Distribution and diversity of type III secretion system-like genes in saprophytic and phytopathogenic fluorescent pseudomonads

FEMS Microbiology Ecology 49 (2004) 455–467

www.fems-microbiology.org

Distribution and diversity of type III secretion system-like genesin saprophytic and phytopathogenic fluorescent pseudomonads

Sylvie Mazurier, M�elanie Lemunier, S�everine Siblot, Christophe Mougel,Philippe Lemanceau *

INRA-CMSE UMR ‘‘Microbiologie et G�eochimie des Sols’’, 17 rue Sully, BP86510, 21065 Dijon cedex, France

Received 2 March 2004; received in revised form 28 April 2004; accepted 29 April 2004

First published online 20 May 2004

Abstract

Type three secretion systems (TTSSs) are protein translocation mechanisms associated with bacterial pathogenicity in host

plants, and hypersensitive reactions in non-host plants. Distribution and diversity of TTSS-like genes within a collection of sap-

rophytic and phytopathogenic fluorescent pseudomonads were characterized. This collection included 16 strains belonging to 13

pathogenic species, and 87 strains belonging to five saprophytic species isolated from plant rhizosphere and soil. Presence of

conserved hypersensitive reaction/pathogenicity (hrp) genes (hrcRST) was assessed both by PCR using primers designed to amplify

the corresponding sequence and by dot-blot hybridization using a PCR-amplified hrcRST fragment as a probe. PCR allowed the

detection of TTSS-like genes in 75% and 32% of the phytopathogenic and saprophytic strains, respectively, and dot-blot hybrid-

ization in 100% and 49% of the phytopathogenic and saprophytic strains, respectively. The restriction fragment length polymor-

phism (RFLP) of 26 amplified hrcRST fragments revealed a considerable diversity. Twenty-one distinct RFLP types were identified

and one hrcRST fragment was sequenced per RFLP type. The obtained hrcRST sequences clustered into three groups. Two of these

groups included both phytopathogenic and saprophytic strains. The diversity of 16S rRNA genes, commonly used as an evolution

marker, was characterized using PCR-RFLP. Polymorphism of the 16S rRNA genes corresponded to that of hrcRST genes, sug-

gesting that these genes have followed a similar evolution. However, the occurrence of few mismatches suggests that sometimes

TTSS-like genes might have undergone horizontal genetic transfer.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Type III secretion system; hrp genes; hrcRST; Rhizosphere; Fluorescent pseudomonads

1. Introduction

A type III protein secretion system (TTSS) is de-

scribed as a macromolecular translocation apparatus

that enables a number of gram-negative pathogenic

bacteria to inject virulence determinants directly into the

cells of their eukaryotic hosts [1]. The importance ofTTSSs in bacterial pathogenicity has been demonstrated

for various genera of animal pathogens (Escherichia,

Pseudomonas, Salmonella, Shigella, and Yersinia) and of

* Corresponding author. Tel.: +33-3-80-69-30-56; fax: +33-3-80-69-

32-24/26.

E-mail address: [email protected] (P. Lemanceau).

0168-6496/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsec.2004.04.019

plant pathogens (Erwinia, Pantoea, Pseudomonas, Rals-

tonia, and Xanthomonas) [2].

In phytopathogenic bacteria, the TTSS is encoded by

hypersensitive reaction/pathogenicity (hrp) genes, which

are associated with the ability to elicit the hypersensitive

reaction (HR) on non-host plants and to cause disease in

the host plant [3]. Depending on their genetic organi-zation and mode of regulation, these genes are found in

clusters of type I for Erwinia spp., Pantoea spp. and

Pseudomonas syringae, and in clusters of type II for

Ralstonia and Xanthomonas [4]. Nine hrp genes, that

code for the core part of the TTSS and are conserved

among all animal and plant pathogens, are called hrc

(for hrp conserved genes) [5]. Eight of the Hrc proteins

(HrcV, N, Q1, Q2, R, S, T, U) have homologues in

. Published by Elsevier B.V. All rights reserved.

mail to: [email protected]

456 S. Mazurier et al. / FEMS Microbiology Ecology 49 (2004) 455–467

flagella biogenesis and are thought to form a specific

inner membrane transport channel [6]. The remaining

Hrc protein (HrcC) belongs to the large family of sec-

retins, which is involved in various processes of trans-

port [7]. HrcC is thought to constitute the outermembrane transport channel [8].

TTSSs have recently been reported in non-pathogenic

bacteria. TTSSs were described in the symbiotic bacte-

ria, Rhizobium sp. parasponia NGR234, Sinorhizobium

fredii, Mesorhizobium loti and Bradyrhizobium japoni-

cum, responsible for biological nitrogen fixation in the

roots of legumes [9]. In these bacteria, TTSSs have been

implicated in the plant-bacteria molecular dialoguemediating the establishment and the activity of the

symbiotic association [9,10].

Fluorescent pseudomonads include strains that im-

prove plant growth and health [11], and strains patho-

genic to plants [12]. Among the pathogenic fluorescent

pseudomonads, other than the species P. syringae, the

presence of a TTSS has only been reported for P. viri-

diflava on the basis of the presence of hrp genes [13,14].The homologues of hrp genes were also described in few

strains of P. fluorescens known to be non-phytopatho-

genic or even beneficial for plant growth and health:

PfG32R [15], C7 [16] and SBW25 [17]. Four of the nine

hrc genes were described in C7 [16]. This strain, previ-

ously reported to suppress fusarium crown and root rot

of tomato [18], was shown to induce a hypersensitive

reaction in this plant species after leaf infiltration [16]. Atype III gene cluster (Rsp) resembling the type III gene

cluster of P. syringae (Hrp) was identified in SBW25

[17]. Using homologues of some of the rsc genes that

encode the TTSS described in P. fluorescens SBW25,

Preston et al. [17] have shown that these genes were also

present in other strains of P. fluorescens and P. putida.

However, the number of strains tested was limited and

these strains only belonged to two saprophytic species.Therefore, data on the distribution of TTSS-like genes

in phytopathogenic and saprophytic fluorescent pseu-

domonads appears to be restricted to few species and,

except for P. syringae, to a limited number of strains.

Furthermore, no information on the polymorphism of

TTSS-like genes in saprophytic fluorescent pseudomo-

nads is available.

