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  • Research article
  • Open Access

Horizontal gene transfer plays a major role in the pathological convergence of Xanthomonas lineages on common bean

  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 2,
  • 3, 4,
  • 4 and
  • 1Email author
Contributed equally
BMC Genomics201819:606

https://doi.org/10.1186/s12864-018-4975-4

  • Received: 11 February 2018
  • Accepted: 31 July 2018
  • Published:

Abstract

Background

Host specialization is a hallmark of numerous plant pathogens including bacteria, fungi, oomycetes and viruses. Yet, the molecular and evolutionary bases of host specificity are poorly understood. In some cases, pathological convergence is observed for individuals belonging to distant phylogenetic clades. This is the case for Xanthomonas strains responsible for common bacterial blight of bean, spread across four genetic lineages. All the strains from these four lineages converged for pathogenicity on common bean, implying possible gene convergences and/or sharing of a common arsenal of genes conferring the ability to infect common bean.

Results

To search for genes involved in common bean specificity, we used a combination of whole-genome analyses without a priori, including a genome scan based on k-mer search. Analysis of 72 genomes from a collection of Xanthomonas pathovars unveiled 115 genes bearing DNA sequences specific to strains responsible for common bacterial blight, including 20 genes located on a plasmid. Of these 115 genes, 88 were involved in successive events of horizontal gene transfers among the four genetic lineages, and 44 contained nonsynonymous polymorphisms unique to the causal agents of common bacterial blight.

Conclusions

Our study revealed that host specificity of common bacterial blight agents is associated with a combination of horizontal transfers of genes, and highlights the role of plasmids in these horizontal transfers.

Keywords

  • Xanthomonas
  • Common bean
  • TAL effectors
  • Host adaptation
  • Horizontal gene transfer

Background

In nature, most pathogens are generalists, meaning that they are able to infect multiple hosts, while other pathogens are specialists, meaning that they are highly adapted to a single or few host species [1]. For plant pathogens, adaptation to a specific host plant is a complex process possibly involving diverse molecular determinants and leading to host specificity [2, 3]. Understanding the molecular basis of host specificity can provide new insights into the evolution and ecology of specialist pathogens, and their potential to shift species and to infect new hosts. Bacteria from the genus Xanthomonas infect at least 392 plant species including important crops and ornamentals [4]. Yet, each individual strain is able to infect only one or few plant species. Strains able to cause the same symptoms on the same host range are grouped into pathovars [5]. Although our understanding of the molecular basis of host specificity is still limited, chemotactic sensors, adhesins and type III effectors emerge as key determinants for shaping host specificity in Xanthomonas [68]. Chemotactic sensors enable the bacteria to detect attractant or repellent molecules and trigger flagellar motility towards entry sites of the host plant, while adhesins allow the attachment on the host plant surface and biofilm formation, and type III effectors are delivered into the plant cells where they can have different functions including providing pathogen-associated molecular pattern triggered immunity (PTI).

The Xanthomonas axonopodis species complex sensu Vauterin [9, 10] groups more than 30 pathovars infecting a wide range of plants including economically important crops and ornamentals, such as Citrus, Anthurium and Dieffenbachia species, as well as pepper, cassava, cotton, mango, soybean, and common bean. Based on repetitive-sequence-based Polymerase Chain Reaction (rep-PCR) fingerprints, X. axonopodis has been subdivided into six subclusters named 9.1 to 9.6 [11]. More recently, this species complex has been split into the four species X. citri, X. euvesicatoria, X. phaseoli and X. axonopodis [12, 13]. Common bacterial blight of bean (CBB) is the most devastating bacterial disease infecting common bean (Phaseolus vulgaris L.). CBB occurs everywhere where common bean is cultivated and may cause up to 75% yield loss in the most severe cases [14, 15]. Xanthomonas strains responsible for CBB are distributed across four different genetic lineages [16]. The fuscous lineage (fuscans) and the non-fuscous lineages 2 (NF2) and 3 (NF3) belong to X. citri pv. fuscans while the non-fuscous lineage 1 (NF1) belongs to X. phaseoli pv. phaseoli [9, 11, 12]. Pathological convergence between the NF1 and fuscans lineages is associated with horizontal gene transfers (HGT) involving dozens of genes [17]. Horizontal transfer of genes encoding Transcription Activator-Like (TAL) type III effectors was also observed between the four lineages of CBB agents [18]. In particular, all strains from the four genetic lineages display an allele of the tal23A gene, suggesting that this gene is important for Xanthomonas adaptation to common bean.

In order to search for Xanthomonas genes putatively involved in the adaptation leading to common bean specificity in Xanthomonas, we have generated the whole genome sequences of 17 X. citri pv. fuscans and X. phaseoli pv. phaseoli strains. A combination of approaches including a comparison between the phylogeny of genes and the phylogeny of organisms, a parsimony approach to infer gene gains and losses, and a genome-wide search for specific k-mers, was used to search for genes presenting common characteristics unique to strains belonging to the four bean-pathogenic lineages of X. citri pv. fuscans and X. phaseoli pv. phaseoli.

Results

Genome sequencing and phylogeny

In order to obtain genomic data representative of the diversity of Xanthomonas strains responsible for CBB, we produced whole genome sequences for 17 strains from the four genetic lineages of X. citri pv. fuscans and X. phaseoli pv. phaseoli that affect beans. In addition, we sequenced two strains of X. citri pv. mangiferaeindicae, one strain of X. citri pv. anacardii, three strains of X. oryzae pv. oryzicola, and used 51 other publically available Xanthomonas genomes for a total of 72 whole genome sequences (Table 1). Stenotrophomonas maltophilia strain R551–3 and Xylella fastidiosa strains 9a5c and Temecula1 were used as outgroups for further analyses [1921]. Annotation revealed from 3209 to 5405 coding sequences (CDS) per Xanthomonas genome (Additional file 1). Among Xanthomonas strains responsible for CBB, chromosome size ranged from 4,957,446 bp in strain CFBP1815 to 5,517,999 bp in strain CFBP6992, with an average GC content ranging from 64.3 to 64.9%. The phylogeny of strains was assessed based on the amino acid sequences of all annotated CDS using CVTree (Fig. 1). The overall topology of this tree was congruent with previous Xanthomonas phylogenies [11, 12, 22]. As described previously, strains responsible for CBB are distributed into four distinct genetic lineages belonging to two different species, X. citri and X. phaseoli [12, 13, 16, 23].
Table 1

Informations on the sequenced strains used in this study

Identifier

Species/Pathovar

Strain

Host of isolation

Country and date of isolation

Contigs

Total size (bp)

GC%

Accession number

Reference

stenma5513

Stenotrophomonas maltophilia

R551-3

Populus trichocarpa

na

1

4,573,969

66.29

GCA_000020665.1

Taghavi et al., 2008 [21]

albiliPC73

X. albilineans

GPE PC73

Saccharum spp. cv. H63-1418.

Guadeloupe (France), 2003

1

3,768,695

62.97

GCA_000087965.1

Pieretti et al., 2009 [77]

aberra6865

X. campestris pv. campestris

CFBP6865

Brassica oleracea var. capitata.

Australia, 1975

4

5,169,518

64.93

Unpublished

Noël, comm. pers

barbar5825

X. campestris pv. barbareae

CFBP5825R

Barbarea vulgaris

USA, 1939

2

5,055,186

65.09

ATNQ00000000

Roux et al., 2015 [78]

campes8004

X. campestris pv. campestris

8004

Brassica oleracea var. botrytis.

