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Keywords:

  • Type III secretion system;
  • hrp genes;
  • hrc RST;
  • Rhizosphere;
  • Fluorescent pseudomonads

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

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 saprophytic 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 (hrc RST) was assessed both by PCR using primers designed to amplify the corresponding sequence and by dot-blot hybridization using a PCR-amplified hrc RST 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 hybridization in 100% and 49% of the phytopathogenic and saprophytic strains, respectively. The restriction fragment length polymorphism (RFLP) of 26 amplified hrc RST fragments revealed a considerable diversity. Twenty-one distinct RFLP types were identified and one hrc RST fragment was sequenced per RFLP type. The obtained hrc RST 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 hrc RST genes, suggesting 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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

A type III protein secretion system (TTSS) is described 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 of TTSSs in bacterial pathogenicity has been demonstrated for various genera of animal pathogens (Escherichia, Pseudomonas, Salmonella, Shigella, and Yersinia) and of plant pathogens (Erwinia, Pantoea, Pseudomonas, Ralstonia, 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 organization 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 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 secretins, which is involved in various processes of transport [7]. HrcC is thought to constitute the outer membrane transport channel [8].

TTSSs have recently been reported in non-pathogenic bacteria. TTSSs were described in the symbiotic bacteria, Rhizobium sp. parasponia NGR234, Sinorhizobium fredii, Mesorhizobium loti and Bradyrhizobium japonicum, responsible for biological nitrogen fixation in the roots of legumes [9]. In these bacteria, TTSSs have been implicated in the plant-bacteria molecular dialogue mediating the establishment and the activity of the symbiotic association [9,10].

Fluorescent pseudomonads include strains that improve plant growth and health [11], and strains pathogenic 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. viridiflava 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-phytopathogenic 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, previously 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]. A type 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 pseudomonads 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 pseudomonads is available.

The objectives of this study were to evaluate the distribution of TTSSs in saprophytic and phytopathogenic 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 hrc RST sequence in these strains, corresponding to a specific hrc gene succession typical of type I hrp clusters was evaluated by PCR with specific primers and by dot-blot hybridization using the P. fluorescens C7 hrc RST sequence as a probe. The polymorphism of the hrc RST 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 by PCR-RFLP in order to analyse the relationships between that gene and the hrc RST sequence.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

2.1Bacterial 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 various 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 phytopathogenic 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 sixteen soil-borne strains were previously shown to be representative of the diversity of a larger bacterial collection (340 strains) isolated from flax and tomato cultivated in soils of Dijon and Châteaurenard (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 PaVé INRA-INH-Université, Angers, France). Altogether this collection included 16 phytopathogenic strains belonging to 13 species and 87 saprophytic strains belonging to five species.

Table 1.  Characteristics of the fluorescent pseudomonads analysed in this study
StrainsaOther designationaHost-plant/OriginSpecies, biovar and pathovarhrc RST16S rDNA
    PCRcDots hybridizationdRFLP GenotypeseRFLP Genotypesf
  1. aCFBP, Collection Française des Bacteries Phytopathogènes, INRA Beaucouzé, France; ATCC, American Type Culture Collection, University Boulevard, Manassas, VA, USA; ICMP, International Collection of Micro-organisms from Plants, Landcare Research, Auckland, New Zealand; NCPPB, National Collection of Plant Pathogenic Bacteria, Central Science Laboratory, York, UK; other strain names are laboratory designations.

  2. bStrains belonging to phytopathogenic species are indicated with an asterisk.

  3. c+, strong PCR product of expected size and hybridizing with the probe C7hrc RST; ±, weak PCR product of expected size hybridizing with the probe C7hrc RST; −, no PCR product and no hybridization signal.

  4. dDot blot hybridization of total DNA with the probe C7hrc RST; +, hybridization signal of intensity comparable to that of the C7 DNA used as a control; ±, hybridization signal of intensity significantly weakest than that of the control; −, no visible hybridization signal.

  5. eNumbers designate the hrc RST types, and letters designate the patterns obtained with the restriction enzymes Alu I, Ava II, Dde I, Hin fI, Msp I, Pst I, Rsa I, and Sph I, respectively.