The objectives of this study were to evaluate thedistribution of TTSSs in saprophytic and phytopatho-

genic fluorescent pseudomonads, and to describe the

diversity of the corresponding TTSS-like genes. We

studied 103 strains of fluorescent pseudomonads. This

collection included 16 strains belonging to 13 species

pathogenic to plants, and 87 strains belonging to five

saprophytic species. Presence of the hrcRST sequence in

these strains, corresponding to a specific hrc gene suc-cession typical of type I hrp clusters was evaluated by

PCR with specific primers and by dot-blot hybridization

using the P. fluorescens C7 hrcRST sequence as a probe.

The polymorphism of the hrcRST sequence was assessed

by restriction fragment length polymorphism (RFLP)

and by sequencing one fragment per RFLP type. The

genetic diversity of the 16S rRNA genes, commonly

used as an evolution marker, was studied in parallel byPCR-RFLP in order to analyse the relationships be-

tween that gene and the hrcRST sequence.

2. Materials and methods

2.1. Bacterial strains and culture conditions

Bacterial stains used in this work are presented in

Table 1. The collection included (i) 17 type-strains, (ii)

five reference strains, (iii) 65 strains isolated from vari-

ous plant rhizospheres (28 from corn, 24 from tomato,

eight from flax, and five from different plant species),

and (iv) 16 strains isolated from bulk soils. The type-

strains included 14 phytopathogenic strains and two

strains isolated from soils. The reference strains included(i) three PGPR and biocontrol agents P. fluorescens

CFBP2392 (syn. A6) [18,19], CFBP5759 (syn. C7) [20]

and CFBP5935 (syn. F113) [21–23], and (ii) two phy-

topathogenic strains (P. syringae SD68 and 2027-37)

used as PCR controls. The type strain P. lini

CFBP5737T, 14 of the rhizosphere strains (CFBP5733 to

CFBP5736, CFBP5738, CFBP5743 to CFBP5746,

CFBP5755 to CFBP5758, and CFBP5760) and the six-teen soil-borne strains were previously shown to be

representative of the diversity of a larger bacterial col-

lection (340 strains) isolated from flax and tomato cul-

tivated in soils of Dijon and Chateaurenard (France),

and from these soils kept uncultivated [24,25]. The 52

remaining rhizosphere strains were described by Bossis

[26] and kindly provided by L. Gardan (UMR77 PaV�eINRA-INH-Universit�e, Angers, France). Altogetherthis collection included 16 phytopathogenic strains be-

longing to 13 species and 87 saprophytic strains be-

longing to five species.

Pseudomonas strains were grown at 25 �C in LB

medium [27]. For solid medium, 15 g l�1 of agar were

added.

2.2. Extraction of total genomic DNA and PCR ampli-

fications

Total DNA extractions were performed using stan-

dard methods [28]. PCR reactions were conducted in a

programmable thermal cycler (PTC-200, MJ Research)

and primers were synthesized by Eurogentec (Angers,

France). DNA sequences of two conserved hrp genes

(hrcR and hrcT) of phytopathogenic bacteria harbour-ing hrp clusters of type I [4] and belonging to different

species (P. syringae, Erwinia amylovora, and Pantoea

agglomerans formerly E. herbicola), were aligned using

Table 1

Characteristics of the fluorescent pseudomonads analysed in this study

Strainsa Other designationa Host-plant/Origin Species, biovar and pathovar hrcRST 16S rDNA

PCRc Dots

hybridizationd

RFLP

GenotypeseRFLP

Genotypesf

Type strains

CFBP2063�b ATCC25941 Agaricus bisporus, New-Zealand P. agarici � + N3 DHBAAAAB

CFBP3279� ATCC23835 Asplenium nidus P. asplenii ) � N4 DHAHAAAK

CFBP3280� ICMP8933 Phaseolus vulgaris, USA P. blatchfordae + + 4 RPBCPAMG N5 AAAFAAAA

CFBP2101� ATCC10857 Cichorium endivia P. cichorii � + 13 EFABDABC

CFBP2431� ATCC29436 Lycopersicon esculentum, UK P. corrugata ) + N6 CAAAAAAA

CFBP3281� ATCC12775 Phaseolus vulgaris, Australia P. flectens � � N7 HIFIFGFH

CFBP2102 ATCC13525 Water tank, UK P. fluorescens bv. II ) ) 1 AAAAAAAA

CFBP2065� St6801 Oryza sativa, Japan P. fuscovaginae ) � N8 DHAAAFAK

CFBP2810� ICMP8872 Agaricus bisporus, UK P. gingeri + + 5 SOHKQAOC N9 DHAAAAAB

CFBP5737 DLE411J Linum usitatissinum rhizosphere, France P. lini ) ) C8 CAABAAAA

CFBP2039� NCPPB2644 Medicago sativa, USA P. marginalis pv. alfalfae + + 6 CEDADAAA 3 AABBAAAA

CFBP2038� ATCC13889 Pastinaca sativa, USA P. marginalis pv. pastinacae + + 6 CEDADAAA 3 AABBAAAA

CFBP2037� ATCC10844 Cichorium intybus, USA P. marginalis pv. marginalis + + 6 CEDADAAA 3 AABBAAAA

CFBP2066 ATCC12633 Soil, USA P. putida bv. A ) ) 11 DBAAAADB

CFBP2022� Allium sativum, France P. salomonii + + 7 DNJJOCND N10 AJAAAAAA

CFBP2068� ATCC33618 Agaricus bisporus, UK P. tolaasii � + 7 ABABBAAA

CFBP2107� ATCC13223 Phaseolus sp., Switzerland P. viridiflava ) � 8 DBABAAAA

Reference strains

2027-37� Pyrus communis leave, France P. syringae pv. syringae + + 1 TQIKRBLE 9 CAABAABA

SD68� Phaseolus vulgaris, France P. syringae pv. phaseolicola + + 2 OLIHMBLE 9 CAABAABA

CFBP5759 C7 Linum usitatissinum rhizosphere, France P. fluorescens bv. II + + 3 NKHGLBKE C9 CAAAAAAE

CFBP2392 A6 Phaseolus vulgaris rhizosphere, France P. fluorescens bv. VI ) ) N1 BAAFAAAG

