Date Published: February 6, 2018
Publisher: Public Library of Science
Author(s): Michael E. Sparks, Kristi D. Bottner-Parker, Dawn E. Gundersen-Rindal, Ing-Ming Lee, Chih-Horng Kuo.
The NJAY (New Jersey aster yellows) strain of ‘Candidatus Phytoplasma asteris’ is a significant plant pathogen responsible for causing severe lettuce yellows in the U.S. state of New Jersey. A draft genome sequence was prepared for this organism. A total of 177,847 reads were assembled into 75 contigs > 518 bp with a total base value of 652,092 and an overall [G+C] content of 27.1%. A total of 733 protein coding genes were identified. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MAPF00000000. This draft genome was used for genome- and gene-based comparative phylogenetic analyses with other phytoplasmas, including the closely related ‘Ca. Phytoplasma asteris’ strain, aster yellows witches’- broom (AY-WB). NJAY and AY-WB exhibit approximately 0.5% dissimilarity at the nucleotide level among their shared genomic segments. Evidence indicated that NJAY harbors four plasmids homologous to those known to encode pathogenicity determinants in AY-WB, as well as a chromosome-encoded mobile unit. Apparent NJAY orthologs to the important AY-WB virulence factors, SAP11 and SAP54, were identified. A number of secreted proteins, both membrane-bound and soluble, were encoded, with many bearing similarity to known AY-WB effector molecules and others representing possible secreted proteins that may be novel to the NJAY lineage.
Phytoplasmas are cell wall-less bacterial pathogens infecting both plants and insects. They are transmitted by leafhoppers, planthoppers and psyllids, and cause several hundred economically important plant diseases worldwide. A vast array of diverse phytoplasma strains are distributed on all continents. Phylogenetic analysis based on 16S rRNA gene sequences indicated that phytoplasmas compose a large, discrete and monophyletic clade paraphyletic to the genus Acholeplasma in the Mollicutes class . Because of the inability to readily cultivate these organisms in cell-free media, the provisional genus ‘Candidatus Phytoplasma’ and ‘Candidatus Phytoplasma spp.’ were proposed to accommodate their classification . For simple and rapid classification of phytoplasmas, a scheme was proposed based on RFLP analysis of 16Sr RNA sequence, which to date includes 32 16S ribosomal (16Sr) groups and more than 200 subgroups [3,4]. More than 30 species have been assigned to the provisionary genus ‘Candidatus Phytoplasma’. Because of the difficulty to obtain cell-free, pure phytoplasma cultures, to date only six fully-assembled genomes [5–10] and eleven partial draft genomes [11–21] have been reported. In the aster yellows group 16SrI, complete genomes of ‘Candidatus Phytoplasma asteris’ strain AY-WB (aster yellows witches’-broom), belonging to subgroup 16SrI-A,and strains OY-M (onion yellows-M), and MBSP (maize bushy stunt phytoplasma) strain M3, both belonging to subgroup16SrI-B, and the draft genome of strain OY-V (onion yellows), belonging to subgroup 16SrI-B, were published [5,6,10,14].
A total of 347,686 genomic reads comprising 141,050,336 bases was generated from a Roche/454 sequencing library. The reference-based assembly incorporated a total of 177,847 reads (~51.2% of sequenced reads) into 75 contigs comprising 652,092 bases and exhibiting an N50 of 21,929 bp. Contig lengths ranged from 518 to 57,687 bp, and the assembly’s overall [G+C] was 27.1%. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MAPF00000000. The version described in this paper is version MAPF01000000. S1 Table summarizes the syntenic regions observed between the chromosomal genome of NJAY and that of the AY-WB and OY-M reference sequences used to guide assembly; 70 NJAY contigs were syntenic with AY-WB and 5 with OY-M. Although also used as reference sequences, no evident syntenic blocks were found between NJAY and either of the Australian (PAa) and strawberry lethal yellows (SLY) strains of ‘Candidatus Phytoplasma australiense’.
Early hybridization studies suggested that a high degree of similarity exists between the genomes of subgroup 16SrI-A phytoplasmas . Comparative genomics results of NJAY and AY-WB strains presented in this study corroborate those earlier findings, demonstrating that high levels of similarity at the molecular level are indeed present between strains within the same subgroup. Phylograms derived from similarity measures based on both whole-genome and protein-coding gene sequences demonstrate the very close placement of NJAY and AY-WB within a broader set of representative phytoplasma taxa. The phylogenetic placement of all taxa considered was consistent with a priori expectations. NJAY and AY-WB exhibit roughly 0.5% dissimilarity at the nucleotide level among their shared genomic segments, which is approximately five-fold less than the levels observed between two strains of ‘Candidatus Phytoplasma australiense’, SLY and PAa (Fig 1A). Interestingly, among conserved orthologous genes, the estimated number of nucleotide substitutions per codon between NJAY and AY-WB is roughly 40-fold higher than that between SLY and PAa (Fig 1B), which may reflect the fact that far fewer sites were considered for the concatenated coding sequence comparison than for full genome comparisons (and thus may reflect some inherent degree of small sample bias) or that NJAY, AY-WB or both are exposed to environments more favorable to diversifying selection at the coding sequence level than those to which SLY and PAa are subjected.
NJAY phytoplasma strain in periwinkle was originally obtained from Dr. C. J. Chen, emeritus professor at Rutgers University in New Jersey, and maintained in the USDA periwinkle collection. This phytoplasma strain was insect transmitted from aster yellows phytoplasma infected lettuce to periwinkle . NJAY phytoplasma DNA was extracted from sieve cell preparations isolated from infected periwinkle plants (Catharanthus roseus) exhibiting aster yellows syndrome, including typical phyllody and witches’-broom symptoms, as previously described [27,28] with the addition of RNase A digestion prior to the final phenol-chloroform extraction. These materials were used to prepare a DNA library for use with 454 sequencing instruments. Assemblies for ‘Ca. Phytoplasma asteris’ strains AY-WB (GenBank accession no, GCA_000012225.1) and OY-M (Genbank accession no. GCA_000009845.1), as well as ‘Candidatus Phytoplasma australiense’ strains PAa (GenBank accession no. GCA_000069925.1) and SLY (GenBank accession no. GCA_000397185.1), were obtained [5,6,8,9]. Using each of these as references, a chromosomal assembly of NJAY was prepared from unassembled 454 reads using Roche’s runMapping utility (version 2.9) with default parameter settings  and a 500 bp floor imposed on resultant contig length. Because the sequencing library consisted of a mixture of DNA from both phytoplasma and the host plant, a reference-based assembly strategy was utilized to minimize the chances of unintentional and erroneous incorporation of viridiplantae sequences into the NJAY genome.