United Kingdom, 1958

1

5,148,708

64.95

GCA_000012105.1

Qian et al., 2005 [79]

campesAT33

X. campestris pv. campestris

ATCC 33913

Brassica oleracea var. gemmifera

United Kingdom, 1957

1

5,076,188

65.06

GCA_000007145.1

da Silva et al., 2002 [80]

campesB100

X. campestris pv. campestris

B100

na

na

1

5,079,002

65.04

GCA_000070605.1

Vorhölter et al., 2008 [81]

incana2527

X. campestris pv. incanae

CFBP2527R

Matthiola incana

USA, 1950

1

4,926,205

65.12

ATNO00000000

Roux et al., 2015 [78]

incana1606

X. campestris pv. incanae

CFBP1606R

Matthiola incana

France, 1974

1

4,967,651

65.17

ATNN00000000

Roux et al., 2015 [78]

musace4381

X. campestris pv. musacearum

NCPPB4381

Musa sp.

Uganda, 2005

115

4,810,038

61.96

ACHT00000000

Studholme et al., 2010 [82]

raphan5828

X. campestris pv. raphani

CFBP5828R

Raphanus sativus

USA, na

2

4,912,709

65.35

ATNP00000000

Roux et al., 2015 [78]

raphan756C

X. campestris pv. raphani

756C

Brassica oleracea var. capitata

east asia, na

1

4,941,214

65.28

GCA_000221965.1

Bogdanove et al., 2011 [83]

cassav4642

X. cassavae

CFBP4642

Manihot esculenta

Malawi, 1951

7

5,278,192

65.22

GCA_000454545.1

Bolot et al., 2013 [84]

axanac2913

X. citri pv. anacardii

CFBP2913

Mangifera indica

Brazil, na

1

5,203,496

64.57

CP024057

this study

axaura1035

X. citri pv. aurantifolii

ICPB10535

Citrus aurantiifolia

Brazil, na

351

5,060,896

64.81

ACPY00000000

Moreira et al., 2010 [85]

axcitr1083

X. citri pv. citri

FDC1083

Citrus reticulata

Brazil, 1980

1

5,218,314

64.72

CCVZ01000000

Gordon et al., 2015 [86]

axcitrJ902

X. citri pv. citri

JF90-2

Citrus aurantiifolia

Oman, 1986

1

5,251,225

64.66

CCWA01000000

Gordon et al., 2015 [86]

axcitrJ238

X. citri pv. citri

JJ238-24

Citrus aurantiifolia

Thailand, 1989

1

5,282,622

64.78

CCVX01000000

Gordon et al., 2015 [86]

axcitrLC80

X. citri pv. citri

LC80

Citrus reticulata x C. sinensis

Mali, 2006

1

5,229,127

64.7

CCWJ01000000

Gordon et al., 2015 [86]

axcitrLE20

X. citri pv. citri

LE20-1

Citrus aurantiifolia

Ethiopia, 2008

1

5,309,240

64.71

CCWK01000000

Gordon et al., 2015 [86]

axcitr9322

X. citri pv. citri

LMG9322

Citrus aurantiifolia

Florida, 1986

1

5,194,131

64.75

CCVY01000000

Gordon et al., 2015 [86]

axcitri306

X. citri pv. citri

306

na

na

1

5,175,554

64.77

GCA_000007165.1

da Silva et al., 2002 [80]

phafus1815

X. citri pv. fuscans (fuscans)

CFBP1815

Phaseolus sp

Greece, 1978

1

4,957,446

64.82

GCA_900234415

this study

phafus4834

X. citri pv. fuscans (fuscans)

4834-R

Phaseolus vulgaris cv. Michelet

France, 1998

1

4,981,995

64.81

FO681494

Darrasse et al., 2013 [26]

phafus6166

X. citri pv. fuscans (fuscans)

CFBP6166

Phaseolus vulgaris

South Africa, 1963

1

4,987,587

64.8

GCA_900234455

this study

phafus6960

X. citri pv. fuscans (fuscans)

CFBP6960

Phaseolus vulgaris

Reunion island, France, 2000

1

4,992,131

64.79

GCA_900234515

this study

phafus6970

X. citri pv. fuscans (fuscans)

CFBP6970

Phaseolus sp

USA, 1990

1

5,006,656

64.9

GCA_900234505

this study

pha2GL6988

X. citri pv. fuscans (NF2)

CFBP6988

Phaseolus vulgaris cv. marla

Reunion island, France, 2000

1

5,132,433

64.64

Deposited

this study

pha2GL6990

X. citri pv. fuscans (NF2)

CFBP6990

Phaseolus vulgaris cv. marla

Reunion island, France, 2000

1

5,124,653

64.63

GCA_900234485

this study

pha2GL6991

X. citri pv. fuscans (NF2)

CFBP6991

Phaseolus vulgaris cv. marla

Reunion island, France, 2000

1

5,342,565

64.35

GCA_900234525

this study

pha3GL6992

X. citri pv. fuscans (NF3)

CFBP6992

Phaseolus vulgaris cv. marla

Reunion island, France, 2000

1

5,517,999

64.48

GCA_900234495

this study

pha3GL6994

X. citri pv. fuscans (NF3)

CFBP6994

Phaseolus vulgaris

Tanzania, 1990

1

5,250,266

64.57

GCA_900234565

this study

pha3GL6996

X. citri pv. fuscans (NF3)

CFBP6996

Phaseolus vulgaris cv. marla

Reunion island, France, 2000

4

5,095,420

64.72

AVET00000000

this study

phafus7766

X. citri pv. fuscans (fuscans)

CFBP7766

Phaseolus vulgaris

Cameroon, 2009

1

5,185,891

64.63

GCA_900234475

this study

phafus7767

X. citri pv. fuscans (fuscans)

CFBP7767

Phaseolus vulgaris

Cameroon, 2009

1

5,107,388

64.7

GCA_900234465

this study

axglyc2526

X. citri pv. glycines

CFBP2526

Glycine hispida

Sudan, 1956

4

5,255,152

64.63

GCA_000495275.1

Darrasse et al., 2013 [26]

axglyc7119

X. citri pv. glycines

CFBP7119

Glycine max

Brazil, 1981

4

5,521,783

64.39

GCA_000488895.1

Darrasse et al., 2013 [26]

axmalv1386

X. citri pv. malvacearum

GSPB1386

na

na

127

4,991,411

64.7

GCA_000309905.1

Hainan University

axmalv2388

X. citri pv. malvacearum

GSPB2388

na

na

61

5,127,016

64.53

GCA_000309925.1

Hainan University

axmalvaX18

X. citri pv. malvacearum

X18

Gossypium spp.

Burkina Faso, 1980s

4

4,993,692

64.7

ATMA00000000

Cunnac et al., 2013 [87]

axmalvaX20

X. citri pv. malvacearum

X20

Gossypium spp.

Burkina Faso, 1980s

4

5,219,648

64.49

ATMB00000000

Cunnac et al., 2013 [87]

axmang5610

X. citri pv. mangiferaeindicae

LG56-10

Mangifera indica

na

4

5,281,952

64.56

PEBY00000000

this study

axmang8127

X. citri pv. mangiferaeindicae

LG81-27

Mangifera indica

na

6

5,209,594

64.68

PEBZ00000000

this study

axmangL941

X. citri pv. mangiferaeindicae

LMG941

Mangifera indica

India, 1957

195

5,144,323

64.84

CAHO01000001

Midha et al., 2012 [88]

axalfa3836

X. euvesicatoria pv. alfalfae

CFBP3836

Medicago sativa

Sudan, na

6

5,081,438

64.74

GCA_000488955.1

Jacques et al., 2013 [3, 22]

axalli6369

X. euvesicatoria pv. allii

CFBP6369

Allium cepa

Reunion island, France, 1996

3

5,427,488

64.36

GCA_000730305.1

Gagnevin et al., 2014 [89]

axmeloniF1

X. euvesicatoria pv. citrumelonis

F1

Citrus sp.