  6. fNumbers designate the 16S rDNA types as described by Laguerre et al. [31], numbers following “C” and “D” designate 16S rDNA types as described 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 Alu I, Dde I, HaeII I, Mse I, Msp I, Nde II, Rsa I, and Taq I, respectively.

Type strains       
CFBP2063*bATCC25941Agaricus bisporus, New-ZealandP. agarici±+ N3 DHBAAAAB
CFBP3279*ATCC23835Asplenium nidusP. asplenii± N4 DHAHAAAK
CFBP3280*ICMP8933Phaseolus vulgaris, USAP. blatchfordae++4 RPBCPAMGN5 AAAFAAAA
CFBP2101*ATCC10857Cichorium endiviaP. cichorii±+ 13 EFABDABC
CFBP2431*ATCC29436Lycopersicon esculentum, UKP. corrugata+ N6 CAAAAAAA
CFBP3281*ATCC12775Phaseolus vulgaris, AustraliaP. flectens±± N7 HIFIFGFH
CFBP2102ATCC13525Water tank, UKP. fluorescens bv. II 1 AAAAAAAA
CFBP2065*St6801Oryza sativa, JapanP. fuscovaginae± N8 DHAAAFAK
CFBP2810*ICMP8872Agaricus bisporus, UKP. gingeri++5 SOHKQAOCN9 DHAAAAAB
CFBP5737DLE411JLinum usitatissinum rhizosphere, FranceP. lini C8 CAABAAAA
CFBP2039*NCPPB2644Medicago sativa, USAP. marginalis pv. alfalfae++6 CEDADAAA3 AABBAAAA
CFBP2038*ATCC13889Pastinaca sativa, USAP. marginalis pv. pastinacae++6 CEDADAAA3 AABBAAAA
CFBP2037*ATCC10844Cichorium intybus, USAP. marginalis pv. marginalis++6 CEDADAAA3 AABBAAAA
CFBP2066ATCC12633Soil, USAP. putida bv. A 11 DBAAAADB
CFBP2022* Allium sativum, FranceP. salomonii++7 DNJJOCNDN10 AJAAAAAA
CFBP2068*ATCC33618Agaricus bisporus, UKP. tolaasii±+ 7 ABABBAAA
CFBP2107*ATCC13223Phaseolus sp., SwitzerlandP. viridiflava± 8 DBABAAAA
Reference strains       
2027–37* Pyrus communis leave, FranceP. syringae pv. syringae++1 TQIKRBLE9 CAABAABA
SD68* Phaseolus vulgaris, FranceP. syringae pv. phaseolicola++2 OLIHMBLE9 CAABAABA
CFBP5759C7Linum usitatissinum rhizosphere, FranceP. fluorescens bv. II++3 NKHGLBKEC9 CAAAAAAE
CFBP2392A6Phaseolus vulgaris rhizosphere, FranceP. fluorescens bv. VI N1 BAAFAAAG
CFBP5935F113Beta vulgaris, IrelandP. fluorescens++21 MCCCKAJJN6 CAAAAAAA
Rhizosphere strains       
CFBP11341M11Zea mays, Anjou, FranceP. putida bv. A± C5 BAAFAAAB
CFBP11342M12.1Zea mays, Anjou, FranceP. putida bv. A± C5 BAAFAAAB
CFBP11343M12.2Zea mays, Anjou, FranceP. fluorescens bv. I C5 BAAFAAAB
CFBP11344M12.3Zea mays, Anjou, FranceP. fluorescens bv. I± C5 BAAFAAAB
CFBP11345M17Zea mays, Anjou, FranceP. fluorescens bv. I++6 CEDADAAA3 AABBAAAA
CFBP11346M18.1Zea mays, Anjou, FranceP. fluorescens bv. II±+ 2 AAABAAAA
CFBP11347M21.1Zea mays, Anjou, FranceP. fluorescens bv. I++8 DABEAACA3 AABBAAAA
CFBP11348M25Zea mays, Anjou, FranceP. fluorescens bv. V++9 AAABAABC4 ABABAAAA
CFBP11349M26Zea mays, Anjou, FranceP. putida bv. B++9 AAABAABC4 ABABAAAA
