Identification of novel virulence genes and metabolic pathways required for full fitness of Pseudomonas savastanoi pv. savastanoi in olive (Olea europaea) knots

Authors

  • Isabel M. Matas,

    1. Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Área de Genética, Facultad de Ciencias, Málaga, Spain
    Search for more papers by this author
  • Lotte Lambertsen,

    1. Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Área de Genética, Facultad de Ciencias, Málaga, Spain
    Search for more papers by this author
  • Luis Rodríguez-Moreno,

    1. Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Área de Genética, Facultad de Ciencias, Málaga, Spain
    Search for more papers by this author
  • Cayo Ramos

    Corresponding author
    • Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Área de Genética, Facultad de Ciencias, Málaga, Spain
    Search for more papers by this author

Author for correspondence:

Cayo Ramos

Tel: +34 95213 1955

Email: crr@uma.es

Summary

  • Comparative genomics and functional analysis of Pseudomonas syringae and related pathogens have mainly focused on diseases of herbaceous plants; however, there is a general lack of knowledge about the virulence and pathogenicity determinants required for infection of woody plants.
  • Here, we applied signature-tagged mutagenesis (STM) to Pseudomonas savastanoi pv. savastanoi during colonization of olive (Olea europaea) knots, with the goal of identifying the range of genes linked to growth and symptom production in its plant host.
  • A total of 58 different genes were identified, and most mutations resulted in hypovirulence in woody olive plants. Sequence analysis of STM mutations allowed us to identify metabolic pathways required for full fitness of P. savastanoi in olive and revealed novel mechanisms involved in the virulence of this pathogen, some of which are essential for full colonization of olive knots by the pathogen and for the lysis of host cells.
  • This first application of STM to a P. syringae-like pathogen provides confirmation of functional capabilities long believed to play a role in the survival and virulence of this group of pathogens but not adequately tested before, and unravels novel factors not correlated previously with the virulence of other plant or animal bacterial pathogens.

Introduction

Gram-negative plant-pathogenic bacteria grouped in the Pseudomonas syringae complex are considered economically and agriculturally important pathogens that produce a broad variety of symptoms in a wide range of herbaceous and woody host plants. This complex includes at least 10 Pseudomonas species and 60 pathovars of P. syringae, distinguished by their host range (Gardan et al., 1999). In general, pathovars of the Psyringae complex cause foliar necrosis, with a minority of strains causing other types of symptoms, such as soft rots of vegetables, wilts, overgrowths, knots, scabs, and cankers (Agrios, 2005). During the last decade, three P. syringae strains, that is, P. syringae pv. tomato DC3000 (Alfano & Collmer, 1996), P. syringae pv. phaseolicola 1448A (Jackson et al., 1999) and P. syringae pv. syringae B728a (Hirano & Upper, 2000), have predominated as model systems, each providing a different perspective into the complex interactions of this pathogen with herbaceous plants. However, there is a general lack of knowledge about the virulence and pathogenicity determinants specific for infection of woody plants.

Pseudomonas savastanoi pv. savastanoi is a model bacterium for the study of the molecular basis of woody plant diseases. Infection of olive (Olea europaea) by P. savastanoi pv. savastanoi results in overgrowth formation (tumors, galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. The disease is thought to reduce both olive yield and productivity (Iacobellis, 2001; Quesada et al., 2010), and few commercial cultivars are significantly tolerant to olive knot (Penyalver et al., 2006). Recently, in vitro-propagated olive plantlets were established as a model system for the analysis of the interaction of P. savastanoi pv. savastanoi with olive, allowing successful evaluation of virulence and in vivo monitoring of bacterial localization inside olive knots (Rodríguez-Moreno et al., 2008, 2009). Additionally, the draft genome sequence and the complete sequence of the plasmid complement of P. savastanoi pv. savastanoi strain NCPPB 3335 (Rodríguez-Palenzuela et al., 2010; Bardaji et al., 2011) are currently available.

Virulence factors contribute to and orchestrate each distinct aspect of bacterial pathogenicity; however, few determinants have been functionally characterized in detail in the P. syringae complex. The type III secretion system (T3SS) has been identified as a key virulence determinant among multiple Gram-negative pathogens of both plants and animals, including P. syringae (Cunnac et al., 2009; Mansfield, 2009) and olive isolates of P. savastanoi (Sisto et al., 2004; Pérez-Martínez et al., 2010). After the T3SS proteins, bacterial toxins are perhaps the best-characterized P. syringae virulence determinants (Lindeberg et al., 2008). The better-characterized virulence factors in tumor-inducing isolates of P. savastanoi are the phytohormones indoleacetic acid (IAA) and cytokinins (CKs; Surico et al., 1985; Powell & Morris, 1986; Glass & Kosuge, 1988; Rodríguez-Moreno et al., 2008). Moreover, exopolysaccharides promote virulence, biofilm formation, and surface adhesion (Yu et al., 1999; Laue et al., 2006). Also, virulence of P. savastanoi pv. savastanoi is critically dependent on quorum sensing regulation (Hosni et al., 2011).