FL, USA, 1984

1

4,967,469

64.91

GCA_000225915.1

Jalan et al., 2011 [90]

axeuve8510

X. euvesicatoria pv. euvesicatoria

85-10

Capsicum annuum

FL, USA, 1985

1

5,178,466

64.74

GCA_000009165.1

Thieme et al., 2005 [91]

perfor9118

X. euvesicatoria pv. perforans

91-118

Lycopersicon esculentum

FL, USA, na

291

5,296,241

65.04

AEQW00000000

Potnis et al., 2011 [92]

gardne1965

X. gardneri

ATCC19865

Lycopersicon esculentum

Yugoslavia, 1953

552

5,594,687

63.68

AEQX00000000

Potnis et al., 2011 [92]

carotaM081

X. hortorum pv. carotae

M081

Daucus carota

OR, USA, na

154

5,039,269

63.83

GCA_000505565.1

Kimbrel et al., 2011 [93]

oryzaeKA10

X. oryzae pv. oryzae

KACC10331

Oryza sativa

Korea, na

1

4,941,439

63.69

GCA_000007385.1

Lee et al., 2005 [94]

oryzaeMA31

X. oryzae pv. oryzae

MAFF 311018

Oryza sativa

Japan, na

1

4,940,217

63.7

GCA_000010025.1

unpublished

oryzaePX99

X. oryzae pv. oryzae

PXO99A

Oryza sativa

Philippines, na

1

5,240,075

63.63

GCA_000019585.1

Salzberg et al., 2008 [95]

oryzicBA15

X. oryzae pv. oryzicola

BAI15

Oryza sativa

Burkina Faso, 2009

1

4,315,327

64.16

GCA_002189395.1

this study

oryzicBA20

X. oryzae pv. oryzicola

BAI20

Oryza sativa

Burkina Faso, 2009

1

4,498,643

64.05

GCA_002189435.1

this study

oryzicBA21

X. oryzae pv. oryzicola

BAI21

Oryza sativa

Burkina Faso, 2009

1

4,419,870

64.07

GCA_002189465.1

this study

oryzicB256

X. oryzae pv. oryzicola

BLS256

Oryza sativa

Philippines, 1984

1

4,831,739

64.05

AAQN01000001

Bogdanove et al., 2011 [83]

axdieff695

X. phaseoli pv. dieffenbachiae

LMG695

Anthurium andreanum

Brazil, 1965

1

5,037,357

64.88

GCA_001564415.1

Robène et al., 2016 [96]

axmaniC151

X. phaseoli pv. manihotis

CIO151

Manihot sp.

Colombia, na

36

5,154,532

64.7

GCA_000265845.1

Rodriguez et al., 2012 [97]

pha1GLC412

X. phaseoli pv. phaseoli (NF1)

CFBP412

Phaseolus vulgaris

USA, na

1

5,028,351

64.94

GCA_900234435

this study

pha1GL6164

X. phaseoli pv. phaseoli (NF1)

CFBP6164

Phaseolus vulgaris

Romania, 1996

1

5,319,061

64.64

GCA_900234535

this study

pha1GL6546

X. phaseoli pv. phaseoli (NF1)

CFBP6546

Phaseolus vulgaris

USA, na

4

4,994,665

64.92

Deposited

this study

pha1GL6984

X. phaseoli pv. phaseoli (NF1)

CFBP6984

Phaseolus vulgaris

Reunion island, France, 2000

1

5,106,663

64.82

GCA_900234425

this study

pha1GL7430

X. phaseoli pv. phaseoli (NF1)

CFBP7430

Phaseolus vulgaris cv. CFO16

Iran, 2006

1

5,085,978

64.89

GCA_900234445

this study

axsyng9055

X. phaseoli pv. syngonii (NF1)

LMG9055

Syngonium podophyllum

na

6

5,010,905

64.82

GCA_001640215.1

Robène et al., 2016 [96]

saccha4393

X. sacchari

NCPPB4393

Musa sp.

Tanzania, 2007

470

4,955,099

69.03

AGDB00000000

Studholme et al., 2011 [98]

cereal2541

X. translucens pv. cerealis

CFBP2541

Bromus inermis

USA, 1941

10

4,524,870

67.37

GCA_000807145.1

Pesce et al., 2015a [99]

trgramXt29

X. translucens pv. graminis

ART-Xtg29

Lolium multiflorum

Switzerland, na

788

4,203,855

68.58

ANGG00000000

Wichman et al., 2013 [101]

gramin2053

X. translucens pv. graminis

CFPB2053

Dactylis glomerata

Switzerland, 1973

2

4,344,936

68.33

LHSI00000000

Pesce et al., 2015b [100]

trtran1874

X. translucens pv. translucens

DSM18974

Hordeum vulgare

USA, 1958

551

4,550,921

67.67

GCA_000331775.1

unpublished

vasculN702

X. vasicola pv. vasculorum

NCPPB702

Saccharum officinarum

Zimbabwe, 1959

97

5,491,457

56.86

ACHS00000000

Studholme et al., 2010 [82]

vesica3537

X. vesicatoria

ATCC35937

Lycopersicon esculentum

New Zealand, 1955

296

5,567,561

64.06

AEQV01000000

Potnis et al., 2011 [92]

xylefaTem1

Xylella fastidiosa subsp. fastidiosa

Temecula1

Vitis vinifera

USA, na

1

2,519,802

51.77

GCA_000007245.1

van Sluys et al., 2003 [102]

xylefa9a5c

Xylella fastidiosa subsp. pauca

9a5c

Citrus sinensis cv. Valencia

Brazil, 1992

1

2,679,306

52.67

GCA_000006725.1

Simpson et al., 2000 [19]

na not available

Fig. 1
Fig. 1

Phylogeny of Xanthomonas strains used in this study. Phylogeny of Xanthomonas strains used in this study with indication of gene gains and losses. The phylogenetic tree is based on whole genome analysis using CVTree [66] with default parameters. Strain aliases are described in Table 1. Stenotrophomonas and Xylella genomes have been used as outgroups. Xanthomonas main phylogenetic groups 1 and 2 [24] and the X. axonopodis species complex [9, 10] are indicated by arrows. Groups 9.2, 9.4, 9.5 and 9.6 [11] are indicated in brackets. Fuscans, NF2, NF3 and NF1 refer to the four genetic lineages of strains responsible for CBB. A parsimony approach was performed to infer gene gains (blue) and losses (red) at levels higher than the pathovar rank, and numbers are displayed at each branch. Red stars highlight cases where gene loss was greater that gene gain. Curved arrows represent horizontal gene transfers (HGT) retrieved by Ks analysis on alignments of 115 candidate genes for bean specificity, with HGT from X. citri pv. fuscans to X. phaseoli pv. phaseoli in green, HGT from X. phaseoli pv. phaseoli to X. citri pv. fuscans in purple, and HGT between X. citri pv. fuscans lineages in red. Numbers in circles correspond to the numbers of candidate genes involved for each HGT. Question marks indicate events for which the origin or end of the HGT was not precise enough to assign any particular lineage

Genome expansion occurred during the evolution of Xanthomonas

To identify the genes shared by different clades of Xanthomonas, we constructed an orthology matrix using OrthoMCL (Additional file 2). Based on this orthology matrix, we performed a parsimony approach to infer gene gains and losses at each branch of the phylogenetic tree (Fig. 1). We did not take into account events occurring on the most distal branches to reduce the bias due to the difference of quality between sequenced genomes. At every branch, one to several hundreds of genes were either gained or lost. A general observation was that gene gains were higher than gene losses, suggesting that genome expansion occurred during the evolution of Xanthomonas (Fig. 1). Only four cases of genome reduction were observed (i) at the origin of the Xylella genus, (ii and iii) along two consecutive branches before and after the split between the X. oryzae species and the X. vasculorum and X. musacearum species, and (iv) at the origin of X. citri pv. malvacearum. The largest gene gain (418) was observed at the origin of Xanthomonas phylogenetic group 2 as defined by Young et al. [24], while the largest gene loss (819) was observed at the origin of the Xylella genus. Few gene losses (9 to 32) were observed before the diversification of each of the four genetic lineages involved in CBB. Of those, the NF1 lineage was the one which gained the most genes (271) followed by the NF2 (225), NF3 (108) and fuscans (83) lineages, respectively.