CFBP5935 F113 Beta vulgaris, Ireland P. fluorescens + + 21 MCCCKAJJ N6 CAAAAAAA

Rhizosphere strains

CFBP11341 M11 Zea mays, Anjou, France P. putida bv. A ) � C5 BAAFAAAB

CFBP11342 M12.1 Zea mays, Anjou, France P. putida bv. A ) � C5 BAAFAAAB

CFBP11343 M12.2 Zea mays, Anjou, France P. fluorescens bv. I ) ) C5 BAAFAAAB

CFBP11344 M12.3 Zea mays, Anjou, France P. fluorescens bv. I ) � C5 BAAFAAAB

CFBP11345 M17 Zea mays, Anjou, France P. fluorescens bv. I + + 6 CEDADAAA 3 AABBAAAA

CFBP11346 M18.1 Zea mays, Anjou, France P. fluorescens bv. II � + 2 AAABAAAA

CFBP11347 M21.1 Zea mays, Anjou, France P. fluorescens bv. I + + 8 DABEAACA 3 AABBAAAA

CFBP11348 M25 Zea mays, Anjou, France P. fluorescens bv. V + + 9 AAABAABC 4 ABABAAAA

CFBP11349 M26 Zea mays, Anjou, France P. putida bv. B + + 9 AAABAABC 4 ABABAAAA

CFBP11350 M27 Zea mays, Anjou, France P. fluorescens bv. V + + 10 EBBCEADA 4 ABABAAAA

CFBP11351 M30 Zea mays, Anjou, France P. fluorescens bv. I + + 11 FFBCFAAB N11 ABAFAAAA

CFBP11352 M31 Zea mays, Anjou, France P. fluorescens bv. V + + 12 GAEBAABC 4 ABABAAAA

CFBP11353 M32.21 Zea mays, Anjou, France P. fluorescens bv. V ) � N1 BAAFAAAG

CFBP11354 M32.22 Zea mays, Anjou, France P. fluorescens bv. I ) ) N1 BAAFAAAG

CFBP11355 M42 Zea mays, Anjou, France P. fluorescens bv. I + + 13 HGFFGAGG 4 ABABAAAA

CFBP11357 Ma1 Zea mays, Anjou, France P. fluorescens bv. I + + 14 ICGCBAEC C9 CAAAAAAE

CFBP11358 Ma2 Zea mays, Anjou, France P. fluorescens bv. I ) ) D3 BAABAAAA

CFBP11359 Ma3 Zea mays, Anjou, France P. fluorescens bv. V ) � N1 BAAFAAAG

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biologyEcology49(2004)455–467

457

Table 1 (continued)

Strainsa Other designationa Host-plant/Origin Species, biovar and pathovar hrcRST 16S rDNA

PCRc Dots

hybridizationd

RFLP

GenotypeseRFLP

Genotypesf

CFBP11361 Ma5 Zea mays, Anjou, France P. fluorescens bv. V � + N11 ABAFAAAA

CFBP11362 Ma6 Zea mays, Anjou, France P. fluorescens bv. V + + 15 BHBAHACC N12 CAAFBAAA

CFBP11363 Ma7 Zea mays, Anjou, France P. fluorescens bv. I + + 16 BDBDCAFD 2 AAABAAAA

CFBP11364 Ma8 Zea mays, Anjou, France P. fluorescens bv. II + + 17 JCCCBAEC C9 CAAAAAAE

CFBP11365 Ma10 Zea mays, Anjou, France P. fluorescens bv. I ) ) 12 BAAAAAAB

CFBP11366 Ma11 Zea mays, Anjou, France P. putida bv. A ) ) C5 BAAFAAAB

CFBP11367 Ma12 Zea mays, Anjou, France P. fluorescens bv. III + + 18 KICCIAHH C9 CAAAAAAE

CFBP11368 Ma13 Zea mays, Anjou, France P. fluorescens bv. V ) � N1 BAAFAAAG

CFBP11369 Ma14 Zea mays, Anjou, France P. fluorescens bv. II ) � C8 CAABAAAA

CFBP11398 M3.1 Zea mays, Tha€ıland P. fluorescens bv. V ) ) N13 DBAFAAAB

CFBP11385 To2 Lycopersicon esculentum, Anjou, France P. fluorescens bv. V ) ) N1 BAAFAAAG

CFBP11386 To3 Lycopersicon esculentum, Anjou, France P. fluorescens bv. V + + 19 LBBCAADB 4 ABABAAAA

CFBP11388 Tp1 Lycopersicon esculentum, Anjou, France P. putida bv. B ) � N14 CAABAABE

CFBP11389 Tp2 Lycopersicon esculentum, Anjou, France P. fluorescens bv. V � � N1 BAAFAAAG

CFBP11390 Tp3 Lycopersicon esculentum, Anjou, France P. fluorescens bv. V � � N11 ABAFAAAA

CFBP11391 T47 Lycopersicon esculentum, Anjou, France P. putida bv. A ) ) N15 DBABAADB

CFBP11392 T66 Lycopersicon esculentum, Anjou, France P. putida bv. A ) ) N15 DBABAADB

CFBP11393 To17 Lycopersicon esculentum, Anjou, France P. fluorescens bv. I + + 16 BDBDCAFD 2 AAABAAAA

CFBP11394 To29 Lycopersicon esculentum, Anjou, France P. putida bv. B ) � N16 BAAABAAA

CFBP11395 To35 Lycopersicon esculentum, Anjou, France P. putida bv. B � + N11 ABAFAAAA

CFBP5738 DTR335 Lycopersicon esculentum, Dijon, France P. lini ) ) D3 BAABAAAA

CFBP5746 DTRp621 Lycopersicon esculentum, Dijon, France P. jessenii ) ) 12 BAAAAAAB

CFBP5760 DTR133 Lycopersicon esculentum, Dijon, France P. chlororaphis ) ) D5 DAABAACA

CFBP5756 CTRp112 Lycopersicon esculentum, Chateaurenard,

France

P. fluorescens bv. VI ) ) C5 BAAFAAAB

CFBP5757 CTR212 Lycopersicon esculentum, Chateaurenard,

France

P. fluorescens bv. II ) ) C8 CAABAAAA

CFBP5758 CTR1015 Lycopersicon esculentum, Chateaurenard,

France

P. fluorescens bv. II ) � C9 CAAAAAAE

CFBP11371 Tg2 Lycopersicon esculentum, French Cara€ıbes P. fluorescens bv. V ) ) N13 DBAFAAAB

CFBP11372 Tg4 Lycopersicon esculentum, French Cara€ıbes P. fluorescens bv. V ) ) N17 GBABAAAB