The advent of genome sequencing, especially next-generation technologies, has seen a revolution in the study of the P. syringae complex. Complete or draft genome sequences are now available for 30 strains representing two species and 18 pathovars of the P. syringae complex (Buell et al., 2003; Feil et al., 2005; Joardar et al., 2005; Almeida et al., 2009; Reinhardt et al., 2009; Studholme et al., 2009; Clarke et al., 2010; Green et al., 2010; Rodríguez-Palenzuela et al., 2010; Baltrus et al., 2011; Cai et al., 2011; Qi et al., 2011), which are predicted to facilitate the identification of a set of pathogenicity determinants and help to define host-specific genes and the virulence metagenome. However, as the number of available genome sequences grows, the number of genes with unknown functions increases substantially. Thus, genome-wide approaches to functionally characterize these genes in the process of infection have acquired great importance (Saenz & Dehio, 2005).

A number of different genetic methods are currently available to unravel novel genes involved in the interaction of bacterial pathogens with their hosts, especially those that cannot be identified by computer-assisted genomic predictions. Signature-tagged mutagenesis (STM) combines the power of conventional gene disruption methods with the ability to trace the fates of individual mutant strains within complex pools. STM has been successfully applied to identify novel virulence genes in a vast number of human and animal bacterial pathogens (Holden & Hensel, 1998; Mecsas, 2002; Saenz & Dehio, 2005; Lawley et al., 2006; Bianconi et al., 2011). However, applications of this strategy to the study of plant–bacterium interactions have been scarce and have been mainly directed to identify bacterial genes relevant to symbiosis and competitiveness in the rhizosphere of plants (Pobigaylo et al., 2006, 2008; Shimoda et al., 2008). In this work, we report the first application of STM to a bacterial phytopathogen belonging to the P. syringae complex, P. savastanoi pv. savastanoi strain NCPPB 3335. Sequence analysis of the genes altered in the selected mutants, along with a detailed study of the knot symptoms generated by individual mutants, revealed novel molecular processes involved in the interaction of P. savastanoi with olive plants. This approach also allowed us to identify metabolic pathways required by this pathogen to survive in olive knots.

Materials and Methods

Bacterial strains, plasmids, media, and growth conditions

The original bacterial strains and plasmids used in this study are listed in Table 1. Pseudomonas savastanoi pv. savastanoi strains were grown at 28°C in King's medium B (KB; King et al., 1954), Luria–Bertani (LB) medium (Miller, 1972), standard succinate medium (SSM; Meyer & Abdallah, 1978), or super optimal broth (SOB) medium (Hanahan, 1983). Escherichia coli strains were grown at 37°C in LB and SOB media. Solid and liquid media were supplemented, when required, with the following antibiotics (μg ml−1) for Pseudomonas/E. coli strains: ampicillin (Amp) 300/100 and kanamycin (Km) 10/50. Gentamicin (Gm) 10 μg ml−1 and nitrofurantoin (Nf) 100 μg ml−1 were used, when required, for P. savastanoi pv. savastanoi strains.

Table 1. Strains and plasmids used in this study
Strains/plasmidsaRelevant characteristicsReference or source
  1. a

    Plasmids constructed in this study are indicated in Supporting Information Table S1.

Strains
Pseudomonas savastanoi pv. savastanoi
NCPPB 3335Wild-type strainPérez-Martínez et al. (2007)
NCPPB 3335-GFPGFP-tagged derivative harboring plasmid pLRM1-GFPRodríguez-Moreno et al. (2009)
Escherichia coli
XL1 BluehsdR17, supE44, recA1, endA1, gyrA46, thi, relA1, lac/F′ [proAB+, lacIq, lacZ M15::Tn10(TcR)]Bullock et al. (1987)
DH5αF, φ 80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1 endA1, hsdR17(rK mK+), phoA, supE44, λ, thi-1, gyrA96, relA1Hanahan (1983)
S17-1λpirthi pro hsdR recA RP4-2 (Tc::Mu Km::Tn7 [TcR StrR], λ-pir lysogenSimon et al. (1983)
CC118λpir∆(ara-leu) araDlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1, λ-pir lysogen, (RifR)Herrero et al. (1990)
Plasmids

pUTminiTn5Km2-

STM

Pool of tagged pUTminiTn5Km2 vectors (AmpR, KmR)Holden & Hensel (1998)
pBluescript II SK(−)Cloning vector; orif1(−), oripUC, Plac, lacZ', (AmpR)Agilent Technologies, Inc. (Santa Clara, CA, USA)

pGEMT Easy

vector

PCR cloning vector. 3′-T ends (AmpR)Promega Cor. (Madison, USA)
pLRM1-GFPpBBR1-MCS5::PA1/04/03-RBSII-GFPmut3*-T0-T1, (GmR)Rodríguez-Moreno et al. (2009)

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 derivatives selected in this study that contained a miniTn5Km2 transposon are listed in Tables 2 and 3. Plasmids constructed and primers used in this study are listed in Supporting Information Tables S1 and S2, respectively.

Table 2. Pseudomonas savastanoi pv. savastanoi NCPPB 3335 signature-tagged mutagenesis (STM) mutants disrupted in metabolic-related genes
NameaIDbGeneFunctionSSMcORF lengthminiTn5 insertiondPossiblepolaritye
  1. a

    Name of the strain and functional group to which it belongs.

  2. b

    ASAP (A Systematic Annotation Package for community Analysis of genomes) ID number for P. savastanoi pv. savastanoi NCPPB 3335.

  3. c

    Ability (+) or inability (−) to grow in standard succinate medium (SSM) minimal medium.

