Occurrence, distribution and possible functional roles of simple sequence repeats in phytoplasma genomes.

Phytoplasmas are unculturable, cell-wall-less bacteria that parasitize plants and insects. This transkingdom life cycle requires rapid responses to vastly different environments, including transitions from plant phloem sieve elements to various insect tissues and alternations among diverse plant hosts. Features that enable such flexibility in other microbes include simple sequence repeats (SSRs) - mutation-prone, phase-variable short DNA tracts that function as 'evolutionary rheostats' and enhance rapid adaptations. To gain insights into the occurrence, distribution and potentially functional roles of SSRs in phytoplasmas, we performed computational analysis on the genomes of five completely sequenced phytoplasma strains, 'Candidatus Phytoplasma asteris'-related strains OYM and AYWB, 'Candidatus Phytoplasma australiense'-related strains CBWB and SLY and 'Candidatus Phytoplasma mali'-related strain AP-AT. The overall density of SSRs in phytoplasma genomes was higher than in representative strains of other prokaryotes. While mono- and trinucleotide SSRs were significantly overrepresented in the phytoplasma genomes, dinucleotide SSRs and other higher-order SSRs were underrepresented. The occurrence and distribution of long SSRs in the prophage islands and phytoplasma-unique genetic loci indicated that SSRs played a role in compounding the complexity of sequence mosaics in individual genomes and in increasing allelic diversity among genomes. Findings from computational analyses were further complemented by an examination of SSRs in varied additional phytoplasma strains, with a focus on potential contingency genes. Some SSRs were located in regions that could profoundly alter the regulation of transcription and translation of affected genes and/or the composition of protein products.

The iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment.

The iPhyClassifier is an internet-based research tool for quick identification and classification of diverse phytoplasmas. The iPhyClassifier simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) patterns. Based on RFLP pattern similarity coefficient scores, the iPhyClassifier gives instant suggestions on group and subgroup classification status of the phytoplasma strains under study. The iPhyClassifier also aligns the query sequences with that of reference strains of all previously described 'Candidatus Phytoplasma' species, -calculates sequence similarity scores, and assigns the phytoplasmas under study into respective 'Ca. Phytoplasma' species as related strains according to the guidelines set forth by the Phytoplasma Taxonomy Group of the International Research Program on Comparative Mycoplasmology. Additional functions of the iPhyClassifier include delineation of potentially new phytoplasma groups and subgroups as well as new 'Ca. Phytoplasma' species. This chapter describes the program components, the operational procedure, and the underlying principles of the iPhyClassifier operation. The chapter also provides hints on how to interpret the results.

Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII).

Phytoplasmas, the causal agents of numerous plant diseases, are insect-vector-transmitted, cell-wall-less bacteria descended from ancestral low-G+C-content Gram-positive bacteria in the Bacillus-Clostridium group. Despite their monophyletic origin, widely divergent phytoplasma lineages have evolved in adaptation to specific ecological niches. Classification and taxonomic assignment of phytoplasmas have been based primarily on molecular analysis of 16S rRNA gene sequences because of the inaccessibility of measurable phenotypic characters suitable for conventional microbial characterization. In the present study, an interactive online tool, iPhyClassifier, was developed to expand the efficacy and capacity of the current 16S rRNA gene sequence-based phytoplasma classification system. iPhyClassifier performs sequence similarity analysis, simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) profiles. Based on calculated RFLP pattern similarity coefficients and overall sequence similarity scores, iPhyClassifier makes instant suggestions on tentative phytoplasma 16Sr group/subgroup classification status and 'Candidatus Phytoplasma' species assignment. Using iPhyClassifier, we revised and updated the classification of strains affiliated with the peach X-disease phytoplasma group. The online tool can be accessed at http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier.html.

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).