The pan and core genomes of Xanthomonas reveal extensive horizontal gene transfers between strains pathogenic on common bean

Individuals that are closely related to each other typically share more orthologs than unrelated individuals. Therefore, groups of closely related individuals tend to have a smaller pan genome and a larger core genome than groups of more divergent individuals. As such, the pan and core genomes for the 72 Xanthomonas strains comprised 32,602 and 1144 CDS, respectively, while the pan and core genomes for the 75 strains including the outgroups comprised 34,723 and 816 CDS, respectively (Fig. 2). Similarly, each Rademaker group alone, i.e. 9.2, 9.4, 9.5 and 9.6, had a smaller pan genome (6578, 8222, 9387 and 9437 CDS, respectively) and a larger core genome (3493, 2949, 3056 and 3213 CDS, respectively) than the X. axonopodis species complex, which had pan and core genomes of 19,010 and 2297 CDS, respectively. Strikingly, when grouping strains responsible for CBB belonging to X. citri pv. fuscans and X. phaseoli pv. phaseoli, both the pan and core genomes (10,750 and 3222 CDS, respectively) were larger than the pan and core genomes from groups 9.4 (8222 and 2949 CDS, respectively) or 9.6 (9437 and 3213 CDS, respectively) (Fig. 2). Thus, strains responsible for CBB, although being phylogenetically diverse, had more genes in common than they had with other strains belonging to their respective clades, which was suggestive of extensive HGT among these strains. This result was reminiscent of previous comparative analyses showing that dozens of genes have been horizontally transferred between the fuscans and NF1 lineages [17].
Fig. 2
Fig. 2

The core- and pan-genome of Xanthomonas. Gene numbers correspond to the number of ortholog groups retrieved for each group of strains. All: all strains used in this study (n = 75); Xantho: strains from the Xanthomonas genus (n = 72); X.axo: strains from the Xanthomonas axonopodis species complex (n = 44). 9.2, 9.4, 9.5, 9.6: strains belonging to rep-PCR groups 9.2 (n = 5), 9.4 (n = 8), 9.5 (n = 16) and 9.6 (n = 15), respectively, as defined in Rademaker et al. [103]. Xcf-Xpp: strains pathogenic on common bean belonging to Xanthomonas citri pv. fuscans or Xanthomonas phaseoli pv. phaseoli (n = 18)

Strains pathogenic on common bean share 115 CDS presenting unique characteristics

To search for genes potentially involved in the convergence between X. phaseoli pv. phaseoli (i.e. the NF1 lineage) and X. citri pv. fuscans (i.e. the NF2, NF3 and fuscans lineages) to infect common bean, we performed a combination of four different analyses. First, within the OrthoMCL matrix, we searched for CDS specifically present in the genomes of CBB agents and absent from any other Xanthomonas genome, or present in the genomes from all Xanthomonas but not in the genomes of CBB agents. No CDS was retrieved by this analysis. We also searched for CDS specifically present or absent when grouping the NF1 lineage to each of the NF2, NF3, or fuscans lineages. Only one CDS was specifically retrieved in the NF1 and fuscans lineages.

Second, we used the results from the CDS gains and losses approach described above to search for genes shared by all strains from X. phaseoli pv. phaseoli and X. citri pv. fuscans, and gained in the ancestor of one pathovar or the other. This approach unveiled nine CDS shared by all strains responsible for CBB, and gained in either X. phaseoli pv. phaseoli or X. citri pv. fuscans (Table 2). We also searched for CDS shared by the NF1 and each of the NF2, NF3, or fuscans lineage and gained in at least one of these lineages. Four CDS were shared by the NF1 and NF2 lineages, or the NF1 and NF3 lineages, while three CDS were shared by the NF1 and fuscans lineages.
Table 2

Numbers of CDS presenting similarities among the lineages of CBB agents

Lineages studied

Presence/absencea

Gainedb

Monophyleticc

24-mersd

NF1/NF2/NF3/fuscans

0

9

28

108

NF1/NF2

0

4

9

33

NF1/NF3

0

4

5

28

NF1/fuscans

1

3

105

231

Total

1

20

147

400

aCDS specifically present or absent in all the lineages studied compared to all other X. axonopodis strains

bCDS present in all the lineages studied and gained in a least one of these lineages

cCDS monophyletic for the lineages studied

dCDS containing 24-mers specifically present or absent in all the lineages studied compared to all other X.axonopodis strains

Third, we used a phylogenetic approach to search for genes for which strains from X. citri pv. fuscans and X. phaseoli pv. phaseoli formed a monophyletic group. For this, we constructed phylogenetic trees on 3202 CDS present in every X. citri pv. fuscans and X. phaseoli pv. phaseoli strain and in at least one additional strain from group 9.2 or 9.4 and one other strain from group 9.5 or 9.6. The additional strains from groups 9.2, 9.4, 9.5 and 9.6 were located inbetween X. citri pv. fuscans and X. phaseoli pv. phaseoli (Fig. 1). Thus, CDS found as monophyletic for CBB strains could be potential traces of HGT between both pathovars. This approach unveiled 28 CDS for which the four genetic lineages formed a monophyletic group, suggesting that they were horizontally transferred among these lineages (Table 2). Nine CDS were specifically monophyletic for the NF1 and NF2 lineages, five for the NF1 and NF3 lineages, and 105 for the NF1 and fuscans lineages, suggesting that most horizontal transfers occurred among the NF1 and fuscans lineages.

Finally, we used the SkIf tool [25] on the 72 Xanthomonas genomes to search for genes containing short 24-bp sequences (24-mers) specific to strains responsible for CBB, or alternatively genes from strains responsible for CBB lacking 24-mers present in all other strains from the X. axonopodis species complex. In all, we identified 108 CDS containing 24-mers either specifically present or absent from the four lineages (Table 2). Moreover, 33 CDS contained 24-mers specific for the NF1 and NF2 lineages, 28 for the NF1 and NF3 lineages and 231 for the NF1 and fuscans lineages. Similarly to the analysis based on phylogeny, this analysis based on k-mers pointed an overrepresentation of CDS with specific 24-mers shared by the NF1 and fuscans lineages compared to NF2 and NF3 lineages.

Together, these four analyses unveiled respectively 0, 9, 28, or 109 CDS presenting features unique to CBB agents. The analysis based on presence/absence seemed to be too stringent for unveiling any CDS, while the analysis based on k-mers was the most sensitive, suggesting that SkIf was an appropriate tool for finding common traits shared by phylogenetically distant strains. Most of these CDS were found redundantly by two or more analyses, for a total of 115 non-redundant CDS (Table 3). The most represented functions encoded by these 115 predicted CDS were hypothetical proteins (26 CDS), followed by membrane-related proteins (10 CDS), two-component system proteins (six CDS), putative secreted proteins (five CDS), reductases (five CDS), RNA-related proteins (five CDS), Type III secretion system-related proteins (five CDS), TonB-dependent proteins (four CDS), Type IV secretion system-related proteins (three CDS), Type VI secretion system-related proteins (three CDS), DNA-related proteins (three CDS) and transcription regulators (three CDS) (Table 3).
Table 3

Overview of the 115 genes putatively involved in bean specificity

Identifiera

Accession numberb

Predicted function

Gene

Gained

Monophyletic

24-mers

Recombinant

HGT

Atypical GC%

Nonsynonymous sites

Aritua et al. [17]c

1

m00100560

TonB-dependent transporter

Yes

Yes

 

