First genomic analysis of a strain of Ralstonia pseudosolanacearum isolated from Mayotte island

TL;DR

This paper presents the first complete genome of a Ralstonia pseudosolanacearum strain from Mayotte, offering insights into its potential role in agricultural disease spread.

Contribution

The study provides the first genomic analysis of a Ralstonia pseudosolanacearum isolate from Mayotte, revealing unique plasmid features.

Findings

The RUN2161 genome includes a megaplasmid with both Type IV and Type II secretion system genes.

The isolate's genome is assembled into one chromosome, one megaplasmid, and one plasmid.

Accessory plasmids are rare in the Ralstonia solanacearum species complex.

Abstract

The Ralstonia solanacearum species complex (RSSC) encompasses phytopathogenic bacteria responsible for bacterial wilt, a devastating disease affecting a wide range of agriculturally important crops. In the South-West Indian Ocean, lineage I-18 of R. pseudosolanacearum has emerged as a particularly destructive pathogen, posing a serious threat to regional food security. In this context, we report the complete genome sequence of isolate RUN2161, collected in Mayotte. This first genome from this island provides a valuable resource for unraveling the evolutionary and epidemiological mechanisms driving the emergence and spread of highly epidemic strains in agriculture..

The genome of strain RUN2161 from Mayotte was sequenced using Illumina short reads and Nanopore long reads. A hybrid assembly was performed resulting in a complete genome of 5,989,529 bp with a G + C content of 66.7%. Functional annotation identified 5,268 CDS, 12 rRNAs, 61 tRNA genes, and 4 ncRNAs, assembled into one chromosome, one megaplasmid and one plasmid. Accessory plasmids are uncommon in RSSC. The RUN2161 plasmid contains Type IV secretion system genes, commonly found on conjugative plasmids, but less commonly, it also carries Type II secretion system genes involved in secretion of toxins and degradative enzymes, which could contribute to epidemiological success.

Full text

Objective

The Ralstonia solanacearum species complex (RSSC), responsible for bacterial wilt in many economically significant crops is distributed globally [1]. RSSC is a species complex divided into species and phylotypes, corresponding to their geographical origins: R. pseudosolanacearum from Asia (Phylotype I) and Africa (Phylotype III), R. solanacearum from Americas (Phylotype IIA and IIB), and R. syzygii from Indonesia-Australia (Phylotype IV) [2–4]. Within these phylotypes, 71 sequevars, based on the similarities in their nucleic sequence that encode egl gene have been defined so far [5].

In the South-West Indian Ocean region, the epidemiological distribution of the RSSC is particularly noteworthy. While phylotype I is dominant in this area [6], distinct prevalence patterns have been reported between sequevars I-18 and I-31. While I-31 strains are predominant in the small islands of this region as well as in East Africa [6–8], I-18 strains are strongly established in Madagascar [9, 10]. Interestingly, recent study highlights genomic diversity within the sequevar I-18 [10, 11].

In Mayotte Island, sequevar I-18 strains have been detected in agricultural fields, raising new concerns for local crop production [7]. Here, we present the first chromosome-level hybrid assembly of a Mahoran I-18 strain, isolated in 2012 from Solanum lycopersicum (tomato), in Dembeni in the east coast of Mayotte.

Data description

High molecular weight genomic DNA of R. pseudosolanacearum RUN2161 strain, was extracted using a protocol adapted for RSSC [11]. Samples were sequenced by the Genewiz-Azenta Laboratory platform (Leipzig, Germany) using Illumina NovaSeq technology. Long-read sequencing was performed using an R10.4.1 MinION flowcell and the SQK-RBK 114.96 Rapid Barcoding Kit (Oxford Nanopore Technology, UK). Basecalling was conducted with Dorado v0.9.0 [12] in the super accuracy mode. The genome assembly was performed using a hybrid pipeline [13]. Short reads and long reads were trimmed using Trimmomatic v0.39 [14] and Filtlong v0.2.1 [12], respectively. An initial hybrid assembly was generated with Unicycler v0.5.1 [15]. The assembly was first polished with long reads using four iterations of Racon v1.4.3 [16], followed by a second long-read polishing with Medaka v1.9.1 [17]. A third polishing step using short reads was performed with four iterations of NextPolish [18]. Finally, circularization of the replicons was performed using Berokka v0.2.3 [12].

