Open Access

Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031

  • Rodrigo Guarischi-Sousa1,
  • Marina Puigvert2,
  • Núria S. Coll2,
  • María Inés Siri3,
  • María Julia Pianzzola3,
  • Marc Valls2 and
  • João C. Setubal1, 4Email author
Standards in Genomic Sciences201611:7

https://doi.org/10.1186/s40793-016-0131-4

Received: 29 September 2015

Accepted: 10 December 2015

Published: 15 January 2016

Abstract

Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain’s virulence.

Keywords

Short genome reportBacterial wilt Ralstonia solanacearum Bacterial plant pathogenMethylomeUruguay

Introduction

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most devastating plant diseases worldwide [1]. It is a highly diversified bacterial plant pathogen in terms of host range, geographical distribution, pathogenicity, epidemiological relationships, and physiological properties [2]. Strains are divided in four phylotypes, corresponding roughly to their geographic origin: Asia (phylotype I), the Americas (II), Africa (III), and Indonesia (IV) [3]. Strain UY031 belongs to phylotype IIB, sequevar 1 (IIB1), the group considered mainly responsible for bacterial wilt of potato in cold and temperate regions [4]. Phylotype IIB, sequevar 1 is also traditionally classified as race 3 biovar 2.

Strain UY031 was isolated in Uruguay from infected potato tubers in 2003 and displays high aggressiveness both on potato and tomato hosts [5]. This strain is being used as a model in plant-pathogen gene expression studies carried out by our group; having its genome available greatly facilitates the identification of pathogenicity-related genes. Four other IIB1 R. solanacearum strains have been partially sequenced: UW551 [6], IPO1609 [7], NCPPB909 [8], and CFIA906 [8]. This is the first genome of this group to be completely sequenced, and the fourth within the R. solanacearum species complex (the other three are strains GMI1000 [9], Po82 [10] , and PSI07 [11]).

Organism information

Classification and features

Ralstonia solanacearum UY031 strain is classified within the order Burkholderiales of the class Betaproteobacteria . It is an aerobic, non-sporulating, Gram-negative bacterium with rod-shaped cells ranging from 0.5 to 1.5 μm in length (Fig. 1, (a) and (b)). The strain is moderately fast-growing, forming 34 mm colonies within 23 days at 28 °C. On a general nutrient medium containing tetrazolium chloride and high glucose content, strain UY031 usually produces a diffusible brown pigment and develops pearly cream-white, flat, irregular, and fluidal colonies with characteristic pink whorls in the centre (Fig. 1, (c)). Strain UY031 was isolated from a naturally infected potato tuber showing typical brown rot symptoms (creamy exudates from the vascular rings and eyes of the tuber). This strain is highly pathogenic in different solanaceous hosts including important crops like tomato and potato [5]. Pathogenicity of this strain was also confirmed in several accessions of Solanum commersonii Dunal, a wild species considered as a valuable source of resistance for potato breeding. Due to its great aggressiveness, strain UY031 is being used for selection of resistant germplasm as part of the potato breeding program developed in Uruguay. This strain has been deposited in the CFBP collection of plant-associated bacteria, and has received code CFBP 8401. Minimum Information about the Genome Sequence of R. solanacearum strain UY031 is summarized in Table 1, and a phylogenetic tree is shown in Fig. 2.
Fig. 1

Images of Ralstonia solanacearum strain UY031 using transmission (a) and scanning (b) electron microscopy, as well as light microscopy to visualize colony morphology on solid media (c)

Table 1

Classification and general features of Ralstonia solanacearum strain UY031according to the MIGS recommendations [27]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [28]

  

Phylum Proteobacteria

TAS [29]

  

Class Betaproteobacteria

TAS [30, 31]

  

Order Burkholderiales

TAS [31, 32]

  

Family Burkholderiaceae

TAS [31, 33]

  

Genus Ralstonia

TAS [34, 35]

  

Species Ralstonia solanacearum

TAS [34, 35]

  

Strain UY031

 
 

Gram stain

Negative

IDA

 

Cell shape

Rod

IDA

 

Motility

Motile

IDA

 

Sporulation

Non sporulating

NAS

 

Temperature range

Mesophile

IDA

 

Optimum temperature

27 °C

IDA

 

pH range; Optimum

5.5 – 8.0; 6.5

NAS

 

Carbon source

Dextrose, lactose, maltose, cellobiose

IDA

MIGS-6

Habitat

potato plants, soil

TAS [5]