2

m00100580

type III effector

avrBs2

Yes

Yes

Yes

3

m00100590

hypothetical protein

Yes

Yes

Yes

4

m00101230

two compoment system sensor protein

Yes

5

m00101980

two component system protein

glnG

Yes

6

m00102200

two component system response regulator

Yes

Yes

Yes

Yes

7

m00104250

type III secretion system protein

hrpF

Yes

Yes

Yes

8

m00104520

type III effector

xopA

Yes

Yes

Yes

9

m00104530

lytic transglycosylase-like protein

hpaH

Yes

Yes

Yes

Yes

10

m00104540

hypothetical protein

Yes

Yes

Yes

11

m00104640

diguanylate cyclase

Yes

Yes

Yes

12

m00104690

4-alpha-glucaNotransferase

malQ

Yes

Yes

13

m00105290

threonine aldolase

Yes

Yes

14

m00105330

flavin reductase

Yes

Yes

Yes

Yes

15

m00105340

anthranilate phosphoribosyl transferase

trpD

Yes

Yes

16

m00105390

S-adenosylmethionine decarboxylase

speD

Yes

Yes

Yes

17

m00105750

hypothetical membrane protein

Yes

18

m00107500

TonB-dependent transporter

Yes

Yes

Yes

19

m00108560

general secretion pathway protein D

xpsD

Yes

Yes

Yes

Yes

Yes

20

m00108570

hypothetical protein

Yes

Yes

Yes

21

m00108600

TonB-dependent transporter

Yes

Yes

Yes

Yes

22

m00109610

TonB-dependent transporter

Yes

Yes

Yes

23

m00109620

phosphoanhydride phosphohydrolase

appA

Yes

Yes

Yes

Yes

Yes

24

m00109670

hypothetical protein

Yes

Yes

25

m00109990

bis(5′nucleosyl)-tetraphosphatase

apaH

Yes

26

m00110970

peptidyl-tRNA hydrolase

Yes

Yes

Yes

Yes

27

m00111160

50S ribosomal protein

rplW

Yes

Yes

28

m00112070

two-component system sensor protein

phoR

Yes

29

m00113470

xanthine dehydrogenase subunit

Yes

Yes

Yes

Yes

Yes

30

m00113490

xanthine dehydrogenase subunit

Yes

Yes

31

m00114150

membrane protein

Yes

Yes

Yes

32

m00114680

two component system sensor protein

Yes

Yes

Yes

33

m00116790

NAD/FAD binding protein

Yes

Yes

34

m00117590

acetyltransferase

Yes

Yes

35

m00117600

transcriptional regulator

Yes

Yes

Yes

Yes

Yes

36

m00117610

hypothetical protein

Yes

Yes

Yes

37

m00118890

hypothetical protein

Yes

Yes

38

m00119700

putative secreted protein

Yes

39

m00119970

phosphomethylpyrimidine kinase

thiD

Yes

Yes

Yes

40

m00121810

threonine synthase

thrC

Yes

Yes

Yes

Yes

41

m00121850

histidyl tRNA synthetase

hisS

Yes

Yes

42

m00121900

histidinol phosphatase

hisB

Yes

Yes

Yes

43

m00123210

recombination factor protein

rarA

Yes

Yes

44

m00126530

ethanolamine permease

eutP

Yes

Yes

Yes

45

m00126540

ethanolamine ammona-lyase

eutB

Yes

Yes

46

m00127140

membrane protein

Yes

Yes

47

m00127150

cGMP specific phosphodiesterase

Yes

Yes

Yes

Yes

48

m00127220

hypothetical protein

Yes

Yes

49

m00127250

ubiquinol-cytochrome c reductase subunit

Yes

Yes

50

m00127260

ubiquinol-cytochrome c reductase subunit

Yes

Yes

51

m00127850

1-phosphofructokinase

fruK

Yes

Yes

52

m00127860

PTS fructose porter IIBC component

fruA

Yes

Yes

53

m00130650

RNA polymerase ECF-type sigma factor

rpoE2

Yes

Yes

Yes

54

m00130660

hypothetical protein

Yes

Yes

Yes

Yes

55

m00130670

RNA-binding protein

Yes

Yes

Yes

56

m00130690

inner membrane protein

Yes

Yes

57

m00132430

DNA topoisomerase IA

Yes

Yes

58

m00135840

membrane fusion protein

Yes

59

m00135870

sulfite reductase (NADPH) subunit

cysJ

Yes

Yes

Yes

60

m00135880

sulfite reductase (NADPH) subunit

cysI

Yes

Yes

61

m00135970

transcriptional regulator protein

cysB

Yes

Yes

62

m00135980

siroheme synthase

cysG

Yes

Yes

Yes

63

m00136050

phosphoglycerate kinase

pgk

Yes

Yes

64

m00136550

hypothetical protein

Yes

65

m00138140

two-compoment system sensor protein

Yes

Yes

66

m00139330

dihydrolipoamide acetyltransferase

aceF

Yes

Yes

Yes

67

m00139350

DNA glycosylase

Yes

Yes

Yes

68

m00139420

membrane protein

Yes

Yes

Yes

Yes

69

m00139860

putative secreted protein

Yes

70

m00139930

flavodoxin protein

Yes

Yes

71

m00140860

peptidoglycan binding protein

lysM

Yes

Yes

72

m00140880

hypothetical protein

Yes

73

m00141310

PhnB like protein

Yes

Yes

Yes

74

m00141340

hypothetical protein

Yes

Yes

75

m00141400

hypothetical protein

Yes

Yes

76

m00141430

transmembrane protein

Yes

Yes

Yes

77

m00141440

hypothetical protein

Yes

Yes

Yes

78

m00141490

dipeptide epimerase

Yes

Yes

79

m00142140

putative glucosyltransferase

Yes

Yes

Yes

80

m00144120

type VI secretion system protein

icmF

Yes

Yes

Yes

Yes

81

m00144240

type VI secretion system kinase protein

tagE

Yes

Yes

Yes

82

m00144270

transmembrane sensor protein

fecR

Yes

Yes

83

m00144420

type VI secretion system virulence protein

impE

Yes

Yes

Yes

84

m00145330

type III effector

xopAD

Yes

Yes

Yes

Yes

85

m00146940

membrane protein

Yes

Yes

Yes

86

m00146970

LysR family transcriptional regulator

Yes

Yes

87

m00146980

short chain dehydrogenase

Yes

Yes

88

m00146990

RNA 2′-phosphotransferase family protein

Yes

Yes

Yes

89

m00147020

hypothetical protein

Yes

Yes

Yes

90

m00147060

esterase family protein

Yes

Yes

91

m00147090

putative secreted protein

Yes

Yes

92

m00147100

putative secreted protein

Yes

Yes

Yes

93

m00147120

ribosomal pseudouridine synthase

Yes

Yes

Yes

Yes

94

m00147130

hypothetical protein

Yes

Yes

Yes

95

m00147140

putative secreted protein

Yes

Yes

Yes

96

m00200020

plasmid partitioning protein

Yes

Yes

na

Yes

97

m00200060

hypothetical protein

Yes

Yes

na

Yes

98

m00200080

hypothetical protein

Yes

Yes

na

Yes

99

m00200130

hypothetical protein

Yes

Yes

Yes

100

m00200150

hypothetical protein

Yes

Yes

na

Yes

101

m00200160

hypothetical protein

Yes

na

Yes

Yes

102

m00200170

hypothetical protein

Yes

na

Yes

103

m00200190

hypothetical protein

Yes

na

Yes

104

m00200260

hypothetical protein

Yes

Yes

Yes

105

m00200790

transposase

tnpA

Yes

Yes

Yes

106

m00200820

resolvase

tnpR

Yes

Yes

107

m00200880

DNA topoisomerase IA

Yes

na

Yes

108

m00200980

type IV secretion system protein

trbI

Yes

Yes

Yes

na

Yes

109

m00201120

membrane protein

Yes

110

m00201220

type IV conjugal transfer protein

traG

Yes

na

Yes

111

m00201230

hypothetical protein

Yes

na

Yes

112

m00201250

lytic transglycolase

Yes

Yes

Yes

na

Yes

113

m00201260

type IV conjugal transfer protein

traF

Yes

Yes

na

Yes

114

m00201270

hypothetical protein

Yes

Yes

Yes

na

Yes

115

m00201290

hypothetical protein

Yes

Yes

Yes

na

Yes

Total

115

9

28

108

18

88

2

44

9

– not found in corresponding analysis

na not applicable due to the absence of corresponding CDS in outgroup

aidentifiers correponding to the numbers shown in Fig. 3

baccession numbers for strain CFBP6456

cgenes with 100% identity over 95% of their length according to Aritua et al. [17]

We hypothesized that the CDS potentially involved in the specific adaptation to common bean should bear nonsynonymous polymorphisms specific to Xanthomonas strains pathogenic on common bean. Analysis of the alignments for the 115 candidate CDS highlighted 44 CDS with nonsynonymous sites retrieved exclusively in X. citri pv. fuscans and X. phaseoli pv. phaseoli (Table 3). More than one third of these CDS (16/44) encoded hypothetical proteins. Among the other CDS, three encoded type IV secretion system proteins TrbI TraG and TraF, two encoded putative secreted proteins, two encoded type III secretion system proteins XopA and XopAD, two encoded DNA topoisomerases, and others encoded proteins of various functions (Table 3).