CFBP11374 Tg8 Lycopersicon esculentum, French Cara€ıbes P. fluorescens bv. V ) � N13 DBAFAAAB


CFBP11379 Tg12 Lycopersicon esculentum, French Cara€ıbes P. putida bv. A ) ) N13 DBAFAAAB



CFBP11384 Tg18 Lycopersicon esculentum, French Cara€ıbes P. putida bv. A ) ) N15 DBABAADB

CFBP5735 DLR426 Linum usitatissinum, Dijon, France P. lini ) ) C8 CAABAAAA

CFBP5736 DLRp214 Linum usitatissinum, Dijon, France P. lini ) ) C8 CAABAAAA

CFBP5743 DLR223 Linum usitatissinum, Dijon, France P. jessenii ) � 12 BAAAAAAB

458

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CFBP5744 DLR228 Linum usitatissinum, Dijon, France P. jessenii ) ) C5 BAAFAAAB

CFBP5745 DLE3216 Linum usitatissinum, Dijon, France P. jessenii ) ) 12 BAAAAAAB

CFBP5733 CLRp812 Linum usitatissinum, Chateaurenard, France P. lini ) ) 9 CAABAABA

CFBP5734 CLE513 Linum usitatissinum, Chateaurenard, France P. lini ) ) 9 CAABAABA

CFBP5755 CLR711 Linum usitatissinum, Chateaurenard, France P. fluorescens bv. VI ) ) C5 BAAFAAAB

CFBP11397 L26.1 Lactuca satvia, Anjou, France P. fluorescens bv. V ) ) N1 BAAFAAAG

CFBP11400 M114 Glycine max, Ireland P. fluorescens bv. I ) ) N1 BAAFAAAG

CFBP11401 CTQMP26 Glycine max, Ireland P. fluorescens bv. V � � C5 BAAFAAAB

CFBP11402 Persica vulgaris, Algeria Pseudomonas sp. + + 20 QJBCJAII N11 ABAFAAAA

CFBP11403 Prunus armeniaca, Algeria Pseudomonas sp. ) ) N13 DBAFAAAB

Soil strains

CFBP5741 DS131 Dijon, France P. jessenii ) � 12 BAAAAAAB

CFBP5742 DS1026 Dijon, France P. jessenii ) ) 12 BAAAAAAB

CFBP5747 DS824 Dijon, France P. jessenii ) ) 12 BAAAAAAB

DS134 Dijon, France P. chlororaphis ) ) D5 DAABAACA

DS222 Dijon, France P. chlororaphis � � D5 DAABAACA

DS321 Dijon, France P. putida bv. A ) ) 12 BAAAAAAB

DS624 Dijon, France P. putida bv. A � � 12 BAAAAAAB

DS924 Dijon, France P. chlororaphis ) ) D5 DAABAACA

CFBP5732 CS611 Chateaurenard, France P. lini ) ) 9 CAABAABA

CFBP5739 CS111 Chateaurenard, France P. putida bv. A ) ) C1 BAABAAAB

CFBP5740 CS413 Chateaurenard, France P. putida bv. A ) ) C1 BAABAAAB

CS215 Chateaurenard, France P. putida bv. A ) ) C1 BAABAAAB

CS411 Chateaurenard, France P. fluorescens bv. II ) � 9 CAABAABA

CS511 Chateaurenard, France P. fluorescens bv. II � � 9 CAABAABA

CS712 Chateaurenard, France P. putida bv. A ) � C1 BAABAAAB

CS2114 Chateaurenard, France P. putida bv. A � � C1 BAABAAABaCFBP, Collection Franc�aise des Bacteries Phytopathog�enes, INRA Beaucouz�e, France; ATCC, American Type Culture Collection, U ersity Boulevard, Manassas, VA, USA; ICMP, In-

ternational Collection of Micro-organisms from Plants, Landcare Research, Auckland, New Zealand; NCPPB, National Collection of Plant P ogenic Bacteria, Central Science Laboratory, York,

UK; other strain names are laboratory designations.b Strains belonging to phytopathogenic species are indicated with an asterisk.cþ, strong PCR product of expected size and hybridizing with the probe C7hrcRST; �, weak PCR product of expected size hybridizing h the probe C7hrcRST; ), no PCR product and no

hybridization signal.dDot blot hybridization of total DNA with the probe C7hrcRST; þ, hybridization signal of intensity comparable to that of the C7 DNA ed as a control; �, hybridization signal of intensity

significantly weakest than that of the control; ), no visible hybridization signal.eNumbers designate the hrcRST types, and letters designate the patterns obtained with the restriction enzymes AluI, AvaII, DdeI, HinfI, spI, PstI, RsaI, and SphI, respectively.f Numbers designate the 16S rDNA types as described by Laguerre et al. [31], numbers following ‘‘C’’ and ‘‘D’’ designate 16S rDNA types as scribed by Latour et al. [25], numbers following ‘‘N’’

designate new 16S rDNA types described in this study, and letters designate the patterns obtained with the restriction enzymes AluI, DdeI, H II, MseI, MspI, NdeII, RsaI, and TaqI, respectively.

S.Mazurier

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CLUSTALW 1.6 [29]. Based on these alignments a pair

of oligonucleotide primers, HRCR8092 50-CCITT(C/T)ATCGT(C/T)AT(C/T)GA(C/T)(C/T)T-30 and HRCT

8986 50-CTGTCCCAGATIAICTGIGT-30 (where I in-

dicates inosine), was designed to amplify a part of theoperon U of the hrp cluster of type I including the 30 endof hrcR (26%), hrcS (100%), and the 50 end of hrcT

(42%) (Fig. 1). PCRs were conducted in a 25 ll reactionvolume. Reaction mixtures contained 150 ng of purified

DNA and 1.25 U of Taq DNA polymerase (Q-Biogen,

Illkirch, France) in the corresponding buffer (10 mM

Tris–HCl, pH 9.0 at 25 �C, 50 mM KCl, 1.5 mMMgCl2,

0.1% Triton X-100, 0.2 mgml�1 BSA, Q-Biogen, Ill-kirch, France). Final concentrations of each primer and

of dNTPs were 0.5 and 200 lM, respectively. Thermal

cycling consisted of an initial denaturation step at 95 �Cfor 3 min followed by 35 cycles of denaturation at 94 �Cfor 1 min, annealing at 41 �C for 1 min, and elongation

at 72 �C for 2 min, with a final elongation step at 72 �Cfor 3 min.