MIGS-6.3

Salinity

<2.0 %

TAS [36]

MIGS-22

Oxygen requirement

Aerobic

IDA

MIGS-15

Biotic relationship

free-living

IDA

MIGS-14

Pathogenicity

Pathogenic

TAS [5]

MIGS-4

Geographic location

Uruguay, San José

TAS [5]

MIGS-5

Sample collection

2003

TAS [5]

MIGS-4.1

Latitude

34°43′58.17”S

NAS

MIGS-4.2

Longitude

56°32′2.87”W

NAS

MIGS-4.4

Altitude

116.7 m

NAS

aEvidence codes - IDA Inferred from direct assay, TAS Traceable author statement (i.e., a direct report exists in the literature), NAS Non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [37]

Fig. 2

Phylogenetic tree highlighting the position of the Ralstonia solanacearum UY031 (shown in bold) relative to other strains from the same species. The phylogenetic tree was constructed using four conserved prokaryotic marker genes, namely: recA, rpoA, rpoB and rpoC. Each gene was aligned individually with MUSCLE [25]; the resulting multiple alignments were concatenated. PhyML [26] was used to perform tree reconstruction using the GTR model and 1,000 bootstrap replicas. Strain names are colour-coded according to the correspondent phylotype. GenBank accession numbers are displayed within brackets. Strains whose genome was completely sequenced are marked with an asterisk. Ralstonia pickettii 12 J (NCBI accession NC_010682) was used as an outgroup

Genome sequencing information

Genome project history

This sequencing project was carried out in 2015; the result is a complete and finished genome. Project data is available from GenBank (Table 2). Accession codes for reads in the Sequence Read Archive are SRP064191, SRR2518086, and SRZ132405.
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Finished

MIGS-28

Libraries used

SMRT library (P5-C3 large insert library)

MIGS 29

Sequencing platforms

PacBio RS II

MIGS 31.2

Fold coverage

138×

MIGS 30

Assemblers

HGAP.2 workflow

MIGS 32

Gene calling method

Prokka v1.10 (ncRNAs search enabled)

 

Locus tag

RSUY

 

Genbank ID

CP012687 (chr), CP012688 (pl)

 

GenBank date of release

September 28, 2015

 

GOLD ID

NA

 

BIOPROJECT

PRJNA278086

MIGS 13

Source material identifier

SAMN03402637

 

Project relevance

Plant pathogen

Table 3

Summary of genome: one chromosome and one plasmid

Label

Size (Mb)

Topology

INSDC identifier

RefSeq ID

Chromosome

3.41

circular

NA

NA

Megaplasmid

1.99

circular

NA

NA

Growth conditions and genomic DNA preparation

R. solanacearum strain UY031 was routinely grown in rich B medium (10 g/l bactopeptone, 1 g/l yeast extract and 1 g/l casaminoacids). Genomic DNA was extracted from a bacterial culture grown to stationary phase to avoid over-representation of genomic sequences close to the origin of replication. Twelve ml of a culture grown for 16 h at 30 °C and shaking at 200 rpm (OD600 = 0.87) were used to extract DNA with Blood & Cell Culture DNA Midi kit (Qiagen), following manufacturer’s instructions for gram-negative bacteria. DNA concentration and quality were measured in a Nanodrop (ND-8000 8-sample spectrophotometer).

Genome sequencing and assembly

Whole-genome sequencing was performed on the PacBio RS II platform at the Duke Center for Genomic and Computational Biology (USA). P5-C3 chemistry and a single SMRTcell were used, and quality control was performed with DUGSIM. The number of Pre-Filter Polymerase Read Bases was greater than 749 million (>130x genome coverage). Reads were assembled using RS_HGAP_Assembly.2 protocol from SMRT Analysis 2.3 [12]. This resulted in one circular chromosome (3,412,138 bp) and one circular megaplasmid (1,999,545 bp). These lengths are very similar to those of the corresponding replicons in R. solanacearum Po82, a IIB sequevar 4 strain, also a potato pathogen and which has also been completely sequenced [10]. The origin of replication was defined for both replicons based on the putative origin for reference strain GMI1000 [9].

An assembly quality assessment was performed before all downstream analyses. All reads were mapped back to the assembled sequences using RS_Resequencing.1 protocol from SMRT Analysis 2.3. This analysis revealed that chromosome and megaplasmid sequences had 100 % of bases called (percentage of assembled sequence with coverage > = 1) and 99.9999 % and 99.9992 %, respectively, of consensus concordance.