Specificity to common bean is associated with successive waves of horizontal gene transfers

Strain CFBP6546 from the NF1 lineage was used as reference for further analyses. Its genome contained one chromosome and three extrachromosomal plasmids formerly described as plasmid a, plasmid b and plasmid c in strain 4834-R [26]. Most candidate genes (95/115) were located on the chromosome, while 20 were located on the plasmid a (Fig. 3). This corresponds to a density of one candidate gene per 50.9 kbp in the chromosome, and one per 3.5 kbp for the plasmid a, while none were retrieved in plasmids b or c. Interestingly, all the CDS found in plasmid a contained specific nonsynonymous sites (Table 3). Thus, plasmid a appeared as an important vector of genes involved in the adaptation to common bean. Another observation was that the chromosome contained regions with various 24-mers shared by the NF1 lineage and any of the fuscans, NF2 or NF3 lineages (in green, blue or black in Fig. 3, respectively). This suggests that the regions shared by the NF1 and the other lineages diverged since the split between the NF2, NF3 and fuscans lineages. By contrast to what was observed for the chromosome, all specific 24-mers found in plasmid a were simultaneously shared by the four genetic lineages of strains responsible for CBB (in purple in Fig. 3), indicating that these regions have been shared between the four lineages recently enough to still have 100% identity between each other. Together, these results suggest that 24-mers retrieved in the chromosome correspond to more ancient HGT events than those retrieved in plasmid a.
Fig. 3
Fig. 3

Mapping of the 24-mers specific for strains pathogenic on common bean. The innermost rings represent the reference chromosome or plasmids with associated coordinates. Colored lines represent 24-mers specifically retrieved in X. citri pv. fuscans and X. phaseoli pv. phaseoli strains (purple), or in the NF1 plus fuscans lineages (green), or in the NF1 plus NF2 lineages (blue), or in the NF1 plus NF3 lineages (black). Red numbers correspond to the identifiers of the 115 genes listed in Table 3

Nucleotide synonymous substitution rates at silent sites (Ks) is an estimation of neutral evolution because it does not take into account the nonsynonymous sites that can be under selection pressure. Therefore, Ks can be used as an approximation of the time of divergence between genes or taxa, with higher Ks value meaning longer time of divergence between two sequences [27, 28]. For each of the 115 candidate genes found in CBB agents, we performed multiple alignments. We could not perform Ks analysis on 15 genes that were lacking outgroups (Table 3), therefore we tested only 100 out of 115 genes. Among these 100 genes, 18 were recombinants according to RDP software analysis (Table 3). For these 18 recombinants, Ks values were independently calculated on both sides of the breakpoints. We calculated pairwise Ks values for different combinations of strains including X. citri pv. fuscans, X. phaseoli pv. phaseoli and closely related strains including X. citri pv. anacardii, X. citri pv. aurantifolii, X. phaseoli pv. syngonii, X. phaseoli pv. dieffienbachiae and X. phaseoli pv. manihotis (Fig. 1, Additional file 3). We then used these Ks as relative time divergence estimations to infer if a HGT occurred between NF1, NF2, NF3 and/or fuscans lineages, as well as the direction of this HGT. For example, for gene m00100580b the mean Ks value between strains from the NF1 and fuscans lineages was 7.40e-03 +/− 2.85e-10. This value was lower than the Ks values when comparing the fuscans lineage to its closest relatives from the NF2 or NF3 lineages (Ks = 4.58e-02 +/− 1.32e-09 or 4.08e-02 +/− 1.54e-09, respectively), or when comparing the NF1 lineage to its closest relative X. phaseoli pv. manihotis (Ks = 5.33e-01 +/− 0.00). These results indicate that m00100580b was more similar between the NF1 and fuscans lineages than between these lineages and their closest relatives, meaning that m00100580b was horizontally transferred between the ancestors of the NF1 and fuscans lineages. Moreover, the Ks value between the NF1 lineage and the NF2 or NF3 lineages (Ks = 4.36e-02 +/− 1.04e-09 or 4.01e-02 +/− 0.00, respectively) was lower than between the NF1 lineage and X. phaseoli pv. manihotis (Ks = 5.33e-01 +/− 0.00). Therefore, m00100580b was closer between NF1 strains and other strains from group 9.6 than from it’s closest relatives, meaning that the horizontal transfer was directed from the fuscans lineage to the NF1 lineage. This analysis confirmed HGT for 88 out of 100 genes tested (Fig. 1, Table 3, Additional file 3). The vast majority of HGT was directed from X. citri pv. fuscans to X. phaseoli pv. phaseoli, while only four HGT occurred from X. phaseoli pv. phaseoli to X. citri pv. fuscans. In particular, 55 HGT events were detected from the fuscans lineage to the NF1 lineage. In addition to having been transferred between distant lineages, 16 and 1 genes were also transferred between the fuscans and the NF2 lineages, or the fuscans and the NF3 lineages, respectively (Fig. 1). Moreover, eight genes had Ks = 0.00 +/− 0.00 between the NF2 and NF3 lineages, and nine between the NF2, NF3 and fuscans lineages, suggesting that HGT events also occurred between these lineages (Fig. 1, Additional file 3). Together, our results show that several more or less important waves of HGT occurred between the ancestors of phylogenetically distant strains responsible for CBB.

Finally, GC content is often used as a mean to detect HGT from foreign origin [29]. Out of the 115 candidate CDS, only two CDS (m00105390 and m00200160) presented an atypical GC content (α < 0.05) within the genome of strain CFBP6546 (Table 3). This result was not unexpected, as all strains from the NF1, NF2, NF3 and fuscans lineages have a similar GC content around 64% (Table 1), therefore HGT between these strains was not expected to result in a shift of GC content.

Discussion

We performed a comparative genomics analysis to detect genes putatively involved in Xanthomonas specificity to common bean. For this, we generated the whole genome sequence from 17 strains representing the diversity of the four genetic lineages belonging to X. citri pv. fuscans and X. phaseoli pv. phaseoli. We used a combination of comparative genomics approaches that led to the discovery of 115 genes bearing features unique to CBB agents. Out of these 115 genes, 108 were retrieved using the SkIf tool based on specific 24-mer search [25]. Previous analyses based on identity percentage unveiled 63 genes sharing 100% identity over at least 95% of their length among strains from the NF1 and fuscans linages [17]. Only nine of these genes were retrieved within our list of 115 genes (Table 3). This difference can be explained by the fact that we discarded most of the genes shared only by the NF1 and fuscans lineages and retained genes similar in all four genetic lineages of CBB agents (Table 2). On the other hand, we unveiled numerous genes that did not share 100% identity among the NF1 and fuscans lineages for their whole length, but instead shared small specific sequences of 24 nucleotides or more. Whether these similarities lie within functionally important domains of the encoded proteins remains to be studied.