CFBP11350M27Zea mays, Anjou, FranceP. fluorescens bv. V++10 EBBCEADA4 ABABAAAA
CFBP11351M30Zea mays, Anjou, FranceP. fluorescens bv. I++11 FFBCFAABN11 ABAFAAAA
CFBP11352M31Zea mays, Anjou, FranceP. fluorescens bv. V++12 GAEBAABC4 ABABAAAA
CFBP11353M32.21Zea mays, Anjou, FranceP. fluorescens bv. V± N1 BAAFAAAG
CFBP11354M32.22Zea mays, Anjou, FranceP. fluorescens bv. I N1 BAAFAAAG
CFBP11355M42Zea mays, Anjou, FranceP. fluorescens bv. I++13 HGFFGAGG4 ABABAAAA
CFBP11357Ma1Zea mays, Anjou, FranceP. fluorescens bv. I++14 ICGCBAECC9 CAAAAAAE
CFBP11358Ma2Zea mays, Anjou, FranceP. fluorescens bv. I D3 BAABAAAA
CFBP11359Ma3Zea mays, Anjou, FranceP. fluorescens bv. V± N1 BAAFAAAG
CFBP11361Ma5Zea mays, Anjou, FranceP. fluorescens bv. V±+ N11 ABAFAAAA
CFBP11362Ma6Zea mays, Anjou, FranceP. fluorescens bv. V++15 BHBAHACCN12 CAAFBAAA
CFBP11363Ma7Zea mays, Anjou, FranceP. fluorescens bv. I++16 BDBDCAFD2 AAABAAAA
CFBP11364Ma8Zea mays, Anjou, FranceP. fluorescens bv. II++17 JCCCBAECC9 CAAAAAAE
CFBP11365Ma10Zea mays, Anjou, FranceP. fluorescens bv. I 12 BAAAAAAB
CFBP11366Ma11Zea mays, Anjou, FranceP. putida bv. A C5 BAAFAAAB
CFBP11367Ma12Zea mays, Anjou, FranceP. fluorescens bv. III++18 KICCIAHHC9 CAAAAAAE
CFBP11368Ma13Zea mays, Anjou, FranceP. fluorescens bv. V± N1 BAAFAAAG
CFBP11369Ma14Zea mays, Anjou, FranceP. fluorescens bv. II± C8 CAABAAAA
CFBP11398M3.1Zea mays, Thaí¨landP. fluorescens bv. V N13 DBAFAAAB
CFBP11385To2Lycopersicon esculentum, Anjou, FranceP. fluorescens bv. V N1 BAAFAAAG
CFBP11386To3Lycopersicon esculentum, Anjou, FranceP. fluorescens bv. V++19 LBBCAADB4 ABABAAAA
CFBP11388Tp1Lycopersicon esculentum, Anjou, FranceP. putida bv. B± N14 CAABAABE
CFBP11389Tp2Lycopersicon esculentum, Anjou, FranceP. fluorescens bv. V±± N1 BAAFAAAG
CFBP11390Tp3Lycopersicon esculentum, Anjou, FranceP. fluorescens bv. V±± N11 ABAFAAAA
CFBP11391T47Lycopersicon esculentum, Anjou, FranceP. putida bv. A N15 DBABAADB
CFBP11392T66Lycopersicon esculentum, Anjou, FranceP. putida bv. A N15 DBABAADB
CFBP11393To17Lycopersicon esculentum, Anjou, FranceP. fluorescens bv. I++16 BDBDCAFD2 AAABAAAA
CFBP11394To29Lycopersicon esculentum, Anjou, FranceP. putida bv. B± N16 BAAABAAA
CFBP11395To35Lycopersicon esculentum, Anjou, FranceP. putida bv. B±+ N11 ABAFAAAA
CFBP5738DTR335Lycopersicon esculentum, Dijon, FranceP. lini D3 BAABAAAA
CFBP5746DTRp621Lycopersicon esculentum, Dijon, FranceP. jessenii 12 BAAAAAAB
CFBP5760DTR133Lycopersicon esculentum, Dijon, FranceP. chlororaphis D5 DAABAACA
CFBP5756CTRp112Lycopersicon esculentum, Châteaurenard, FranceP. fluorescens bv. VI C5 BAAFAAAB
CFBP5757CTR212Lycopersicon esculentum, Châteaurenard, FranceP. fluorescens bv. II C8 CAABAAAA
