Computational protocol: Complete Genome and Plasmid Sequences for Rhodococcus fascians D188 and Draft Sequences for Rhodococcus Isolates PBTS 1 and PBTS 2

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Protocol publication

[…] Rhodococcus fascians, the sole characterized plant pathogen in the genus Rhodococcus, is a Gram-positive pleomorphic bacterium that causes disease through the production and modulation of plant growth regulators, such as cytokinins and auxins (). R. fascians infects a large variety of plants, including monocots and dicots, often causing cryptic symptomology (). R. fascians D188, which has intensively been studied for decades, has revealed a wealth of knowledge about this plant pathogen, including chromosomal and plasmid localized virulence genes necessary for symptomatic host colonization and cytokinin production (, ). The virulence plasmid pFiD188 was previously sequenced using Sanger technology, and fragmented draft genome sequences for multiple R. fascians isolates, including D188, are available (, ). Pistachio bushy top syndrome (PBTS) describes a suite of symptoms affecting clonally propagated UCB-1, an interspecific hybrid rootstock planted in California, Arizona, and New Mexico (, ). Two distinct Rhodococcus species isolates (PBTS 1 and PBTS 2) were isolated from diseased trees and shown to cause stunting and abnormal root and shoot development in pathogenicity assays. Given the global scale of nursery plant production and the diverse population structure of R. fascians, as indicated by initial genome sequences (), high-quality complete reference sequences are needed to fully characterize newly discovered isolates. The reference R. fascians strain D188 (from the private collection of D. Vereecke) and PBTS isolates () were stored in glycerol stocks at -80°C. Cultures were grown in 50 ml of LB broth, and DNA was extracted using a Wizard genomic DNA purification kit, according to the manufacturer’s instructions (Promega, Madison, WI). DNA was shipped on dry ice to the National Center for Genomic Resources (NCGR, Santa Fe, NM) for single-molecule real-time (SMRT) sequencing. Libraries with a 10-kb insert were prepared, and each isolate was sequenced using one SMRT cell on the PacBio RS II instrument (Pacific Biosciences, Menlo Park, CA). Hierarchical Genome Assembly Process (HGAP) was used for de novo assembly () (RS_HGAP assembly.2; Pacific Biosciences). For strain D188, an additional round of polishing was performed using only reads with a quality of ≥84. The genomes were annotated with Prokka version 1.12-beta (), protein-coding features were predicted using Prodigal version 2.6 (), tRNA was predicted by ARAGORN version 1.2 (), and rRNA was predicted by RNAmmer version 1.2 (). The genome annotations reveal a wealth of predicted secondary metabolism-related genes, including novel antibiotic synthesis, heavy metal resistance, siderophore production, pilus-like assembly mechanisms, secreted proteins, transposons and other mobile elements, and prophage sequences. […]

Pipeline specifications

Software tools HGAP, Prokka, Prodigal, ARAGORN, RNAmmer
Application Genome annotation
Organisms Rhodococcus fascians, Rhodococcus fascians D188
Diseases Brain Stem Infarctions