the current study, pathological data of infected trees with single or multiple phytoplasma were analyzed and compared with molecular data based on a hflB gene of the infecting phytoplasmas. Single-strand conformation polymorphism and sequence analysis of a variable hflB gene fragment revealed that mild and severe strains can be distinguished by their SSCP profiles and their phylogenetic clustering. Analysis of cloned sequences from mild and severe single-strain accessions resulted in two groups of reads that clustered, according to their virulence, distantly in the phylogram. Based on this data, the clustering patterns of multiple-strain accession sequences indicated that nearly all of them were composed of mild and severe strains. Our data indicate that the virulence of multiple-strain accessions is determined by the ratio of the occurring mild and severe strains in that mild accessions were characterized by the predominance of sequences representing mild strains and vice versa. There is evidence that shifts in the population may occur that drastically alter virulence of multiple-strain accessions

First data obtained by shotgun proteomics from Nicotiana occidentalis infected by &apos;Candidatus Phytoplasma mali&apos;

Abstract The protein content of Nicotiana occidentalis infected by the non-cultivable phytopathogenic mollicute &apos;Candidatus Phytoplasma mali&apos; strain AT was determined by shotgun proteomics. 102 out of 497 predicted phytoplasma proteins were identified as expressed in shoot tissue. In addition, 940 proteins of N. occidentalis were detected. Results demonstrate the successful application of LTQ Orbitrap XL ETD™ mass spectrometer in detecting phytoplasma-specific proteins in protein mixtures. A high portion of proteins with unknown function was identified beside prominent proteins involved in translation. Several of the proteins with unknown function contain a signal peptide suggesting a potential pathogen-host interaction

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