CFBP5758CTR1015Lycopersicon esculentum, Châteaurenard, FranceP. fluorescens bv. II± C9 CAAAAAAE
CFBP11371Tg2Lycopersicon esculentum, French Caraí¨besP. fluorescens bv. V N13 DBAFAAAB
CFBP11372Tg4Lycopersicon esculentum, French Caraí¨besP. fluorescens bv. V N17 GBABAAAB
CFBP11374Tg8Lycopersicon esculentum, French Caraí¨besP. fluorescens bv. V± N13 DBAFAAAB
CFBP11377Tg10Lycopersicon esculentum, French Caraí¨besP. fluorescens bv. V N13 DBAFAAAB
CFBP11379Tg12Lycopersicon esculentum, French Caraí¨besP. putida bv. A N13 DBAFAAAB
CFBP11380Tg13Lycopersicon esculentum, French Caraí¨besP. fluorescens bv. V N13 DBAFAAAB
CFBP11382Tg16Lycopersicon esculentum, French Caraí¨besP. fluorescens bv. V N13 DBAFAAAB
CFBP11384Tg18Lycopersicon esculentum, French Caraí¨besP. putida bv. A N15 DBABAADB
CFBP5735DLR426Linum usitatissinum, Dijon, FranceP. lini C8 CAABAAAA
CFBP5736DLRp214Linum usitatissinum, Dijon, FranceP. lini C8 CAABAAAA
CFBP5743DLR223Linum usitatissinum, Dijon, FranceP. jessenii± 12 BAAAAAAB
CFBP5744DLR228Linum usitatissinum, Dijon, FranceP. jessenii C5 BAAFAAAB
CFBP5745DLE3216Linum usitatissinum, Dijon, FranceP. jessenii 12 BAAAAAAB
CFBP5733CLRp812Linum usitatissinum, Châteaurenard, FranceP. lini 9 CAABAABA
CFBP5734CLE513Linum usitatissinum, Châteaurenard, FranceP. lini 9 CAABAABA
CFBP5755CLR711Linum usitatissinum, Châteaurenard, FranceP. fluorescens bv. VI C5 BAAFAAAB
CFBP11397L26.1Lactuca satvia, Anjou, FranceP. fluorescens bv. V N1 BAAFAAAG
CFBP11400M114Glycine max, IrelandP. fluorescens bv. I N1 BAAFAAAG
CFBP11401CTQMP26Glycine max, IrelandP. fluorescens bv. V±± C5 BAAFAAAB
CFBP11402 Persica vulgaris, AlgeriaPseudomonas sp.++20 QJBCJAIIN11 ABAFAAAA
CFBP11403 Prunus armeniaca, AlgeriaPseudomonas sp. N13 DBAFAAAB
Soil strains       
CFBP5741DS131Dijon, FranceP. jessenii± 12 BAAAAAAB
CFBP5742DS1026Dijon, FranceP. jessenii 12 BAAAAAAB
CFBP5747DS824Dijon, FranceP. jessenii 12 BAAAAAAB
DS134 Dijon, FranceP. chlororaphis D5 DAABAACA
DS222 Dijon, FranceP. chlororaphis±± D5 DAABAACA
DS321 Dijon, FranceP. putida bv. A 12 BAAAAAAB
DS624 Dijon, FranceP. putida bv. A±± 12 BAAAAAAB
DS924 Dijon, FranceP. chlororaphis D5 DAABAACA
CFBP5732CS611Châteaurenard, FranceP. lini 9 CAABAABA
CFBP5739CS111Châteaurenard, FranceP. putida bv. A C1 BAABAAAB
CFBP5740CS413Châteaurenard, FranceP. putida bv. A C1 BAABAAAB
CS215 Châteaurenard, FranceP. putida bv. A C1 BAABAAAB
CS411 Châteaurenard, FranceP. fluorescens bv. II± 9 CAABAABA
CS511 Châteaurenard, FranceP. fluorescens bv. II±± 9 CAABAABA
CS712 Châteaurenard, FranceP. putida bv. A± C1 BAABAAAB
CS2114 Châteaurenard, FranceP. putida bv. A±± C1 BAABAAAB