Genome annotation

Genome annotation was done using Prokka [13] with the option for ncRNA search. Type III effectors of strain UY031 were identified and annotated in three steps: First, 17 of the T3Es from the R. solanacearum species complex [14] were identified based on the Prokka annotations. Second, the 15 T3Es annotated as “Type III Effector Protein”, “Probable Type III Effector Protein” or “Putative Type III Effector Protein” by Prokka were manually annotated using the first BLAST [15] hits (usually 100 % identity) of their DNA sequences against genome sequences of phylotype IIB strains MOLK2 and Po82. Third, the UY031 genome was uploaded to the “ Ralstonia T3E” web interface tool [14] to search for additional T3Es not annotated as such with Prokka. The additional 28 T3E genes identified were manually annotated as above. Homologous Gene Group clustering was performed with get_homologues [16] using the orthoMCL program [17] and requiring a minimum sequence identity in BLAST query/subject pairs of 30 %.

The sequencing plataform used to assemble the genome (PacBio RS II) also gives kinectics information about the sequenced genome. The presence of a methylated base in the DNA template delays the incorporation of the complementary nucleotide; such modifications in the kinectics may be used to characterize modified bases by methylation including: 6-mA, 5-mC and 4-mC [18]. The analysis of these modifications in a genome-wide and single-base-resolution scale allowed us to characterize the ‘methylome’ of this strain. These epigenetic marks are commonly used by bacteria, and its implications vary from a defense mechanism, protecting the cell from invading bacteriophages or other foreign DNA, to the bacterial virulence itself [1921]. We performed methylome analysis and motif detection using RS_Modification_and_Motif_analysis.1 protocol from SMRT Analysis 2.3. Such epigenetic marks arise from DNA methyl-transferases, sometimes coupled with a restriction endonuclease (a Restriction-Modification System). We further characterized which genes give rise to the modified motifs using tools available at REBASE [22].

Genome properties

The genome of R. solanacearum strain UY031 has one chromosome (3,412,138 bp) and one circular megaplasmid (1,999,545 bp) (Table 3). The average GC content of the chromosome is 66.5 % while that of the megaplasmid is 66.7 %. A total of 4,778 genes (4,683 CDSs and 95 RNAs) were predicted. Of the protein-coding genes, 3,566 (76.1 %) had functions assigned while 1,212 were considered hypothetical (Table 4). Of all CDSs, 76.6 % could be assigned to one COG functional category and for 83.1 % one or more conserved PFAM-A domains were identified (Table 5).
Table 4

Genome statistics

Attribute

Value

% of total

Genome size (bp)

5,411,683

100.00

DNA coding (bp)

4,737,274

87.5

DNA G + C (bp)

3,604,487

66.6

DNA scaffolds

2

100.00

Total genes

4,778

100.00

Protein coding genes

4,683

98.0

RNA genes

95

1.9

Pseudo genes

NA

NA

Genes in internal clusters

NA

NA

Genes with function prediction

3,566

74.6

Genes assigned to COGs

3,586

76.6

Genes with Pfam domains

3,892

83.1

Genes with signal peptides

501

10.6

Genes with transmembrane helices

1132

24.1

CRISPR repeats

0

-

Table 5

Number of genes associated with general COG functional categories

Code

Value

%

Description

J

160

3.4

Translation, ribosomal structure and biogenesis

A

2

<0.1

RNA processing and modification

K

273

5.8

Transcription

L

240

5.1

Replication, recombination and repair

B

3

<0.1

Chromatin structure and dynamics

D

28

0.6

Cell cycle control, Cell division, chromosome partitioning

V

45

1.0

Defense mechanisms

T

162

3.5

Signal transduction mechanisms

M

237

5.1

Cell wall/membrane biogenesis

N

119

2.5

Cell motility

U

61

1.3

Intracellular trafficking and secretion

O

154

3.3

Posttranslational modification, protein turnover, chaperones

C

226

4.8

Energy production and conversion

G

165

3.5

Carbohydrate transport and metabolism

E

342

7.3

Amino acid transport and metabolism

F

75

1.6

Nucleotide transport and metabolism

H

154

3.3

Coenzyme transport and metabolism

I

177

3.8

Lipid transport and metabolism

P

176

3.8

Inorganic ion transport and metabolism

Q

73

1.6

Secondary metabolites biosynthesis, transport and catabolism

R

352

7.5

General function prediction only

S

362

7.7

Function unknown

-

1097

23.4

Not in COGs

The total is based on the total number of protein coding genes in the genome

Insights from the genome sequence

We performed a pan-genome analysis of the R. solanacearum UY031 genome, comparing it to four other genomes: two closely-related R. solanacearum strains (UW551 and IPO1609) and two others with complete genome sequences available (GMI1000 and Po82). The pan-genome consists of 7,594 HGGs while the core genome consists of 2,958 HGGs; the variable genome consists of 2,643 HGGs, and the number of strain-specific HGGs ranges from 193 to 774 (Fig. 3). We identified 193 HGGs that are UY031-specific; 75.1 % of them were annotated as hypothetical proteins.
Fig. 3