16S rRNA genes were amplified with primer pair fD1(50-AGAGTTTGATCCTGGCTCAG-30) and rD1 (50-AAGGAGGTGATCCAGCC-30) [30]. Reactions were

performed in a total volume of 50 ll, by mixing 50 ng of

DNA with 0.2 lM of each primer, 20 lM each of dATP,

dCTP, dGTP, dTTP, 2.5 U of Taq DNA polymerase in

the corresponding buffer (see above). Amplification re-

actions started with an initial denaturation step (3 min

at 95 �C) followed by 35 cycles of 1 min at 94 �C, 1 minat 55 �C, and 2 min at 72 �C, with a final extension of 3

min at 72 �C.Aliquots (5 ll) of the PCR products were analysed by

electrophoresis in 0.9% agarose gels stained with ethi-

dium bromide and photographed underUV illumination.

2.3. DNA hybridization

Hybridizations were performed on Pall Biodyne Plus

membranes (VWR, France). For DNA dot blots, 1 lg oftotal DNA was spotted onto the membrane after a de-

naturation of 10 min in a boiling water bath. For PCR

products, DNA was transferred from agarose gels by

vacuum blotting. DNA was fixed on membranes during

30 min at 80 �C. The C7hrcRST probe used for dot-blot

HRCR8092

HRCT8986

hrcR hrcThrcS

897 bp

Fig. 1. Scheme showing the position and orientation of the primers

HRCR8092 and HRCT8986 (black arrowheads) with respect to hrcR,

hrcS, and hrcT of P. syringae pv. syringae NPS3121 (GenBank Ac-

cession No. AF043444).

hybridization and for checking the specificity of PCR

amplifications was obtained by labeling the hrcRST

fragment yielded by PCR amplification of DNA from P.

fluorescens C7 (syn. CFBP5759). Digoxigenin labeling of

the DNA probe, DNA hybridization and probe detectionwere done using a non-radioactive DNA labeling and

detection kit (Roche Molecular Biochemicals, Meylan,

France) and applying high stringency conditions (hy-

bridization at 68 �C, first washing step in 0.1% SDS and

SSC2�, secondwashing step in 0.1%SDSandSSC0.1�).

2.4. RFLP of hrcRST and 16S rRNA genes

Aliquots (6 ll) of PCR products were digested over

night with 10 U of the following restriction endonuc-

leases purchased from Q-Biogen (Illkirch, France): AluI,

AvaII, DdeI, HinfI, MspI, PstI, RsaI, and SphI for

hrcRST, and AluI, DdeI, HaeIII, MseI, MspI, NdeII,

RsaI, and TaqI for 16S rDNA. The eight enzymes used

for hrcRST PCR fragments were selected on the basis of

restriction maps. They were chosen to discriminate allknown hrcRST sequences of the phytopathogens

aligned for primer design and the hrcRST sequence of

C7 (syn. CFBP5759) (EMBL Accession No. AJ271105).

The eight enzymes used for the amplified 16S rRNA

gene fragments were selected among the 13 used by

Laguerre et al. [31] for their ability to discriminate

strains belonging to different species of fluorescent

pseudomonads. The restriction fragments were sepa-rated by electrophoresis in TAE buffer (40 mM Tris–

HCl, pH 7.9, containing 4 mM sodium acetate and 1

mM EDTA) using 3% (w/v) Small Fragment agarose

(Appligene, Illkirch, France). Electrophoresis was car-

ried out at 100 V. After staining with ethidium bromide,

gels were photographed under UV illumination. For

each gene, each strain was assigned a composite type

defined by the combination of the patterns obtainedwith the different restriction endonucleases.

The computer program TREECON for Windows

1.3b [32] was used to estimate the relationships between

the hrcRST sequences on the basis of CLUSTALW 1.6

[29] alignments and for bootstrap analysis. Distance

estimation was computed according to Jukes and Can-

tor [33]. The matrix of distances was displayed as a

dendrogram using the UPGMA method [34]. Thecomputer program NTSYSpc 2.02k [35] was used to

compare the 16S rDNA types from the fragments ob-

tained with the different restriction endonucleases. The

pairwise Jaccard coefficients of similarity were computed

[36]. The matrix of similarities was displayed as a den-

drogram using the UPGMA method.

2.5. Cloning and sequencing PCR fragments

Before cloning, PCR fragments were purified by

electrophoresis in a 0.9% agarose gel in TAE, excised

S. Mazurier et al. / FEMS Microbiology Ecology 49 (2004) 455–467 461

and extracted from the gel by electro-elution using a

Biotrap BT 1000 (Schleicher & Schuell, Dassel, Ger-

many) according to the instructions of the manufac-

turer. The pGEM-T Easy Vector System II (Promega,

Charbonni�eres, France) was used for cloning as rec-ommended. Nucleotide sequences of the cloned frag-

ments were determined by Genome Express (Meylan,

France). Nucleotide sequence homology searches

against major sequence databases were done with pro-

grams BLAST 2.0 and FASTA version 2.0X.

2.6. Nucleotide sequence accession numbers

Newly obtained sequences of hrcRST fragments were

deposited in EMBL under the following accession num-

bers: P. syringae pv. syringae 2027-37 (AJ605515), P. sy-

ringae pv. phaseolicola SD68 (AJ605516), P. salomonii

CFBP2022T (AJ605517), P. gingeri CFBP2810T

(AJ605518), P. blatchfordae CFBP3280T (AJ605519), P.

fluorescens bv. II reference strain CFBP5759 (syn. C7)

(AJ271105), P. fluorescens reference strain CFBP5935(syn. F113) (AJ605533), P. fluorescens bv. I CFBP11345

(AJ605520), P. fluorescens bv. I CFBP11347 (AJ605521),

P. fluorescens bv. V CFBP11348 (AJ605522), P. fluores-

cens bv. V CFBP11350 (AJ605523), P. fluorescens bv. I

CFBP11351 (AJ605524),P. fluorescens bv. VCFBP11352

(AJ605525), P. fluorescens bv. I CFBP11355 (AJ605526),

P. fluorescens bv. I CFBP11357 (AJ605527), P. fluores-

cens bv. V CFBP11362 (AJ605528), P. fluorescens bv. ICFBP11363 (AJ605529),P. fluorescensbv. IICFBP11364

(AJ605530), P. fluorescens bv. III CFBP11367

(AJ605531),P. fluorescens bv. VCFBP11386 (AJ605532),

Pseudomonas sp. CFBP11402 (AJ605534).