  4. d

    MiniTn5Km2 insertion point. Numbers indicate the exact position (bp) of the transposon in the disrupted gene, assuming the first nucleotide of the start codon = 1.

  5. e

    Possible polarity of miniTn5Km2 insertions in fitness-attenuated mutant (FAM) strains. Operon predictions were performed following the criteria defined by Dam et al. (2007). Intergenic distances of < 40 nt between the genes interrupted by the transposons and the next downstream genes were considered to affect operonic pairs (I); > 40 but < 200 nt were considered to affect operonic pairs with intermediate probability (II); at > 200 nt, mutations were considered to affect probable single loci (III).

Biotin biosynthesis
FAM-101AER-0005006 bioB Biotin synthase+1059525I
FAM-023AER-0005001 bioD Dethiobiotin synthetase+681116III
Cobalamin biosynthesis
FAM-156AER-0002272 cobB Cobyrinic acid-diamide synthase1296580I
FAM-147AER-0002279 cobV Alpha-ribazole-5′-phosphate phosphatase573313I
Thiamine metabolism
FAM-111AER-0000150 thiE Thiamin-phosphate pyrophosphorylase61874III
Arginine metabolism
FAM-098AER-0002156 argA N-acetylglutamate synthase1299781III
FAM-148AER-0004412 argB Acetylglutamate kinase906330III
Glutamate metabolism
FAM-172AER-0001174 gltB Glutamate synthase (NADPH) large chain44463910I
FAM-178AER-0001174 gltB Glutamate synthase (NADPH) large chain44463056I
Histidine metabolism
FAM-022AER-0004017 hisD Histidinol dehydrogenase13471213I
FAM-053AER-0004356 hisF1 Imidazole glycerol phosphate synthase cyclase771380III
FAM-096AER-0004356 hisF1 Imidazole glycerol phosphate synthase cyclase771568III
FAM-183AER-0004356 hisF1 Imidazole glycerol phosphate synthase cyclase771681III
FAM-116AER-0004016 hisG ATP phosphoribosyltransferase catalytic638600I
FAM-063AER-0001745 hutG N-formylglutamate deformylase+801311III
Isoleucine, leucine and valine metabolism
FAM-094AER-0000496 ilvB Acetolactate synthase large subunit17251360I
FAM-195AER-0000496 ilvB Acetolactate synthase large subunit1725823I
FAM-016AER-0000498 ilvC Ketol-acid reductoisomerase1017ndII
FAM-001AER-0000894 ilvD Dihydroxy-acid dehydratase1848519III
FAM-078AER-0003433 leuD 3-Isopropylmalate dehydratase small subunit+64267II
Methionine metabolism
FAM-138AER-0004647 metG Methionyl-tRNA synthetase+20492017II
FAM-188AER-0000890 metW Methionine biosynthesis protein621151III
FAM-146AER-0000889 metX Homoserine O-acetyltransferase+1140183I
FAM-186AER-0000889 metX Homoserine O-acetyltransferase1140469I
FAM-095AER-0002900 metZ O-acetylhomoserine sulfhydrylase1212418II
FAM-136AER-0002900 metZ O-acetylhomoserine sulfhydrylase121254II
Proline metabolism
FAM-171AER-0002315 proB Glutamate 5-kinase1119ndI
FAM-168AER-0000887 proC Pyrroline-5-carboxylate reductase855114I
FAM-192AER-0000887 proC Pyrroline-5-carboxylate reductase855619I
Tryptophan metabolism
FAM-196AER-0003214 trpE Anthranilate synthase1482110III
Transport systems
FAM-140AER-0004392 citN Citrate transporter+1308692III
FAM-120AER-0004505 cysT Sulfate transport system permease protein+822213I
FAM-069AER-0004717 gltP Proton/glutamate symporter+285135III
Benzoate degradation
FAM-083AER-0004134 catJ 3-Oxoadipate CoA-transferase subunit B+780450I
Others
FAM-151AER-0000904 metF 5,10-Methylene-tetrahydrofolate reductase+876156III
FAM-181AER-0000630 ptsP Phosphocarrier protein kinase/phosphorylase+22801642III
FAM-079AER-0001487 rsvA Pseudouridine synthase A891853III
FAM-058AER-0004257 tal Transaldolase+927117III
Table 3. Pseudomonas savastanoi pv. savastanoi NCPPB 3335 nonmetabolic mutants obtained by signature-tagged mutagenesis (STM)
NameaIDaGenebFunctiondSSMaORF lengthminiTn5 insertionaPossible polaritya
  1. a

    See descriptions in Table 2.

  2. b

    Mutations in genes whose closest homologs are currently unnamed are indicated as follows: HP, hypothetical protein-coding gene; EEP, endonuclease/exonuclease/phosphatase family protein-coding gene; DBP, DNA-binding protein-coding gene; PMP, putative membrane protein-coding gene; MCP, methyl-accepting chemotaxis protein; GGDEF, GGDEF/EAL domain protein-coding gene.

  3. c

    Plasmid-encoded gene; pA, pPsv48A; pB, pPsv48B (Bardaji et al., 2011).

  4. d

    Putative pseudogenes (Rodríguez-Palenzuela et al., 2010).