The complete genome assembly (DataSet 3; GCA_052747835.1), has a size of 5,989,529 bp. Assembly metrics and quality control, performed using Quast v.5.2.0 [19], BUSCO v.5.8.3 [20] and CheckM v.1.2.2 [21], revealed the high quality of this assembly with a very high level of completeness (99.8%) and no evidence of gene duplication or contamination (DataFile 1). The functional annotation was performed using the NCBI PGAP pipeline [22]. The entire genome encodes 5,161 coding genes and 77 RNAs (including 12 rRNAs, 61 tRNAs, and 4 ncRNAs) and comprise a circular chromosome of 3,758,906 bp with 66.7% GC content and 3,403 coding genes, a megaplasmid of 2,178,702 bp with 66.5% GC content and 1,692 coding genes, and a plasmid of 51,921 bp with 61.5% GC content and 66 coding genes (DataSet 3, DataFile 2).

While a megaplasmid is consistently found across RSSC, accessory plasmids are not ubiquitous and majority of those described are small (< 50 kb) [23, 24], although larger plasmids over 100 kb can also be found [25]. We screened the RUN2161 plasmid sequence against 864 RSSC genomes available in the NCBI database using BLASTN. Ten significant matches were identified, showing 63–87% nucleotide identity. Nine of these plasmids originated from phylotype I strains from China, South Korea, Thailand, and Israel, while one match corresponded to a phylotype IV strain from Indonesia (DataFile 3). The RUN2161 plasmid contains 66 genes, of which 41% correspond to hypothetical proteins with no assigned function. We reported Type IV secretion system (T4SS) genes and a toxin-antitoxin system that are commonly found on conjugative plasmids (DataFile 2).

Surprisingly, the plasmid also carries three proteins associated with the type II secretion system (T2SS) of which an ATPase and a GspD protein. T2SS have been described as a major element implicated in toxins delivery including hydrolytic enzymes and toxins, including proteases, lipases. It often includes a cytoplasmic ATPase [26]. Interestingly, we have found two lytic transglycosylases, a phospholipase D and a peptidase S24. These enzymes, potentially secreted by the T2SS, are thus high valuable candidates to study the molecular mechanisms of virulence and epidemic success.

Table 1. Overview of data files/data setsLabelName of data file/data setFile types(file extension)Data repository and identifier (DOI or accession number)DataSet 1Raw short Illumina sequencing reads 1 and 2Sequence file (.fastq)NCBI Sequence Read Archive (https://identifiers.org/ncbi/insdc.sra:SRR35491076) [27]DataSet 2Raw long nanopore sequencing readsSequence file (.fastq)NCBI Sequence Read Archive (https://identifiers.org/ncbi/insdc.sra:SRR35404195) [28]DataSet 3Complete genome and annotation of R. pseudosolanacearum RUN2161GenBank/Annotation filesNCBI GenBank (http://identifiers.org/assembly:GCA_052747835.1) [29]DataFile 1Quality control of assembly (QUAST, BUSCO and CheckM)MS Excel file (.xlsx)Figshare, 10.6084/m9.figshare.30272398 [30]DataFile 2Circular visualization of RUN2161 genomePortable Document Format fileFigshare, 10.6084/m9.figshare.30273385 [31]DataFile 3Alignments of plasmidsPortable Document Format fileFigshare, 10.6084/m9.figshare.30273451 [32]

Limitations

This study provides a high-quality genome assembly, combining short reads, which offer low error rates, with long reads, which enhance contiguity. This approach provides the best genome quality achievable with available technologies. However, the high genetic diversity of strains circulating in Mayotte highlights the need to investigate other genomes to gain a more comprehensive understanding of variability within RSSC. Moreover, genome annotation still requires improvement, as 18% of the predicted proteins remain of unknown function. Finally, functional validation of these candidate genes will be essential to fully understand their biological roles and potential implications.