Venn diagram of the Ralstonia solanacearum homologous gene groups. The R. solanacearum genomes compared were as follows: strains Po82, GMI1000, IPO1609, UW551, and UY031

Type III effector genes are among the most important for virulence determinants in bacterial plant pathogens such as R. solanacearum [14]. Based on comparisons with effector gene sequences in public databases (see above) we have identified 60 T3Es (Table 6), of which 11 appear to be nonfunctional due to frameshifts or other mutations that disrupt the coding sequence. For example, the effector RipS5 is encoded by a gene that has been clearly interrupted by a 34 kbp prophage. Table 6 also shows the orthologs of these genes in the related strains GMI1000, Po82, IPO1609, and UW551. In the table it can be seen that the genes that code for RipAA and RipAR have frameshifts or truncations in strain UY031 only. The absence of a particular effector may be enough for a pathogen to avoid host defenses, and therefore cause disease. These two genes are therefore a good starting point for additional investigations of phenotypic differences between these strains. Other effector genes of interest are those that are present and do not have disrupting mutations in UY031 but are absent or appear to be nonfunctional in other strains. We have found several such cases (Table 6), but in all cases there is at least one other strain that also has the same gene in what appears to be a functional state.
Table 6

List of T3E genes identified in R. solanacearum UY031 genome and their orthologs

Former effector name

New effector namea

UY031

(RSUY_)

GM1000

(RS)

Po82

(RSPO_)

IPO1609

(RSIPO_)

UW551

(RRSL_)