Secretion systems
FAM-117AER-0002563 hrpR T3SS transcriptional regulator+921659II
FAM-170AER-0002927 ppiD Peptidyl-prolyl cis-trans isomerase+1884458III
FAM-082AER-0001448 secG Preprotein translocase subunit+429102III
FAM-109AER-0000339 traY Type IVB secretion system proteindpA+201617II
FAM-025AER-0000607 virB4 Type IVA secretion system proteindpB+25532129I
Cell-surface structures
FAM-184AER-0004572 algT RNA polymerase sigma-22 factor582148I
FAM-103AER-0004067 ampG Muropeptide permease+15511038I
FAM-118AER-0000830 cls Cardiolipin synthetase+14401069III
FAM-179AER-0000128 mltB Membrane-bound lytic murein transglycosylase B+1008207II
FAM-046AER-0001183 ponA Multimodular transpeptidase-transglycosylase+24451881III
Stress tolerance
FAM-167AER-0002425 arsB Arsenical pump membrane protein+82291III
FAM-019AER-0000755 cioA Cyanide insensitive terminal oxidase, subunit I+1440930I
FAM-119AER-0002177 msrA Peptide methionine sulfoxide reductase+648359III
FAM-089AER-0003040 pqiB Paraquat-inducible protein B+1680398I
FAM-068AER-0000777 rubB Rubredoxin-NAD(+) reductase+114940II
DNA-related protein
FAM-108AER-0002142EEPEndonuclease/exonuclease/phosphatase family protein+876465I
FAM-008AER-0000594 ssb Single-stranded DNA-binding protein dpB+573286III
FAM-059AER-0005415DBPDNA-binding proteind+1132248III
Hypothetical proteins
FAM-189AER-0000598HPHypothetical proteindpB2412442II
FAM-149AER-0001621HPHypothetical protein+89754III
FAM-158AER-0001474HPPossible exported protein+38162376II
FAM-124AER-0004038HPPutative lipoprotein+18151523I
FAM-143AER-0004655PMPPutative membrane protein+2643998I
FAM-099AER-0004655PMPPutative membrane protein+26431749I
FAM-157AER-0004655PMPPutative membrane protein+26431114I
Others
FAM-005AER-0001304MCPMethyl-accepting chemotaxis protein+19861933I
FAM-030AER-0004088GGDEFGGDEF domain/EAL domain protein+20521989III
FAM-110AER-0003642 iaaH-1 Indole acetamide hydrolase+11461118III
FAM-057AER-0004487 spmAB Spore maturation protein AB+12421161III

Generation of a unique-tag marked library of P. savastanoi pv. savastanoi NCPPB 3335 mutants

A library of signature-tagged transposon mutants of P. savastanoi pv. savastanoi NCPPB 3335 was constructed as described by Hensel et al. (1995), with minor modifications. The pool of tagged pUTminiTn5Km2 vectors was transferred from E. coli S17 λpir to P. savastanoi pv. savastanoi NCPPB 3335 by plate conjugation mating as previously described (Pérez-Martínez et al., 2007). The constructed random transposition library consisted of 55 different 96-well microtiter trays, containing a total of 4778 P. savastanoi pv. savastanoi NCPPB 3335 mutants. Individual colonies were challenged on LB-Amp plates to discard P. savastanoi pv. savastanoi transconjugants harboring the plasmid vector integrated into their replicons; 37 AmpR mutant strains (0.77%) were discarded.

Agarose gel electrophoresis and other standard recombinant DNA techniques were performed as described previously (Sambrook & Russell, 2001). Genomic DNA was extracted using the Jet Flex Extraction Kit (Genomed, Löhne, Germany) according to the manufacturer's instructions. Single and random transposon insertions into the genomes of the mutant strains were confirmed by Southern hybridization (Pérez-Martínez et al., 2007).

Colony blots

To fix total DNA from colonies, overnight cultures of P. savastanoi pv. savastanoi NCPPB 3335 mutant strains grown on LB-Km microtiter plates were transferred onto nylon membranes placed on LB-Km agar plates using a 48-pin replicator (Sigma-Aldrich, Inc., St Louis, MO, USA). Colony blots were performed as previously described by Holden & Hensel (1998).

Signature-tagged mutagenesis (STM) screening

STM screening was carried out by testing pools of c. 45 mutants. The input pools were generated by mixing 150 μl of mutant cultures grown for 48 h at 28°C on LB-Km microtiter plates. After this, the mixtures were washed twice with 10 mM Cl2Mg and adjusted to an optical density (OD600) of 0.01 (c. 106 CFU ml−1). Three in vitro olive (Olea europaea L.) plants were inoculated with 2 μl of each bacterial suspension as described by Rodríguez-Moreno et al. (2008). At 30 d post-inoculation (dpi), mutant cells were recovered from the developed olive knots to generate the output pool. Two rounds of PCR were performed to generate a probe of each input and output pool (Holden & Hensel, 1998). Finally, the 40-bp probes were purified using MiroSpin™ G-50 columns (GE Healthcare, Buckinghamshire, UK). Hybridizations of DNA on colony blots to 32P-labeled probes were carried out as described by Holden & Hensel (1998). A schematic representation of the STM selection process is shown in Fig. S1.