Ks comparisons, showed that a majority of these genes were involved in HGT between X. citri pv. fuscans and X. phaseoli pv. phaseoli. Therefore, HGT was the predominant force leading to similarities between the genomes of X. citri pv. fuscans and X. phaseoli pv. phaseoli. Finding HGT events within these genes validated our approach. In particular, SkIf was an interesting tool because in addition to being more sensitive than gene gain and loss or phylogenetic approaches, it was not based on gene alignments, thus less sensitive to annotation and/or sequencing biases. HGT events occurred at different moments of the evolution of Xanthomonas strains having common bean as host. The vast majority of these HGT were directed from X. citri pv. fuscans to X. phaseoli pv. phaseoli. This strongly suggest that X. citri pv. fuscans was originally pathogenic on common bean, and that X. phaseoli pv. phaseoli subsequently acquired the ability to cause CBB on bean due to successive acquisitions of novel genes and/or novel alleles coming from the three X. citri pv. fuscans lineages. This can be compared to our knowledge on the origin of the lineages and their genetic diversity. The causal agent of CBB was first isolated and identified by Smith in 1897 [30] as a yellow pigmented strain, later shown as belonging to the NF1 lineage. Burkholder later isolated the first fuscous strains from beans grown in Switzerland in 1924 [31]. However there are no data to document a putative pre-existence of one, the other, or both types of strains prior to their first identifications. The genetic diversity of the yellow and fuscous strains was revealed by various methods. Amplified or restriction fragment length polymorphism analyses [16, 3234], amplified polymorphic DNA fragments [32, 35], pulse field gel electrophoresis [33], and multilocus sequence analysis [23] all revealed that both types of strains are more or less equivalent in terms of genetic diversity. This suggests that diversification of both lineages occurred around the same time, and thus that the ancestors of these two lineages may have coexisted. As a consequence, X. citri pv. fuscans may be the descendant of the original CBB agent that had transferred determinants useful for adaptation on common bean to the ancestor of X. phaseoli pv. phaseoli. Therefore, the ancestor of X. phaseoli pv. phaseoli appears as a recombinant that emerged as a new common bean pathogen through the acquisition of novel genes and alleles. In quarantine areas such as Europe, Turkey, Barhain, Azerbaijan and Israel, seed lots are routinely tested using a method from the International Seed Testing Association involving isolation of bacterial strains, pathogenicity tests, and specific PCR assays [36, 37]. Our results could serve for improving PCR-based monitoring of CBB agents by designing PCR primers on genes presenting sequences unique to CBB agents and potentially important for common bean specificity. Such primers could potentially detect novel HGT of these genes in strains unrelated to X. phaseoli pv. phaseoli and X. citri pv. fuscans, thus allowing to forecast new threads potentially dangerous for common bean production.

Very diverse functions were retrieved among the proteins encoded by the 115 candidate genes. Interestingly, 44 genes contained nonsynonymous polymorphisms specific for strains responsible for CBB, suggesting that they may play an important role in common bean specificity. Although 17 of these 44 genes encoded hypothetical proteins, it appears that most other genes encoded proteins involved in pathogenicity or in the interaction with the plant environment. The type IV secretion system was particularly represented with genes encoding TraF, TraG and TraI, and is involved in the translocation of macromolecules such as proteins important for pathogenicity, or DNA for mediating HGT [38, 39]. Thus, sharing similar proteins of the type IV secretion system could favour HGT among strains responsible for CBB. Indeed, strains from the NF1, NF2, NF3 and fuscans lineages have been found in La Réunion Island in 2000 (Table 1), indicating that sympatry exists among all these lineages, rendering further HGT events possible [18]. Three genes encoding proteins related to the type III secretion system were retrieved. XopA [40] and HpaH [41] are two proteins that may be involved in the structure of the type III secretion system, while XopAD is a type III effector of unknown function consisting of multiple semi-conserved 42 amino acids SKW repeats [42, 43]. The type III secretion system is pivotal for the virulence of most Gram negative plant pathogenic bacteria, and repertoires of type III effectors have been described as potentially important factors for host specificity and host adaptation in Xanthomonas [7, 44, 45] and other genera such as Pseudomonas [46]. Moreover, our analysis pointed out one diguanilate cyclase and one cGMP specific phosphodiesterase, two proteins involved in the metabolism of cyclic di-GMP that may play a role in biofilm formation [47] and pathogenicity [48]. One TonB-dependent transporter was also retrieved. TonB-dependent transporter are outer membrane receptors involved in molecule uptake such as iron siderophore complexes or nutrients and may play a role in host specificity [3, 49]. Other proteins putatively involved in pathogenicity were retrieved, such as ThrC, a threonine synthase involved in the virulence of X. oryzae pv. oryzicola in rice [50], XpsD, an outer membrane protein from the type II secretion system that is putatively involved in the secretion of cell wall degradative enzymes during infection [51], or IcmF, a protein of the type VI secretion system, which is involved in the interaction with other bacteria and may participate in pathogenicity [52, 53]. One flavine reductase and one xanthine dehydrogenase, two proteins putatively involved in oxydoreduction pathways were retrieved, and may be involved in the response to stress during the interaction with common bean. In addition to genes putatively involved in pathogenicity or virulence, our analysis unveiled genes involved in more general metabolism pathways, such as PhnB involved in the biosynthesis of tryptophan [54], or two DNA topoisomerases involved in the relaxation of the supercoiled DNA molecule during transcription, replication or recombination [55]. On one hand, our analysis unveiled only one or few genes within a given function, while the functions retrieved correspond to pathways often involving dozens of genes. This suggested that slight modifications within a given pathway would be sufficient to impact host specificity. On the other hand, the genes retrieved here encompass almost all the stages of host plant colonization by the bacteria, from the ability to mobilize trophic resources for multiplication to the interaction with other microorganisms, biofilm formation, response to oxidative stress, and inhibition of plant defences. Therefore, the ability to infect a particular plant seems to require not just one or a few adaptative determinants but an arsenal of factors allowing a global adaptation to a specific niche including the plant and, as a consequence a fine tuning and coordination of the activity of these determinants.

Interestingly, 19 of these 44 candidate genes were retrieved on plasmid a, suggesting that this plasmid played a major role for pathological convergence of CBB agents. Plasmid a carries an additional type III effector gene encoding an effector from the Transcription Activator-Like (TAL) family that was horizontally transferred between the NF1, NF2, NF3 and fuscans lineages [18]. Plasmids are genetic elements that favour HGT, but transfers of whole plasmids often induce a fitness cost for the bacteria [56]. More generally, horizontally transferred genes tend to be lost if not providing selective advantages for recipient strains [57]. Interestingly, the nine candidate genes retrieved by the gain and loss approach were all located on plasmid a (Table 3). The maintenance of these novel genes in the four genetic lineages of CBB agents is a testament to the importance of these genes for the bacteria. Except for two genes encoding proteins involved in the type IV secretion system, the other seven genes encoded proteins of unknown function. It would be interesting to perform functional characterization of these genes, and further analyse their implication in common bean specificity. Analysing the expression patterns during infection would be a natural extension of this study, and a first step towards functional validation of these genes.

Conclusion

Together, our results indicate that consecutive waves of HGT occurred between phylogenetically distant Xanthomonas strains able to cause the same symptoms on the same host plant: common bean. These HGT led to specific combinations of genes only retrieved in strains responsible for CBB, which provided new insights into the evolution of these bacteria towards infecting common bean. Mining for candidate genes for host specificity could be generalized to other polyphyletic pathovars such as pathovars euvesicatoria, vesicatoria, perforans, and gardneri forming a group of strains pathogenic on pepper and tomato [8]. Such analyses could both give new information on the molecular bases of host specificity, and provide new tools for enhancing epidemiological surveillance of strains pathogenic on a given host, or detecting recombinant strains presenting a high potential of emergence through the acquisition of novel genes.