AWR2

RipA2

32720

p0099

m00080

03169

03418

AWR3

RipA3

40320

p0846

m01165

03901 + 05027b

-

AWR4

RipA4

40330/40b

p0847

m01166b

03902/3b

-

AWR5

RipA5_1

41860

p1024

m01289/90b

04049

01071

AWR5

RipA5_2

19780

-

c01821

01281

00546

Rip2

RipB

30390

c0245

c03161

00263

02573

Rip62

RipC1

42590

p1239

m01371

04123

03371

Rip34

RipD

33840

p0304

m01520

04484

00947

Rip26

RipE1

01190

c3369

c00070

03083

00852

-

RipE2

35100

-

c02513

04353

03923

PopF1

RipF1_1

45370

p1555

m01541

03403

04777

PopF2

RipF2

45510

-

m01557

05028/9b

04764

Gala2

RipG2

38790

p0672

m01007

04892

02264

Gala3

RipG3

32420

p0028

m00035

03202

00752

Gala4

RipG4

19910

c1800

c01835

01266/68b

00532

Gala5

RipG5

19920

c1801

c01836

01264

00531

Gala6

RipG6

17940

c1356

c01999

01463

01561

Gala7

RipG7

17950

c1357

c01998

01462

01562

HLK1

RipH1

19380

c1386

c01846

01319

00426

HLK2

RipH2

35470

p0215

m00201/2c

04317

03559

HLK3

RipH3

33320

p0160

m00157

03105

00041b

Rip1

RipI

00490 + 32050b

c0041

c03319

00098b

02976 + 02040b

Rip22

RipJ

24610b

c2132

c02749

-

-

Rip16

RipM

19180

c1475

c01871/2/3

01339 + 05024b

00705

Rip58

RipN

43290

p1130

m00869

04184

04736

Rip35

RipO1

34050

p0323

m01496

04463

00926

Rip63

RipQ

44390b

p1277

m00717

04287b

02855b

PopS

RipR

42640

p1281

m01376

04127

03375

SKWP1

RipS1

00860

c3401

c00036

00017

04182

SKWP2

RipS2

44630

p1374

m00690

04310

-

SKWP3

RipS3

41210

p0930

m01229

03993/4b

00237b

SKWP5

RipS5

10370 + 10840b

p0296

c02546b

-

-

SKWP7

RipS7

35110b

-

m00383

04352b

03921

Rip59

RipU

43920

p1212

m00805

04243

04660

Rip12

RipV1

17880

c1349

c02006

01470

01554

-

RipV2

19160b

-

c01875/76b

01341

00703

PopW

RipW

07010

c2775

c00735

02524

02682

PopA

RipX

40640

p0877

m01196

03933

02443

Rip3

RipY

30260

c0257

c03153

00276

01439

Rip57

RipZ

42040

p1031

m01312

04067

00271b

AvrA

RipAA

26380b

c0608

c02748

00659

01581

PopB

RipAB

40630

p0876

m01195

03932

02442

PopC

RipAC

40620

p0875

m01194

03931

02441

Rip72

ripAD

45790

p1601

m01585

03364

02518

Rip4

RipAE

29570

c0321

c03085

00343

01625

Rip41

RipAI

40230

p0838

m01156

03894

01021

Rip21

RipAJ

13300

c2101

c01332

04893

01260

Rip38

RipAL

39210b

-

m01053

-

02221

Brg40

RipAM

02270

c3272

c00191

02968

02810

Rip43

RipAN

40310

p0845

m01164

03900

01013

Rip50

RipAO

40750

p0879

m01206

03944

03105

Rip60

RipAP

43960

p1215b

m00800

04247

04655

Rip51

RipAQ

40810

p0885

-

03951

03113

Rip61

RipAR

44220b

p1236

m00770

04270

01136

Rip39

RipAV

39280

p0732

m01061

-

02213

Brg13

RipAX1

02040

c3290

m01221

02991

-

Rip55

RipAY

41810

p1022

m01283

04046

01066

-

RipBH

45880

-

m01600

03355

00782

-

RipBI

45200b

-

m00718

03419

00326

-

RipTPS

39290

p0731

m01062b

-

02212

aAccording to Peeters et al. [14]; b: these genes appear to be nonfunctional due to various reasons (frameshift, truncation, etc.); genes in other columns that appear in the form locus tag x + locus tag y are genes which also appear to be nonfunctional due to frameshifts. c:this gene is duplicated

Our modification analysis revealed two motifs that are essentially always methylated, namely: CAACRAC and GTWWAC. Both are fairly frequent in the genome, occurring respectively 2144 and 716 times. Motif CAACRAC is associated with the product of gene RSUY_11320 (R. Roberts, personal communication), which is hypothesized to be an enzyme of the Restriction-Modification System, with a restriction nuclease and a DNA methyltransferase role. This gene does not have homologs in other R. solanacearum strains and is located close to a region containing phage-related genes. This region contains gene RSUY_11410, which has been annotated as encoding a zonular occludens toxin. The provenance of this annotation is an enterotoxin gene found in Vibrio cholera [23]; in R. solanacearum the role of this toxin gene is still unclear [24]. Motif GTWWAC is probably associated with the product of gene RSUY_22890 (R. Roberts, personal communication), which is hypothesized to be a solitary DNA methyltransferase (no restriction endonuclease linked). This gene does have homologs in other R. solanacearum strains (GMI1000, IPO1609, Po82 and PSI07). To our knowledge this is the first R. solanacearum genome with a methylome profile available.

Conclusions

The complete sequence of R. solanacearum UY031 strain presented here should provide a rich platform upon which additional plant-pathogen studies can be carried out. Even though this is the fifth phylotype IIB1 sequenced, we found many differences with respect to the genomes of the other strains. In particular, the repertoire of T3E genes has many variations among these strains, and this may help explain some of the most relevant pathogenicity-related phenotypes described in the literature, opening the way to new control methods for bacterial wilt.

Abbreviations

IIB1: 

Phylotype IIB, sequevar 1

T3E: 

Type III effectors

HGG: 

Homologous gene groups

Declarations

Acknowledgements

We thank Carlos Balsalobre and Cristina Madrid for their helpful advice and for kindly providing materials and protocols; and Carlos Morais for help with NCBI submission. We also thank COST action Sustain from the European Union for funding and Nemo Peeters and Stéphane Genin for hosting MP for a short stay to carry out UY031 effector annotation. RGS has a Ph.D. fellowship from FAPESP, Brazil. JCS has an investigator fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Instituto de Química, Universidade de São Paulo
(2)
Department of Genetics, University of Barcelona and Centre for Research in Agricultural Genomics (CRAG)
(3)
Departamento de Biociencias, Cátedra de Microbiología, Facultad de Química, Universidad de la República
(4)
Biocomplexity Institute, Virginia Tech

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