3. Results

3.1. Distribution of hrp genes homologues in fluorescent

pseudomonads

The distribution of hrcRST sequences in the 103

strains was assessed by PCR with the primers

HRCR8092 and HRCT8986, and by hybridization of

total DNA dot blots with the C7hrcRST probe (Table 1).

PCR allowed the amplification of a single DNAfragment of the expected size (ca. 897 bp) in 40 of the

103 strains tested, these fragments being further showed

to be specific by hybridization with the C7hrcRST

probe. Positive dot-blot hybridization was recorded in

59 of the 103 strains. PCR products and positive hy-

bridization by dot blot were obtained in 75% and 100%

of the phytopathogenic strains, and in 32% and 49% of

the saprophytic strains, respectively. Among the sapro-phytic strains, PCR products and positive hybridization

by dot blot were obtained in 35% and 52% of the rhi-

zosphere strains, and in 22% and 39% of the non-rhi-

zosphere strains, respectively. All strains, for which a

PCR fragment was obtained, gave a positive signal by

dot-blot hybridization.

3.2. Polymorphism of hrcRST PCR fragment and

sequence information

Fourteen of the 40 positive strains gave PCR products

that were too weak to allow RFLP analysis. Analysis

of the PCR fragments was performed with the 26 re-

maining strains that included (i) eight of the 16 phyto-

pathogenic strains (P. blatchfordae CFBP3280T, P.

gingeri CFBP2810T, P. salomonii CFBP2022T, the threeP. marginalis strains CFBP2037T, CFBP2038T,

CFBP2039T, and both P. syringae reference strains 2027-

37 and SD68), and (ii) 18 of the 87 saprophytic strains all

isolated from plant rhizospheres including the reference

strains P. fluorescensC7 (syn. CFBP5759) and F113 (syn.

CFBP5935). Restriction digests were performed with 8

endonucleases on hrcRST PCR fragments. The com-

bined RFLP patterns obtained for the 26 strains studiedgave 21 distinct hrcRST types (Table 1). Each hrcRST

type was represented by a single strain except the types 9,

6 and 16 which included 2, 4 and 2 strains, respectively.

For each hrcRST type, a PCR fragment has been cloned

and sequenced. The strains for which a hrcRST fragment

was sequenced included 16 of the 87 saprophytic strains

and five of the 16 phytopathogenic strains.

The analysis of the Jukes and Cantor [33] pairwisedistance matrix (data not shown) indicated that genetic

distances between the hrcRST sequences ranged be-

tween 0.003 and 0.50, showing a high polymorphism of

hrcRST among the strains. The dendrogram presented

in Fig. 2 indicates the relationships between the se-

quences of the 21 hrcRST fragments and of those ex-

tracted from databases for P. syringae pv. syringae

NSP3121 (Accession No. AF043444), P. fluorescens

SBW25 (Accession No. AF292566), E. amylovara 321

(Accession No. L25828), Yersinia enterolitica A127/90

(Accession No. AY150843), and Rhizobium sp. NGR

234 (Accession No. AE000107).

According to their level of similarity, hrcRST se-

quences were clustered in three groups (Fig. 2). The first

group was made of sequences showing a maximum of

0.25 nucleotide substitution per site, and included threephytopathogenic strains and the biocontrol strain P.

fluorescens C7 (syn. CFBP5759). The second group was

made of sequences of four saprophytic strains isolated

from rhizospheres, including the biocontrol strain P.

fluorescens F113 (syn. CFBP5935), showing a maximum

of 0.12 nucleotide substitution per site. The third group

was made of sequences showing a maximum of 0.29

nucleotide substitution per site, and included 11 sapro-phytic isolates from plant rhizospheres and the biocon-

trol strain P. fluorescens SBW25, plus five

phytopathogenic strains.

Fig. 2. Dendrogram showing the relationships among the hrcRST sequences. Distance estimations according to the coefficient of Jukes and Cantor

[33] were clustered using the UPGMA method of TREECON for windows. hrcRST types are indicated in front of the strain designations. Strains

followed by an accession number under brackets have been included after extraction of the corresponding hrcRST fragment from the sequence cited.

Bootstrap analyses were performed with 1000 resamplings, percent values are shown at the branching points. For each hrcRST type, only the

fragment amplified from the first strain has been sequenced, the other strains belonging to this hrcRST type are indicated after a slash. R, reference

strain; T, type-strain; *, phytopathogenic strain; **, animal pathogenic strain, P. gi. ¼ P. gingeri; P. fl.¼P. fluorescens; P. sy.¼P. syringae; P.

pu.¼P. putida; P. sp¼P. species; P. ma.¼P. marginalis; P. bl.¼P. blatchfordae; P. sa.¼P. salomonii.


3.3. Restriction fragment length polymorphism of 16S

rRNA genes

The combined RFLP patterns of the 16S rRNA genes

obtained with eight restriction endonucleases for the 103

studied strains plus for the sequences of E. coli S17.1, P.

aeruginosa PAO1 and ATCC10145T, and P. fluorescens

SBW25 (from accessions V00348, NC_002516,

AF094713 and NC_002948, respectively) digested in sil-

ico gave 36 distinct ribotypes that are presented in the

dendrogram in Fig. 3. Twelve ribotypes (1, 2, 3, 4, 7, 8, 9,11, 12, 13, 18 and 20) were previously described by La-

guerre et al. [31] and 6 (C8,D3, C9, C5, C1,D5) byLatour

et al. [25]. According to the Jaccard pairwise similarity

matrix, the level of similarity between the different ribo-

types ranged from 0.17 to 0.97 indicating a high poly-

morphism of the 16S rRNA genes. The ribotypes 20, N7,

18 andN2 includingE. coli S17.1, the phytopathogenicP.

flectens CFBP3281T, and the human pathogenic P.

aeruginosa PAO1 and ATCC10145T, respectively, were

the most distant from the others and were then used as

outgroups to root the dendrogram. The ribotype 13 (P.

cichorii CFBP2101T) was also distant from the others.

