The architecture of the Plasmodiophora brassicae nuclear and mitochondrial genomes

Abstract

Plasmodiophora brassicae is a soil-borne pathogen that attacks roots of cruciferous plants causing clubroot disease. The pathogen belongs to the Plasmodiophorida order in Phytomyxea. Here we used long-read SMRT technology to clarify the P. brassicae e3 genomic constituents along with comparative and phylogenetic analyses. Twenty contigs representing the nuclear genome and one mitochondrial (mt) contig were generated, together comprising 25.1 Mbp. Thirteen of the 20 nuclear contigs represented chromosomes from telomere to telomere characterized by [TTTTAGGG] sequences. Seven active gene candidates encoding synaptonemal complex-associated and meiotic-related protein homologs were identified, a finding that argues for possible genetic recombination events. The circular mt genome is large (114,663 bp), gene dense and intron rich. It shares high synteny with the mt genome of Spongospora subterranea, except in a unique 12 kb region delimited by shifts in GC content and containing tandem minisatellite- and microsatellite repeats with partially palindromic sequences. De novo annotation identified 32 protein-coding genes, 28 structural RNA genes and 19 ORFs. ORFs predicted in the repeat-rich region showed similarities to diverse organisms suggesting possible evolutionary connections. The data generated here form a refined platform for the next step involving functional analysis, all to clarify the complex biology of P. brassicae.

Figure 1

Contigs and telomeres in Plasmodiophora brassicae e3 strain. Contig sizes of the nuclear (no. 1 to 20) and mitochondrial genome (no. 21). Telomeres are marked with black ends. Density of coding (red) and repeat (blue) sequences in non-overlapping sliding 10 kbp windows. The color intensity is proportional to the given feature density.

Figure 2

Alignment between four Plasmodiophora brassicae mitochondrial genomes. The illustration is based on mitochondrial genome synteny using the e3 strain (114,663 bp) as a template to which homologous regions identified in eH (102,962 bp), Pb3 (101,103 bp) and the ZJ-1 (93,640 bp) strain were aligned. For details, see Supplementary Fig. S9. The upper line represents sequence coordinates of the e3 mitochondrial genome.

Figure 3

Physical maps and GC content of the Plasmodiophora brassicae e3 and Spongospora subterranea mitochondrial genomes. The two mitochondrial genomes have circular structure but are here linearized to facilitate comparisons. Boxes represent genes and exons separated by introns illustrated as lines. Colors correspond to specified gene functional groups. Genes transcribed from right to left have names marked with an asterisk. (a) Physical map of the P. brassicae e3 mitochondrial genome. (b) GC content of the P. brassicae e3 mitochondrial genome in non-overlapping sliding 20 bp windows. The horizontal line represents 50% GC. (c) Sequence coordinates of the P. brassicae e3 mitochondrial genome. (d) Sequence coordinates of the S. subterranea mitochondrial genome. For comparison, the S. subterranea sequence was opened at position 21,000 bp and two fragments (21,001-1 bp and 37,699–21,000 bp) were merged. (e) GC content of the S. subterranea mitochondrial genome in non-overlapping sliding 20 bp windows. The horizontal line represents 50% GC. (f) Physical map of the S. subterranea mitochondrial genome.

Figure 4

Synteny between the Plasmodiophora brassicae e3 and Spongospora subterranea mitochondrial genomes. The image illustrates aligned mitochondrial sequences of P. brassicae e3 (upper panel) and S. subterranea (lower panel). Identified homologous regions are displayed as two locally collinear blocks (LCB), green and red. The LCBs with matching colors between the genomes are connected by lines. Inside each LCB, a sequence similarity profile is shown, with its height representing the average level of sequence conservation. White areas within a LCB indicate sequences that are unique to a particular genome. The region outside LCBs (P. brassicae e3 sequence from 42,547 to 55,095 bp) has no detectable homology with the S. subterranea mitochondrial genome.

Figure 5

Maximum likelihood tree inferred from mitochondrial-encoded proteins. The tree was constructed from a concatenated alignment of 12 mitochondrial protein sequences from 67 organisms (63 from major eukaryotic groups and 4 α-protebacteria used as outgroup species). The super-matrix was analyzed with RAxML 8.2.11 using GAMMA and substitution models specified for each partition. For details, see Supplementary Table S15. Rapid bootstrap analysis was done with 250 iterations. Branches with support values <45 were collapsed. The scale bar shows the inferred number of amino acid substitutions per site. The long branch was reduced to 50% of its original length, indicated by a crossed double line.