Pseudomonas strains were grown at 25 °C in LB medium [27]. For solid medium, 15 g l−1 of agar were added.

2.2Extraction of total genomic DNA and PCR amplifications

Total DNA extractions were performed using standard 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 (hrc R and hrc T) of phytopathogenic bacteria harbouring hrp clusters of type I [4] and belonging to different species (P. syringae, Erwinia amylovora, and Pantoea agglomerans formerly E. herbicola), were aligned using CLUSTALW 1.6 [29]. Based on these alignments a pair of oligonucleotide primers, HRCR8092 5′-CCITT(C/T)ATCGT(C/T)AT(C/T)GA(C/T)(C/T)T-3′ and HRCT8986 5′-CTGTCCCAGATIAICTGIGT-3′ (where I indicates inosine), was designed to amplify a part of the operon U of the hrp cluster of type I including the 3′ end of hrc R (26%), hrc S (100%), and the 5′ end of hrc T (42%) (Fig. 1). PCRs were conducted in a 25 μl reaction volume. 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 mM MgCl2, 0.1% Triton X-100, 0.2 mg ml−1 BSA, Q-Biogen, Illkirch, France). Final concentrations of each primer and of dNTPs were 0.5 and 200 μM, respectively. Thermal cycling consisted of an initial denaturation step at 95 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 41 °C for 1 min, and elongation at 72 °C for 2 min, with a final elongation step at 72 °C for 3 min.

image

Figure 1. Scheme showing the position and orientation of the primers HRCR8092 and HRCT8986 (black arrowheads) with respect to hrc R, hrc S, and hrc T of P. syringae pv. syringae NPS3121 (GenBank Accession No. AF043444).

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16S rRNA genes were amplified with primer pair fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rD1 (5′-AAGGAGGTGATCCAGCC-3′) [30]. Reactions were performed in a total volume of 50 μl, by mixing 50 ng of DNA with 0.2 μM of each primer, 20 μM each of dATP, dCTP, dGTP, dTTP, 2.5 U of Taq DNA polymerase in the corresponding buffer (see above). Amplification reactions started with an initial denaturation step (3 min at 95 °C) followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C, with a final extension of 3 min at 72 °C.

Aliquots (5 μl) of the PCR products were analysed by electrophoresis in 0.9% agarose gels stained with ethidium bromide and photographed under UV illumination.

2.3DNA hybridization

Hybridizations were performed on Pall Biodyne Plus membranes (VWR, France). For DNA dot blots, 1 μg of total DNA was spotted onto the membrane after a denaturation 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 C7hrc RST probe used for dot-blot hybridization and for checking the specificity of PCR amplifications was obtained by labeling the hrc RST fragment yielded by PCR amplification of DNA from P. fluorescens C7 (syn. CFBP5759). Digoxigenin labeling of the DNA probe, DNA hybridization and probe detection were done using a non-radioactive DNA labeling and detection kit (Roche Molecular Biochemicals, Meylan, France) and applying high stringency conditions (hybridization at 68 °C, first washing step in 0.1% SDS and SSC 2×, second washing step in 0.1% SDS and SSC 0.1×).

