Location: Molecular Plant Pathology LaboratoryTitle: Evaluation of the DNA-dependent RNA polymerase ß-subunit gene (rpoB) for phytoplasma classification and phylogeny ) Author
Submitted to: International Journal of Systematic and Evolutionary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/9/2013
Publication Date: 10/5/2013
Citation: Valiunas, D., Jomantiene, R., Davis, R.E. 2014. Evaluation of the DNA-dependent RNA polymerase ß-subunit gene (rpoB) for phytoplasma classification and phylogeny. International Journal of Systematic and Evolutionary Microbiology. 63:3904-3914. Interpretive Summary: Phytoplasmas are wall-less bateria that cause many serious diseases of plants in agricultural and natural ecosystems. To understand and manage these diseases, it is important to be able to identify and classify the different phytoplasmas that cause the many different diseases, some of which are of high concern in plant quarantine regulations. Currently, it is difficult to distinguish between closely related phytoplasmas based solely on the gene (the 16S rRNA gene) that is used as the major factor in distinguishing phytoplasma species. Distinctions among closely related phytoplasma species and strains based on 16S rRNA gene sequences alone has limitations imposed by the high degree of rRNA nucleotide sequence conservation across diverse phytoplasma lineages, and by the presence in a phytoplasma genome of two, sometimes sequence-heterogeneous, copies of the 16S rRNA gene. Because of limitations in the usefulnes of the 16S rRNA gene, we explored the use of a gene that exists as a single copy in the phytoplasma genome, and that codes for a protein involved in the cellular process of copying a DNA gene to a RNA copy. We designed tools for isolation of this gene (DNA-dependent RNA polymerase (DpRp) ß-subunit gene, rpoB) from diverse phytoplasmas, subjected the gene to several types of analysis, and found that it distinguished related phytoplamas more clearly than did the 16S rRNA gene. Our findings indicated that the rpoB gene provides a very useful marker for phytoplasma classification and identification, and that its use should facilitate studies of phytoplasmal disease etiology and epidemiology, and efforts aimed at effective disease management.
Technical Abstract: Phytoplasmas are classified into 16Sr groups, subgroups, and ‘Candidatus Phytoplasma’ species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related ‘Candidatus Phytoplasma’ species and strains based on 16S rRNA gene sequences alone has limitations imposed by the high degree of rRNA nucleotide sequence conservation across diverse phytoplasma lineages, and by the presence in a phytoplasma genome of two, sometimes sequence-heterogeneous, copies of the 16S rRNA gene. Since the DNA-dependent RNA polymerase (DpRp) ß-subunit gene (rpoB) exists as a single copy gene in the phytoplasma genome, we explored the use of rpoB for phytoplasma classification and phylogenetic analysis. We cloned and sequenced a clover phyllody (CPh) phytoplasma genetic locus containing ribosomal protein genes rplA (rpl1), rplJ (rpl10), and rplL (rpl7/rpl12), a complete rpoB gene, and a partial rpoC gene encoding the b‘-subunit of DpRp. Primers and reaction conditions were designed for PCR-mediated amplification of rpoB gene fragments from diverse phytoplasmas. The rpoB gene sequences from phytoplasmas classified in groups 16SrI, 16SrII, 16SrIII, 16SrX, 16SrXII were subjected to sequence similarity and phylogenetic analyses. Based on nucleotide sequence similarities, rpoB gene sequences were more variable than 16S rRNA gene sequences, more clearly distinguishing among phytoplasma lineages. Phylogenetic trees based on 16S rRNA and rpoB gene sequences had similar topologies, but branch lengths were longer, and distinctions among closely related phytoplasmas were more pronounced, in the rpoB tree. Virtual RFLP analysis of rpoB gene sequences also improved distinctions among closely related phytoplasma lineages. The results of this study indicate that the rpoB gene provides a useful additional marker for phytoplasma classification that should facilitate studies of disease etiology and epidemiology.