The level of similarity among the remaining 31 ribo-

types (from 102 strains) was between 0.72 and 0.97, in-

dicating a low polymorphism among the corresponding

strains of fluorescent pseudomonads. These 102 strainswere clustered into three groups (Fig. 3). A first group

(a) showing a level of similarity at least equal to 0.83

included 13 ribotypes representing: (i) three phytopath-

ogenic strains, (ii) 42 saprophytic strains from variousrhizospheres, and (iii) all the soil isolates except P.

putida CFBP2066T. A second group (b) of nine ribo-

types showing a level of similarity at least equal to 0.83

was delineated. This group clustered six phytopatho-

genic strains including, P. blatchfordae CFBP3280T, P.

marginalis CFBP2037T-2038T-2039T, P. salomonii

CFBP2022T, and P. tolaasii CFBP2068T, and 18 sap-

rophytic strains including P. fluorescens CFBP2102T

plus 17 rhizosphere strains. Finally, the nine remaining

ribotypes, encompassing 18 isolates and sharing a sim-

ilarity at least equal to 0.82, were described in a third

group (c); this group included six phytopathogenic

strains, a saprophytic strain isolated from soil (P. putida

CFBP2066T) and 12 saprophytic strains isolated from

rhizospheres.

3.4. Relations between TTSS-like genes and 16S rRNA

gene polymorphism

Strains with TTSS-like genes as detected by PCR and/

or by dot-blot hybridization were distributed in 28 of the

36 ribotypes. Strains harbouring TTSS-like genes were

randomly distributed in the different ribotypes (Fig. 3).

Ten ribotypes included both strains with and withoutthe hrcRST sequence.

Fig. 3. Dendrogram showing the relationships among the 16S rRNA gene types. The pairwise coefficients of similarity [36] were clustered with the

UPGMA algorithm of NTSYS-pc 2.02. Strains followed by an accession number under brackets have been included after in silico digestion of the

corresponding 16S rRNA gene fragment extracted from the sequence cited. R, reference strain; T, type-strain; P. sy.¼P. syringae; P. pu.¼P. putida;

P. li.¼P. lini; P. fl.¼P. fluorescens; P. co.¼P. corrugata; P. je.¼P. jessenii; P. chl.¼P. chlororaphis; P. bl.¼P. blatchfordae; P. ma.¼P. marginalis;

P. sa.¼P. salomonii; P. to.¼P. tolaasii; P. sp.¼Pseudomonas sp.; P. ag.¼P. agarici; P. gi.¼P. gingeri; P. as¼P. asplenii; P. fu¼P. fuscovaginae;

P. vi¼P. viridiflava; P. ci.¼P. cichorii; P. flec.¼P. flectens. Bacterial strains, in which hrcRST sequence was detected by PCR and/or dot-blot

hybridization, are underlined.


Relationships were established between the hrcRST

sequence and ribotype groups delineated in Figs. 2 and

3, respectively (Fig. 4). Twelve of the 21 hrcRST frag-

ments sequenced, all belonging to homology group III,

were found in strains included in ribotypes of group b.

Eight of the 21 hrcRST sequenced fragments, all except

Fig. 4. Correspondence between the homology groups of hrcRST sequences (1) and of the ribotypes (2) as delineated in Figs. 2 and 3, respectively.

Dashed lines link hrcRST sequences and ribotypes of strains belonging to corresponding groups of homology (hrcRST sequence groups I and II/

ribotype group a, and hrcRST sequence groups III/ribotype group b). Black lines indicate mismatches in the correspondence between the homology

groups of hrcRST sequences and ribotypes referred above.


one belonging to homology groups I and II, were foundin strains included in ribotypes of the group a. Mis-

matches were only recorded for two strains: P. gingeri

CFBP2810T belonged to ribotype group c and to

hrcRST homology group I and P. fluorescens

CFBP11362 belonged to ribotype group a and to

hrcRST homology group III (Fig. 4).

4. Discussion

4.1. Distribution of TTSS-like genes

TTSSs have been extensively studied in P. syringae

[3,13] and hrp genes were described in P. viridiflava [14].

Presence of TTSS-like genes has been recently reported

in few strains that belonged only to two saprophyticspecies (P. fluorescens and P. putida) [15–17]. However

within the fluorescent pseudomonad group, further in-

formation on the distribution of TTSS-like genes in

other phytopathogenic species and in a larger number of

strains belonging to various saprophytic species clearly

remained to be gained. In the present study, we have

then assessed the distribution of these genes in a large

collection of fluorescent pseudomonads (103 strains)belonging to 13 phytopathogenic and five saprophytic

species.

The presence of TTSS-like genes in these strains was

confirmed by the detection of hrcRST sequences chosen

for their high level of conservation since they are a part

of the nine hrc genes coding for the core part of TTSS.

The presence of the hrcRST sequence detected by dot-

blot hybridization of total DNA using C7hrcRST as aprobe was revealed in 100% of the phytopathogenic

strains. These data allowed extending to a wider range

of phytopathogenic species the observation of TTSS-like

genes previously made in P. syringae and P. viridiflava

[13,14]. Furthermore, these data showing the presence of

TTSS-like genes in all tested phytopathogenic strains are

in agreement with the previous demonstration in several

phytopathogenic bacteria that TTSS is implicated in thepathogenesis process [2], and then validate our strategy

based on dot blot using C7hrcRST as a probe for de-

tecting TTSS-like genes in fluorescent pseudomonads.

TTSS-like genes in fluorescent pseudomonads were

found at a lower frequency by PCR amplification than

by dot-blot hybridization. This type of discrepancy be-

tween the data obtained by the two methods has been


commonly reported and may be ascribed to mismatches

between the template and the primers.

Dot-blot hybridization of total DNA allowed the

detection of TTSS-like genes in 49% of the strains dis-

tributed in the five saprophytic species tested. Theseresults indicate that, although the frequency of TTSS

genes is significantly lower in saprophytic than in phy-

topathogenic species, they remain widely distributed

among saprophytic species of fluorescent pseudomo-

nads. These data support and extend those obtained

previously with a small number of strains belonging

only to P. fluorescens and to P. putida [15–17]. This wide

distribution of TTSS-like genes in these strains raisesquestions about their function in saprophytic species,

since TTSSs are known to be dedicated to the translo-

cation of bacterial effector proteins into the cytosol of

eukaryotic cells during the hypersensitive reaction on

non-host plants and disease expression in host plants [3].