2.4RFLP of hrcRST and 16S rRNA genes

Aliquots (6 μl) of PCR products were digested over night with 10 U of the following restriction endonucleases purchased from Q-Biogen (Illkirch, France): Alu I, Ava II, Dde I, Hin fI, Msp I, Pst I, Rsa I, and Sph I for hrc RST, and Alu I, Dde I, Hae III, Mse I, Msp I, Nde II, Rsa I, and Taq I for 16S rDNA. The eight enzymes used for hrc RST PCR fragments were selected on the basis of restriction maps. They were chosen to discriminate all known hrc RST sequences of the phytopathogens aligned for primer design and the hrc RST 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 separated 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 carried 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 obtained with the different restriction endonucleases.

The computer program TREECON for Windows 1.3b [32] was used to estimate the relationships between the hrc RST sequences on the basis of CLUSTALW 1.6 [29] alignments and for bootstrap analysis. Distance estimation was computed according to Jukes and Cantor [33]. The matrix of distances was displayed as a dendrogram using the UPGMA method [34]. The computer program NTSYSpc 2.02k [35] was used to compare the 16S rDNA types from the fragments obtained with the different restriction endonucleases. The pairwise Jaccard coefficients of similarity were computed [36]. The matrix of similarities was displayed as a dendrogram using the UPGMA method.

2.5Cloning and sequencing PCR fragments

Before cloning, PCR fragments were purified by electrophoresis in a 0.9% agarose gel in TAE, excised and extracted from the gel by electro-elution using a Biotrap BT 1000 (Schleicher & Schuell, Dassel, Germany) according to the instructions of the manufacturer. The pGEM-T Easy Vector System II (Promega, Charbonnières, France) was used for cloning as recommended. Nucleotide sequences of the cloned fragments were determined by Genome Express (Meylan, France). Nucleotide sequence homology searches against major sequence databases were done with programs BLAST 2.0 and FASTA version 2.0X.

2.6Nucleotide sequence accession numbers

Newly obtained sequences of hrc RST fragments were deposited in EMBL under the following accession numbers: P. syringae pv. syringae 2027–37 (AJ605515), P. syringae 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. fluorescens bv. V CFBP11350 (AJ605523), P. fluorescens bv. I CFBP11351 (AJ605524), P. fluorescens bv. V CFBP11352 (AJ605525), P. fluorescens bv. I CFBP11355 (AJ605526), P. fluorescens bv. I CFBP11357 (AJ605527), P. fluorescens bv. V CFBP11362 (AJ605528), P. fluorescens bv. I CFBP11363 (AJ605529), P. fluorescens bv. II CFBP11364 (AJ605530), P. fluorescens bv. III CFBP11367 (AJ605531), P. fluorescens bv. V CFBP11386 (AJ605532), Pseudomonas sp. CFBP11402 (AJ605534).

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

3.1Distribution of hrp genes homologues in fluorescent pseudomonads

The distribution of hrc RST 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 C7hrc RST probe (Table 1).

PCR allowed the amplification of a single DNA fragment 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 C7hrc RST probe. Positive dot-blot hybridization was recorded in 59 of the 103 strains. PCR products and positive hybridization 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 saprophytic strains, PCR products and positive hybridization by dot blot were obtained in 35% and 52% of the rhizosphere strains, and in 22% and 39% of the non-rhizosphere strains, respectively. All strains, for which a PCR fragment was obtained, gave a positive signal by dot-blot hybridization.

3.2Polymorphism 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 remaining strains that included (i) eight of the 16 phytopathogenic strains (P. blatchfordae CFBP3280T, P. gingeri CFBP2810T, P. salomonii CFBP2022T, the three P. 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. fluorescens C7 (syn. CFBP5759) and F113 (syn. CFBP5935). Restriction digests were performed with 8 endonucleases on hrc RST PCR fragments. The combined RFLP patterns obtained for the 26 strains studied gave 21 distinct hrc RST types (Table 1). Each hrc RST type was represented by a single strain except the types 9, 6 and 16 which included 2, 4 and 2 strains, respectively. For each hrc RST type, a PCR fragment has been cloned and sequenced. The strains for which a hrc RST 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] pairwise distance matrix (data not shown) indicated that genetic distances between the hrc RST sequences ranged between 0.003 and 0.50, showing a high polymorphism of hrc RST among the strains. The dendrogram presented in Fig. 2 indicates the relationships between the sequences of the 21 hrc RST fragments and of those extracted 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. NGR234 (Accession No. AE000107).