Determination of transposon insertion sites

Genomic DNA from selected mutants was digested with EcoRI or KpnI and ligated into pBluescript II SK digested with the same restriction enzyme. Ligation reactions were used to transform DH5α by heat shock (Hanahan, 1983), and single Km-resistant colonies were selected. Plasmids showing DNA fragments of at least 1.8 kb were purified using a NucleoSpin Plasmid Quick Pure kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), and the DNA regions flanking the transposons were sequenced using primer P7 (Table S2; Hensel et al., 1995). Automated DNA sequencing was performed by Secugen (Madrid, Spain). The raw sequences were analyzed by general BLASTn searches against National Center for Biotechnology Information (NCBI)-deposited sequences and the complete genome sequence of P. savastanoi pv. savastanoi NCPPB 3335 (Rodríguez-Palenzuela et al., 2010) using the ASAP (A Systematic Annotation Package for Community Analysis of Genomes) Database (http://www.genome.wisc.edu/tools/asap.htm). Specific primers were designed to determine the exact insertion point of the miniTn5Km2 transposon in each selected mutant. The sequences of the interrupted genes and of the putative proteins encoded by those genes were further analyzed using the ASAP Database, the Kyoto Encyclopedia for Genes and Genomes (KEGG; http://www.genome.jp/kegg/pathway.html) and the NCBI platform (http://www.ncbi.nlm.nih.gov/).

Plant infection and isolation of bacteria from olive knots

Olive plants derived from seeds germinated in vitro (originally collected from a cv Arbequina plant) were micropropagated, rooted and maintained as previously described (Rodríguez-Moreno et al., 2008, 2009).

Micropropagated olive plants were infected in the stem wound with bacterial suspension (c. 103 CFU) and incubated for 30 d in a growth chamber as described by Rodríguez-Moreno et al. (2008). Bacteria were recovered from the knots using a mortar containing 1 ml of 10 mM MgCl2, and serial dilutions were plated onto LB medium (to detect the wild type or the total amount of wild-type and mutant cells) and LB-Km medium (to detect miniTn5Km2 mutants) or LB-Gm medium (to detect GFP-tagged strains). Population densities were calculated from at least three independent replicates. The morphology of the olive plants infected with bacteria was visualized using a stereoscopic microscope (Leica MZ FLIII; Leica Microsystems, Wetzlar, Germany).

To analyze the pathogenicity of P. savastanoi pv. savastanoi isolates in 1-yr-old olive explants (woody plants), micropropagated olive plants were transferred to soil and maintained in a glasshouse at 27°C with a relative humidity of 58% under natural daylight. The plants were wounded in the stem and infected with c. 106 CFU of P. savastanoi pv. savastanoi as previously described (Penyalver et al., 2006; Pérez-Martínez et al., 2007). Morphological changes, scored at 90 dpi, were captured with a high-resolution digital camera (Nikon DXM 1200; Nikon Corporation, Tokyo, Japan).

Evaluation of olive knot symptoms

Virulence of P. savastanoi pv. savastanoi strains in woody olive plants was quantified by comparing the symptoms generated by a specific P. savastanoi pv. savastanoi strain at 90 dpi with a visual symptoms scale ranging from 1 to 6: 1, absence of symptoms (mock control); 2, mild thickening of the wound; 3, small knot at the base of the wound; 4, small tumors at both the base and the top of the wound; 5, knot covering the wound completely; and, 6, knot larger than the wound. Symptoms generated by the wild-type strain P. savastanoi pv. savastanoi NCPPB 3335 ranged from 5 to 6, depending on the plant and the inoculation site. Symptom categories 2–4 corresponded to inoculations with hypovirulent P. savastanoi pv. savastanoi mutants. In general, the severity of the symptoms generated decreased with the proximity of the wound to the apex of the plant. To evaluate symptoms, five to 35 different inoculations were used per strain. Statistical data were analyzed by analysis of variance followed by Fisher's least significance difference test (= 0.05) using spss software (SPSS Inc., Chicago, IL, USA).

In vitro and in planta competition assays

In vitro and in planta competition assays were performed as previously described (Rodríguez-Moreno et al., 2008). A competitive index (CI) was calculated by dividing the output ratio (CFU mutant : CFU wild-type) by the input ratio (CFU mutant : CFU wild-type). The in vitro and in planta competition indexes shown (CILB and CIP, respectively) are the mean of three replicates showing typical results from three independent experiments.

Epifluorescence microscopy

To visualize bacterial infection of tumors, the wild-type strain and a collection of selected mutants were tagged with green fluorescent protein (GFP) using plasmid pLRMl-GFP and knots generated by tagged strains were examined by epifluorescence microscopy as previously described (Rodríguez-Moreno et al., 2009).

Results

Selection of P. savastanoi pv. savastanoi mutants with reduced competitiveness in olive plants by signature-tagged mutagenesis

A mutant library of 4741 miniTn5Km2-tagged derivatives of P. savastanoi pv. savastanoi NCPPB 3335 was constructed (see the 'Materials and Methods' section) and screened in a first STM round in in vitro-micropropagated olive plants. For this purpose, dot blot hybridization using probes generated from both the input and the output pools were used. Mutants showing stronger hybridization signal with the input than with the output probe probably had an altered ability to grow or survive in the host. Thus, these strains were selected for further characterization and named fitness-attenuated mutants (FAMs). A total of 442 FAM strains were selected. To reduce the number of false-positive candidates, the selected mutants were grouped in new pools, mixed with other random mutants, and re-tested in a second round of STM screening to determine whether they would be re-identified. After this second STM round, the number of mutants was reduced to 193 FAM strains (Fig. S1). Single insertions of the transposon into the genome of each mutant were selected by hybridization of EcoRI-digested total DNA against a transposon probe (aphA gene). Out of the selected 161 strains containing a single insertion, 127 were able to grow on minimal SSM plates (SSM+ mutants), and the remaining 34 were unable to grow in this medium (SSM mutants).