Methods

Bacterial genomes and strains

All strains used in this study are listed in Table 1. The strains used for genome sequencing were provided by the CIRM-CFBP (International Center for Microbial Ressources - French Collection for Plant-associated Bacteria, https://www6.inra.fr/cirm_eng/). Genome sequencing was performed using the following procedure. Genomic DNA was prepared from overnight liquid cultures of bacteria previously grown on 10% TSA medium (tryptone at 1.7 g/L, soybean peptone at 0.3 g/L, glucose at 0.25 g/L, NaCl at 0.5 g/L, K2HPO4 at 0.5 g/L, agar at 15 g/L, pH 7.2) for 2 days at 28 °C. DNA was extracted and purified by the method of Klotz and Zimm [58]. Illumina sequencing was performed by Genoscope (20 strains, paired end reads of 300/500 bp) or GATC Biotech (three strains, with combined paired-end reads of ca. 250 bp and 3 kb mate-pair reads). Genome assembly was performed using a combination of SOAPdenovo (version 2.04) [59], SOAPGapCloser (version 1.12) [60] and Velvet (version 1.2.02) [61] assemblers. Sequenced genomes were estimated to be > 93% complete and < 3% contaminated (Additional file 4) using CheckM (version 1.0) [62]. The pathogenicity of all CBB strains was confirmed on common bean plants from cultivar Flavert as described in Ruh et al. [18]. The seeds from cultivar Flavert were kindly provided by Vilmorin (La Ménitré, France) and are available at the bean collection of the CIAT (Center for Tropical Agriculture, Colombia, http://genebank.ciat.cgiar.org/genebank/main.do).

Annotation and phylogenomic analyses

Structural and functional annotation of whole genome assemblies was performed using the automated pipeline Eugene-PP (version 1.2) [63], using SWISS-PROT as protein database and training protein database (http://www.uniprot.org/). Additional functional annotation of all predicted CDS was performed with InterProScan (version 4) [64]. A presence/absence matrix of ortholog groups was constructed using OrthoMCL (version 2.0) on amino acid sequences from all predicted CDS at an inflation index of 1.5 [65]. This matrix was then used for defining core and pan genomes. Phylogenetic trees were constructed using CVTree (version 4.2) [66] using the aminoacid sequences of all predicted CDS from the 75 genomes used in this study. CDS gains and losses were analysed using the Most Parcimonious Reconstruction function from the APE package (version 3.2) [67] to search for the most parsimonious succession of events explaining the repertoire of ortholog groups at each node of the phylogenetic tree.

Searching for genes monophyletic for X. citri pv. fuscans and X. phaseoli pv. phaseoli strains

A phylogenetic approach was used to search for genes for which strains from X. citri pv. fuscans and X. phaseoli pv. phaseoli form a monophyletic group. For this we selected 3202 CDS using an R script to search the orthology matrix for genes that were present in all X. citri pv. fuscans and X. phaseoli pv. phaseoli strains and in at least one strain from Rep-PCR group 9.2 or 9.4 [11] plus one another strain from Rep-PCR group 9.5 or 9.6, in order to avoid getting trees were X.citri pv. fuscans and X. phaseoli pv. phaseoli appear monophyletic due to a lack of correspondig genes in the strains inbetween. CDS were aligned using MAFFT (version 7) with L-INS-I strategy [68]. Neighbour-joining trees were constructed using APE (version 3.2) under the Kimura 80 model [67]. CDS monophyletic for all X. citri pv. fuscans and X. phaseoli pv. phaseoli strains, or alternatively for the NF1 and another lineage (i.e. NF2, NF3, or fuscans), were retrieved using the APE package (version 3.2) [67].

Searching for genes containing k-mers specific for X. citri pv. fuscans and X. phaseoli pv. phaseoli strains

A k-mer-based approach was used to search for genes containing short specific sequences present in all strains from X. citri pv. fuscans and X. phaseoli pv. phaseoli but absent in other strains. For this, we used SkIf (version 1.0) [25] with a k-mer size of 24 (or 24-mer), and using X. citri pv. fuscans strain 4834-R genome as reference. The same approach was used to search for genes containing 24-mers absent in strains belonging to X. citri pv. fuscans and X. phaseoli pv. phaseoli but conserved in all other strains from the X. axonopodis species complex, using X. citri pv. anacardii strain CFBP2913 genome as reference.

Recombination and HGT analyses

The 115 genes presenting specific traits of adaptation to common bean were aligned using MAFFT (version 7) with L-INS-I strategy [68]. Intragenic recombination events were then searched using a suite of programs implemented in RDP (version 4.16) [69], RDP [70], Geneconv [71], MaxChi [72], Chimaera [73], Bootscan [74] and 3seq [75]. Default parameters were used for each method except for Bootscan (window = 150, step = 20, neighbor joining trees, 200 replicates, 95% cut-off, J&N model with Ti:Tv = 2, coefficient of variation = 2). Ks was calculated using DNAsp (version 5) [76]. For each gene, the occurrence and dating of HGT events were estimated by comparing Ks values from 28 different pairwise combinations listed in Additional file 3. For example, NF1 and fuscans strains belong to phylogenetically distant strains, thus if the Ks between strains from genetic lineages NF1 and fuscans was lower than the mean Ks between other lineages, it was indicative of recent HGT between the ancestors of NF1 and fuscans. Direction of events were assessed by comparing the Ks values for outgroups belonging to Rep-PCR groups 9.4 and 9.6 (Fig. 1). For recombinants, separate analyses were performed for each region on both sides of the recombination point.

Notes

Abbreviations

CBB: 

Common bacterial blight of bean

CDS: 

Coding sequence

HGT: 

Horizontal gene transfer

Ks

Nucleotide synonymous substitution rate at silent sites

NF1, 2 or 3: 

Non-fuscous lineages 1, 2 or 3

PTI: 

Pattern triggered immunity (PTI)

rep-PCR: 

Repetitive-sequence-based Polymerase Chain Reaction

TAL: 

Transcription activator-like

Declarations

Acknowledgements

The authors thank Sébastien Carrère for his help on genome annotations and Laurent D. Noël for sharing the genome sequence of strain CFBP6865. The authors also thank the French Network on Xanthomonads (FNX) (https://www.reseau-xantho.org/) for recurrent scientific exchanges on Xanthomonas. Authors benefited from interactions promoted by COST Action FA 1208 (https://www.cost-sustain.org). We thank the CIRM-CFBP (Beaucouzé, France) for strain preservation and supply.

Funding

The research leading to these results has received grants from the Genoscope (3X 154/AP2006-2007, XANTHOMICS 18/AP2009-2010) and the French National Research Agency (XANTHOMIX ANR-2010-GENM-013-02). LSG was funded by a postdoctoral grant from the XANTHOMIX ANR project, and another postdoctoral grant from Angers-Loire Metropole, France. MR was funded by a PhD grant from Angers-Loire Metropole, France. This work was supported by France Génomique National infrastructure, funded as part of “Investissement d’avenir” program managed by Agence Nationale pour la Recherche (contrat ANR-10-INBS-0009).

Availability of data and materials

The datasets (genome sequences) generated through various projects and used in this study have been deposited in GenBank under accession numbers listed in Table 1.

Authors’ contributions

MAJ, LSG and NWGC designed the study. This study was partly lead by LSG during her post-doc and is part of the doctoral research of MR supervised by MAJ and NWGC. MAJ, NWGC, LG and RK obtained grants to fund the study. VB managed the genome sequencing at Genoscope. NWGC, LSG, MAJ, MB, MR, SB, AD, and VB contributed to data analyses and interpretation. NWGC, MR and MAJ wrote the manuscript with inputs from all co-authors. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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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)
IRHS, INRA, AGROCAMPUS OUEST, Université d’Angers, SFR4207 QUASAV, 42, rue Georges Morel, 49071 Beaucouzé, France
(2)
CEA/DSV/IG/Genoscope, 2 rue Gaston Crémieux, BP5706, 91057 Evry, France
(3)
CIRAD, UMR PVBMT, F-97410 Saint-Pierre, La Réunion, France
(4)
IRD, CIRAD, Université de Montpellier, IPME, Montpellier, France

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