image

Figure 2. Dendrogram showing the relationships among the hrc RST sequences. Distance estimations according to the coefficient of Jukes and Cantor [33] were clustered using the UPGMA method of TREECON for windows. hrc RST 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 hrc RST fragment from the sequence cited. Bootstrap analyses were performed with 1000 resamplings, percent values are shown at the branching points. For each hrc RST type, only the fragment amplified from the first strain has been sequenced, the other strains belonging to this hrc RST type are indicated after a slash. R, reference strain; T, type-strain; *, phytopathogenic strain; **, animal pathogenic strain, P. gi. =P. gingeri; P. fl. =P. fluorescens; P. sy. =P. syringae; P. pu.=P. putida; P. sp=P. species; P. ma. =P. marginalis; P. bl.=P. blatchfordae; P. sa. =P. salomonii.

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According to their level of similarity, hrc RST sequences 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 three phytopathogenic 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 saprophytic isolates from plant rhizospheres and the biocontrol strain P. fluorescens SBW25, plus five phytopathogenic strains.

3.3Restriction 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 silico 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 Laguerre et al. [31] and 6 (C8, D3, C9, C5, C1, D5) by Latour et al. [25]. According to the Jaccard pairwise similarity matrix, the level of similarity between the different ribotypes ranged from 0.17 to 0.97 indicating a high polymorphism of the 16S rRNA genes. The ribotypes 20, N7, 18 and N2 including E. coli S17.1, the phytopathogenic P. 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.

image

Figure 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. =P. syringae; P. pu. =P. putida; P. li. =P. lini; P. fl. =P. fluorescens; P. co. =P. corrugata; P. je. =P. jessenii; P. chl. =P. chlororaphis; P. bl. =P. blatchfordae; P. ma. =P. marginalis; P. sa. =P. salomonii; P. to. =P. tolaasii; P. sp. =Pseudomonas sp.; P. ag. =P. agarici; P. gi. =P. gingeri; P. as=P. asplenii; P. fu=P. fuscovaginae; P. vi=P. viridiflava; P. ci. =P. cichorii; P. flec. =P. flectens. Bacterial strains, in which hrc RST sequence was detected by PCR and/or dot-blot hybridization, are underlined.

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The level of similarity among the remaining 31 ribotypes (from 102 strains) was between 0.72 and 0.97, indicating a low polymorphism among the corresponding strains of fluorescent pseudomonads. These 102 strains were 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 phytopathogenic strains, (ii) 42 saprophytic strains from various rhizospheres, and (iii) all the soil isolates except P. putida CFBP2066T. A second group (b) of nine ribotypes showing a level of similarity at least equal to 0.83 was delineated. This group clustered six phytopathogenic strains including, P. blatchfordae CFBP3280T, P. marginalis CFBP2037T-2038T-2039T, P. salomonii CFBP2022T, and P. tolaasii CFBP2068T, and 18 saprophytic strains including P. fluorescens CFBP2102T plus 17 rhizosphere strains. Finally, the nine remaining ribotypes, encompassing 18 isolates and sharing a similarity 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.

3.4Relations 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 without the hrc RST sequence.

Relationships were established between the hrc RST sequence and ribotype groups delineated in Figs. 2 and 3, respectively (Fig. 4). Twelve of the 21 hrc RST fragments sequenced, all belonging to homology group III, were found in strains included in ribotypes of group b. Eight of the 21 hrc RST sequenced fragments, all except one belonging to homology groups I and II, were found in strains included in ribotypes of the group a. Mismatches were only recorded for two strains: P. gingeri CFBP2810T belonged to ribotype group c and to hrc RST homology group I and P. fluorescens CFBP11362 belonged to ribotype group a and to hrc RST homology group III (Fig. 4).