FAM strains, presumably selected by their reduced fitness in vivo compared with other mutant strains included in the same pool, potentially have modifications in essential genes or in genes required for competitive survival in the host. To differentiate between these two possibilities, the growth of each of the selected mutants was tested individually in competition with the wild-type strain, both in LB medium and in planta. A total of 40 out of the selected 127 SSM+ strains showed competition indexes in planta (CIP) and in LB medium (CILB) significantly less than 1 and not significantly different from 1, respectively (CIP < 1 and CILB = 1), indicating that they were outcompeted by the wild-type strain only in planta but grew at the same rate as the parental strain in LB medium. CI assays were also performed for a subset of 18 mutants out of the 34 selected SSM strains. All 18 strains showed CIP < 1 and CILB = 1 (Fig. 1). Based on these results, and although CI assays were not carried out for the remaining 16 mutants, all 34 SSM strains were selected for further characterization, including virulence assays in olive plants. At the end of the screening process, a total of 73 FAM strains, 33 of which were SSM, were selected and further characterized.

Figure 1.

In vitro and in planta competition assays of Pseudomonas savastanoi fitness-attenuated mutant (FAM) strains. Competitive index values are shown for mixed inoculations of P. savastanoi pv. savastanoi NCPPB 3335 and its derivative FAM strains in LB medium (CILB) and in micropropagated olive plants (CIP). CI results are indicated after the number of the corresponding FAM strain and the name of the closest homolog to the gene interrupted in each strain (Tables 2 and 3). CI assays of FAM strains disrupted in metabolic-related (a) and nonmetabolic-related genes (b) were performed. Error bars indicate the standard errors from the average of three assays. In all cases, CIP is significantly < 1.0. Asterisks indicate mean CIP significantly lower than CILB. Mutant : wild-type input ratios < 0.5 (35%) or > 2.0 (65%) are indicated by triangles, apex up or triangles, apex down, respectively. Statistical analyses were performed using Student's t-test at = 0.1.

Pseudomonas savastanoi pv. savastanoi genes required for full fitness in olive plants

Genomic DNA fragments flanking the transposon insertions within the 73 selected FAM strains were cloned, sequenced, and used to search the GenBank and ASAP databases for homologous genes. Taking into account that several identical siblings and independent insertions in the same gene were detected (Table S3), the number of FAM strains carrying transposon insertions at different sites was reduced to 67, which had disruptions in a total of 58 different genes. FAM strains were classified into seven categories according to the gene function of the highest-quality BLAST alignment (Tables 2 and 3).

In general, FAM strains with disrupted metabolic-related genes showed the most variable CIP values, ranging from 10−6 (catJ and hisD mutants) to c. 10−1. Interestingly, CIP values slightly > 10−1 were only shown by the three FAM strains with disrupted transporters. However, most strains included in this functional category showed CIP values < 10−2 (Fig. 1a). In relation to FAM strains included in the other functional categories established in this study, and with the exception of the strain disrupted in the alternative sigma factor encoded by algT, which showed a CIP value < 10−6, all other FAM strains showed CIP values > 10−2 (Fig. 1b).

The DNA context surrounding each of the genes interrupted by the transposons in FAM strains was analyzed using the ASAP platform against the genome of P. savastanoi pv. savastanoi NCPPB 3335. The interrupted genes were classified into three groups based on the intergenic distances between the genes interrupted by the transposons and the next downstream genes, following the criteria defined for operon prediction in E. coli and Bacillus subtilis (Dam et al., 2007). Out of the 58 transposon-containing insertions into different genes, 31 were considered to possibly form operons, and could therefore have a polar effect on the transcription of downstream genes (Fig. S2, Tables 2, 3).

In silico reconstruction of metabolic pathways required by P. savastanoi pv. savastanoi for full fitness in olive knots

Metabolic pathways and transporters altered in the 38 mutants (31 different genes) included in this category are presumably essential for growth and survival of this pathogen in planta. Metabolic-related genes disrupted in these strains included 18 genes encoding putative enzymes involved in the biosynthetic pathways of nine of the 20 amino acids commonly found in proteins, five genes possibly involved in the biosynthesis of vitamins (biotin, cobalamin, and thiamine) and three genes encoding putative sulfate, citrate and amino acid transporters. The following genes were also included in this category: catJ, metF, tal, ptsP, and rsvA (Table 2).