The saprophytic strains harbouring TTSS-like genes

included three (P. fluorescens C7, F113 and SBW25) of

the four strains of fluorescent pseudomonads, known asbiocontrol agents, confirming the previous observation

made for P. fluorescens SBW25 [17]. This observation

could raise the hypothesis of the possible involvement of

TTSS in the elicitation of the defence reactions known

to play a major role in the disease suppression by fluo-

rescent pseudomonads [37]. More generally, one may

suggest that TTSS might be involved in the crosstalk

between bacteria and plant as showed for plant-bacterialmolecular dialogue mediating the establishment of

symbiotic associations [9,10]. This is supported by rather

high frequency of the TTSS-like genes in rhizosphere

strains (52%). However, these genes were also found in

non-rhizosphere strains (39%), although at a lower fre-

quency, and their presence was not restricted to specific

bacterial genotypes since they were distributed ran-

domly in most of the ribotypes.Further studies should be performed to determine if

the soil strains harbouring TTSS-like genes, although

they were not isolated from plants, show a higher ability

to elicit plant defence reactions and are more rhizo-

sphere competent than the soil strains without TTSS-

like genes.

4.2. Diversity of TTSS-like genes

The PCR method developed in the present study

provided a series of hrcRST fragments, which allowed

us to characterize their diversity in eight and 18 strains

belonging to various phytopathogenic and saprophytic

species. Comparable studies have so far been performed

for P. syringae and not for saprophytic Pseudomonas

species.PCR-RFLP analysis of the hrcRST sequence allowed

the identification of 21 hrcRST types. One fragment per

type was sequenced, and their diversity was analysed

together with five sequences extracted from a data bank.

The hrcRST sequences have shown a high level of di-

versity. This level was possibly even underestimated

since only 44% of the sequences detected by dot-blot

hybridization gave a PCR product that was strong en-ough for RFLP analysis. Furthermore, only 36% of the

detected hrcRST genes were sequenced. A notably high

diversity was recorded within strains belonging to P.

fluorescens, which contrasts the hypothesis of Preston

et al. [17]. Conversely, a low diversity was recorded

within the two phytopathogenic species P. syringae and

P. marginalis. The three strains of P. marginalis be-

longed to the same hrcRST type despite their distribu-tion in three different pathovars, and the three strains of

P. syringae (pathovars syringae and phaseolicola) har-

boured TTSS-like genes that were closely related. The

low diversity of TTSS-like genes in P. syringae recorded

in the present study is in agreement with the study of

Sawada et al. [38].

The hrcRST sequences were clustered in three groups

according to their level of similarity. The strains be-longing to saprophytic species were distributed

throughout the groups, and the strains belonging to

phytopathogenic species were distributed in the groups I

and III, that included most of the saprophytic strains.

These data indicate that hrcRST sequences in strains

belonging to different phytopathogenic species are dis-

tant from each other, and are even more distant from

each other than from some of the strains belonging tosaprophytic species.

Alfano et al. [39] have shown that in P. syringae the

hrp genes encoding the TTSS machinery are the con-

served center region (CCR) of a tripartite pathogenicity

island that includes exchangeable (EEL) and conserved

(CEL) effector loci. According to these authors, Hrp-

mediated pathogenicity in P. syringae seems to be

dependent on a set of genes that are universal amongdivergent pathovars and on another set that varies

among strains in the same pathovar. This observation

suggests that strains, presenting various pathogenic

properties, would carry a universal TTSS. This sugges-

tion was recently supported by further studies based on

a mutational approach [40,41]. In the present study,

TTSS-like genes belonging to the same homology group

were described in distinct bacterial species and in bac-teria having different effects on plants; either negative

(pathogenic), neutral (saprophytic) or positive (biocon-

trol). These observations suggest that the proposal by

Alfano et al. [39] for P. syringae could possibly be ex-

tended to saprophytic and beneficial fluorescent pseu-

domonads. This hypothesis could be assessed by

comparing the genomic regions of hrc genes of these

types of fluorescent pseudomonads together with thoseknown in P. syringae.

The polymorphism of hrcRST genes within the fluo-

rescent pseudomonads was related to that of the 16S


rRNA genes. 16S rRNA genes are commonly used for

diversity analyses of bacterial populations [25,31,42–44].

A correspondence was established between hrcRST se-

quences and the ribotypes for all the strains, except for

two, when comparing the topology of the correspondingdendrograms. This observation suggests that TTSS-like

and 16S rRNA genes have followed a similar evolution.

According to our data this would apply both to the

saprophytic and phytopathogenic strains. This sugges-

tion is in agreement with the report of Sawada et al. [38]

on the evolutionary stability of hrp gene cluster in P.

syringae when analysing the phylogeny of hrpS and hrpL

of strains belonging to different pathovars. However,two mismatches were observed in the relationship be-

tween 16S rDNA types and hrcRST sequences. These

mismatches involved a phytopathogenic (P. gingeri

CFBP2810T) and a saprophytic strain (P. fluorescens

CFBP11362), and suggest that some TTSS-like genes

might have experienced horizontal gene transfer. This

hypothesis would be consistent with the implication of

horizontal transfer in the evolution of TTSS-like geneclusters, which was previously shown for phytopatho-

genic pseudomonads [1,39,45,46]. Our results also sug-

gest that TTSS gene transfer might not only occur in

phytopathogenic strains but also in saprophytic strains

of fluorescent pseudomonads. Further work on phy-

logeny would be required to further assess the evolution

history of TTSS-like genes within phytopathogenic and

saprophytic strains.

5. Conclusions

TTSS-like genes were distributed in all tested phy-

topathogenic strains belonging to 13 species of fluo-

rescent pseudomonads and were also widely distributed

in strains belonging to saprophytic species. In fact,phytopathogenic and saprophytic strains could be dis-

criminated neither on the basis of the presence of

TTSS-like genes nor on the polymorphism of the cor-

responding sequences. Within saprophytic strains,

TTSS-like genes were more frequently detected in

strains associated with plants but were also common in

strains isolated from soil. Collectively, these data

question the role of these genes in saprophytic strainsincluding strains not associated with eukaryotes.

Comparison of the polymorphisms of hrcRST and of

16S rRNA genes suggests that they have followed a

similar evolution even if horizontal transfer might have

occurred. Further studies are required to support these

hypotheses and to evaluate the possible implication of

the TTSS in plant–microbe interactions. These studies

would be based on phylogeny analysis and on thecomparison of plant–microbe interactions when using

strains harbouring these sequences and the corre-

sponding targeted mutants.

Acknowledgements

The authors are grateful to K. Klein for input to the

English and to S. Delorme, V. Edel, and J. Raaijmakers

for helpful discussions.

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