image

Figure 4. Correspondence between the homology groups of hrc RST sequences (1) and of the ribotypes (2) as delineated in Figs. 2 and 3, respectively. Dashed lines link hrc RST sequences and ribotypes of strains belonging to corresponding groups of homology (hrc RST sequence groups I and II/ribotype group a, and hrc RST sequence groups III/ribotype group b). Black lines indicate mismatches in the correspondence between the homology groups of hrc RST sequences and ribotypes referred above.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

4.1Distribution 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 saprophytic species (P. fluorescens and P. putida) [15–17]. However within the fluorescent pseudomonad group, further information 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 hrc RST 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 hrc RST sequence detected by dot-blot hybridization of total DNA using C7hrc RST as a probe 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 the pathogenesis process [2], and then validate our strategy based on dot blot using C7hrc RST as a probe for detecting 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 between 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 distributed in the five saprophytic species tested. These results indicate that, although the frequency of TTSS genes is significantly lower in saprophytic than in phytopathogenic species, they remain widely distributed among saprophytic species of fluorescent pseudomonads. 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 raises questions about their function in saprophytic species, since TTSSs are known to be dedicated to the translocation 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 as biocontrol 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 fluorescent pseudomonads [37]. More generally, one may suggest that TTSS might be involved in the crosstalk between bacteria and plant as showed for plant-bacterial molecular 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 frequency, and their presence was not restricted to specific bacterial genotypes since they were distributed randomly 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 rhizosphere competent than the soil strains without TTSS-like genes.

4.2Diversity of TTSS-like genes

The PCR method developed in the present study provided a series of hrc RST 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 hrc RST sequence allowed the identification of 21 hrc RST types. One fragment per type was sequenced, and their diversity was analysed together with five sequences extracted from a data bank. The hrc RST sequences have shown a high level of diversity. This level was possibly even underestimated since only 44% of the sequences detected by dot-blot hybridization gave a PCR product that was strong enough for RFLP analysis. Furthermore, only 36% of the detected hrc RST 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 belonged to the same hrc RST type despite their distribution in three different pathovars, and the three strains of P. syringae (pathovars syringae and phaseolicola) harboured 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 hrc RST sequences were clustered in three groups according to their level of similarity. The strains belonging 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 hrc RST sequences in strains belonging to different phytopathogenic species are distant from each other, and are even more distant from each other than from some of the strains belonging to saprophytic species.

Alfano et al. [39] have shown that in P. syringae the hrp genes encoding the TTSS machinery are the conserved 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 among divergent 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 suggestion 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 bacteria having different effects on plants; either negative (pathogenic), neutral (saprophytic) or positive (biocontrol). These observations suggest that the proposal by Alfano et al. [39] for P. syringae could possibly be extended to saprophytic and beneficial fluorescent pseudomonads. This hypothesis could be assessed by comparing the genomic regions of hrc genes of these types of fluorescent pseudomonads together with those known in P. syringae.

The polymorphism of hrc RST genes within the fluorescent 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 hrc RST sequences and the ribotypes for all the strains, except for two, when comparing the topology of the corresponding dendrograms. 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 suggestion 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 hrp S and hrp L of strains belonging to different pathovars. However, two mismatches were observed in the relationship between 16S rDNA types and hrc RST 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 gene clusters, which was previously shown for phytopathogenic pseudomonads [1,39,45,46]. Our results also suggest that TTSS gene transfer might not only occur in phytopathogenic strains but also in saprophytic strains of fluorescent pseudomonads. Further work on phylogeny would be required to further assess the evolution history of TTSS-like genes within phytopathogenic and saprophytic strains.

5Conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

TTSS-like genes were distributed in all tested phytopathogenic strains belonging to 13 species of fluorescent pseudomonads and were also widely distributed in strains belonging to saprophytic species. In fact, phytopathogenic and saprophytic strains could be discriminated neither on the basis of the presence of TTSS-like genes nor on the polymorphism of the corresponding 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 strains including strains not associated with eukaryotes. Comparison of the polymorphisms of hrc RST 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 the comparison of plant–microbe interactions when using strains harbouring these sequences and the corresponding targeted mutants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References

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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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. 5Conclusions
  8. Acknowledgements
  9. References
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