In silico reconstruction of P. savastanoi pv. savastanoi metabolic pathways was performed using Pathways Tools Version 15.0 (http://bioinformatics.ai.sri.com/ptools/), the maps available at the KEGG Pathway Platform and the genome sequence of P. savastanoi pv. savastanoi NCPPB 3335 (Rodríguez-Palenzuela et al., 2010). Additionally, P. syringae pv. phaseolicola 1448A, the closest phylogenetic strain to NCPPB 3335 whose entire genome has been sequenced (Joardar et al., 2005), was used as a template for the reconstructions of the NCPPB 3335 pathways. Metabolic pathways predicted to involve genes disrupted in FAM strains were compared step by step against the genomes of both NCPPB 3335 and 1448A, and all genes involved in these pathways were individually identified (Table S4). Fig. 2 shows the reconstruction of the amino acid and vitamin pathways required for full growth of P. savastanoi pv. savastanoi in olive plants. In addition, the predicted pathway for the catabolism of anthranilate via catechol, involving catJ, is shown. Although the genes responsible for this pathway are not found in the genome of 1448A (Rodríguez-Palenzuela et al., 2010), an ortholog of the NCPPB 3335 catJ gene was found in the genome of 1448A. In addition, pathways involved in glycolysis, fatty acid biosynthesis and methanol metabolism were found (Fig. 2). All these pathways included 26 out of the 31 metabolic-related genes disrupted in FAM strains, most of which resulted in the SSM phenotype (Fig. 2, Table 2). With the exception of a strain containing the transposon that inserted into rsvA, the remaining four FAM strains not included in the reconstructed pathways were SSM+ and contained a transposon inserted in genes sharing homology with ptsP or genes encoding proteins possibly involved in the transport of citrate (citN ), sulfate (cysT ) or glutamate (gltP ) (Table 2).

Figure 2.

In silico reconstruction of metabolic pathways required for full fitness of Pseudomonas savastanoi in olive knots based on sequence analysis of fitness-attenuated mutant (FAM) strains. Red and blue frames indicate genes whose alteration results in mutants able and unable to grow in standard succinate medium (SSM) minimal medium, respectively. Amino acids are indicated in black frames. Circles represent the position in the network of the indicated compounds. Each of the solid arrows shown in this figure represents a single step, the genes responsible for which have been found in the genomes of P. savastanoi pv. savastanoi NCPPB 3335 and P. syringae pv. phaseolicola 1448A. Red arrows indicate single steps encoded in the genome of P. savastanoi pv. savastanoi NCPPB 3335 but not in P. syringae pv. phaseolicola 1448A (for a detailed description, see Supporting Information Table S4). Dashed arrows correspond to multiple steps.

Most P. savastanoi pv. savastanoi fitness-attenuated mutants are hypovirulent in olive plants

We developed a semiquantitative disease phenotype scale to score olive knot disease symptoms, ranging from a value of 1 (no symptoms) to 6 (maximum symptoms induced by the wild-type strain; Fig. 3a). For these assays, symptoms generated by P. savastanoi pv. savastanoi strains in 1-yr-old woody olive plants were evaluated at 90 dpi. A total of 56 of the 67 FAM strains containing the miniTn5Km2 transposon at different sites were tested (Tables 2, 3). The remaining 11 strains, disrupted in the same gene or in the same pathway as some of the 56 strains selected for this assay, were discarded. These analyses revealed that only seven out of the 56 strains tested, which had disruptions in, respectively, three hypothetical proteins, a DNA-binding protein, metX, ppiD and ptsP, caused symptoms as severe as those induced by the wild-type strain. Therefore, these seven strains were classified as virulent. By contrast, the severity of the symptoms generated by the remaining 49 strains was in all cases significantly lower than that induced by the wild-type strain. Among these 49 strains, mutants for the T3SS regulatory gene hrpR or the amino acid biosynthetic gene ilvB or metZ (two different insertions) induced symptoms showing a severity index < 2 and were classified as nonvirulent. The remaining 45 strains, which induced symptoms with severity values ranging from 2 to 5, were considered hypovirulent (Fig. 3b). Interestingly, most olive plants showing symptoms with severity values < 4 were infected with FAM strains with disrupted metabolic-related genes (21 strains of a total of 27 FAM strains; Fig. 3b, Table 2). Among the remaining six FAM strains, only two had mutations in virulence factors previously described in P. savastanoi pv. savastanoi strains (iaaH and hrpR). Thus, genes interrupted by the transposon in the remaining four strains, mutated in a methyl-accepting chemotaxis protein (MCP)-coding gene, a putative membrane protein (PMP)-coding gene, the ssb gene, and the cioA gene, were revealed as novel virulence factors of P. savastanoi pv. savastanoi in its interaction with olive plants. Other fitness and virulence genes identified in this study include pqiB and rubB, involved in tolerance and detoxification of reactive oxygen species (ROS); the Sec pathway and T4SS genes secB and traY, respectively; as well as a muropeptide permease encoded by the ampG gene and a cardiolipin synthetase encoded by the cls gene (Fig. 3b, Table 3). Fig. 4 shows a schematic representation of the novel molecular processes involved in the virulence of P. savastanoi pv. savastanoi identified in this study.

Figure 3.

Virulence of Pseudomonas savastanoi pv. savastanoi fitness-attenuated mutant (FAM) strains in 1-yr-old woody olive (Olea europaea) plants. (a) Visual scale of symptoms generated by P. savastanoi pv. savastanoi strains in woody olive plants (for details, see 'Evaluation of olive knot symptoms' in the Materials and Methods section). (b) Symptom severity generated by FAM strains in comparison to the wild-type strain P. savastanoi pv. savastanoi NCPPB 3335 (WT). (c) Population density reached by FAM strains in olive tissues at 90 d post-inoculation (dpi). FAM strains are indicated by their corresponding numbers and by the name of the gene interrupted by the transposon in each (for details, see Tables 2 and 3). Error bars indicate the standard error from the average of three different assays. Asterisks indicate strains inducing symptom severity not significantly lower than that induced by the wild-type strain at = 0.05, according to analysis of variance followed by Fisher's least significant difference test. nd, not determined.

Figure 4.

Schematic representation of virulence-associated mechanisms in Pseudomonas savastanoi pv. savastanoi identified in this study. Functional categories are represented in different colors: blue, secretion systems; green, cell-surface structures; orange, stress tolerance; gray, transporters; and red, others. OM, outer membrane; PG, peptidoglycan; IM, inner membrane; T3SS, type III secretion system; T4SS, type IV secretion system; MCP, methyl-accepting chemotaxis protein; GGDEF, GGDEF/EAL domain protein-coding gene; IAA, indole-3-acetic acid. See Table 3 for functions related with CioA, RubB, MrsA, PqiB, ArsB, CitN, CysT, GltP, SpmAB, TraY, VirB4, HrpR, SecG, Cls, PonA, AmpG and MltB.

Multiplication and persistence in olive tissues were tested in most of the selected FAM strains in comparison with the wild-type strain. Although both of the nonvirulent mutants tested, with disruptions in the hrpR and ilvB genes, reached population densities approximately two orders of magnitude lower than that of the wild-type strain at 90 dpi, no direct correlation was found between the virulence of the remaining mutants tested and their ability to grow and persist individually in olive knots. Bacterial counts obtained at 90 dpi for both virulent and hypovirulent strains varied widely between c. 103 and 106 CFU per knot (Fig. 3c). Despite the fact that all strains tested in this assay were less competitive than the parental strain on young micropropagated olive plants (Fig. 1), these results were not unexpected because P. savastanoi pv. savastanoi mutants with disruptions in other virulence genes, such as those involved in the biosynthesis of the phytohormones IAA and CKs, reach similar cell densities to the wild-type strain in olive knots (Iacobellis et al., 1994; Rodríguez-Moreno et al., 2008). Moreover, strain FAM-059, disrupted in a DNA-binding protein and classified as virulent, reached a population density at 90 dpi approximately two orders of magnitude lower than that of the wild-type strain (Fig. 3c).

Knots induced by P. savastanoi pv. savastanoi FAM strains show an altered spatial distribution of pathogen cells and a reduced lysis of host cells

The infection process of olive plants by hypovirulent P. savastanoi pv. savastanoi FAM strains was monitored in real time using young micropropagated olive plants. A total of 13 different FAM strains, representing all seven functional categories established in this study, were GFP-tagged with plasmid pLRM1-GFP (Rodríguez-Moreno et al., 2009) and used in these assays. Whole knots developed on in vitro olive plants by each of these strains were visualized at 28 dpi. With the exception of the ilvC mutant, which did not induce a visible knot at 28 dpi, and the catJ and iaaH mutants, which induced the formation of knots clearly smaller than those induced by the wild-type strain, the remaining 10 strains tested induced the formation of visible knots, most of which showed an irregular morphology (Fig. 5a). In agreement with previous data (Rodríguez-Moreno et al., 2009), knots induced by P. savastanoi pv. savastanoi NCPPB 3335 contained 107–108 CFU per knot and exhibited green fluorescent clusters that spanned the entire surface of the knot at 28 dpi. No GFP fluorescence was observed in olive tissue infected with the hrpR or ilvC mutant, whose cell density at 28 dpi was reduced by two to three or four to five orders of magnitude, respectively, in comparison with the wild-type strain. Although knots induced at 28 dpi by strains disrupted in rubB, pqiB or AER-0004655 (encoding a PMP) contained CFU levels similar to that of the wild-type strain and exhibited green fluorescence that covered the entire knot surface, a direct correlation between cell density and GFP emission was not found for the remaining eight strains (Fig. 5b). An unevenly distributed fluorescence pattern across the knot surface was observed for these eight strains, which included spots with increased GFP fluorescence (Fig. 5). These results suggest a spatial distribution of mutant cells inside olive knots totally different from that of wild-type cells.

Figure 5.

Real-time monitoring of the infection of olive (Olea europaea) plants by hypovirulent Pseudomonas savastanoi pv. savastanoi strains. (a) Images of knots induced by the indicated strains on in vitro olive plants at 28 d post-inoculation (dpi) and complementary epifluorescence microscopy images. Bars, 1 mm. (b) Total number of bacteria extracted from knots at 28 dpi of the indicated strains. Data represent the average of three independent experiments. Error bars indicate the standard deviation from the average. Asterisks indicate significant differences (= 0.05) between wild-type and mutant strains. FAM strains are indicated by the name of the gene interrupted by the transposon (Table 3). EEP, endonuclease/exonuclease/phosphatase family protein; WT, wild-type NCPPB 3335; MCP, methyl-accepting chemotaxis protein; PMP, putative membrane protein.

In the light of these results, the spatial distribution of bacterial cells inside olive tissues and the histological structure of the knots were analyzed for a selection of hypovirulent mutants in comparison with the parental strain. In agreement with previously reported data (Rodríguez-Moreno et al., 2009), knots induced by NCPPB 3335 showed hypertrophic parenchymal tissue that expanded from the vascular cylinder and exhibited internal open fissures surrounded by plasmolyzed cells. Transverse sections of knots induced by NCPPB 3335 at 28 dpi clearly showed expanded areas of green fluorescent spots colonizing the internal open cavities and periphery of the knot tissues. By contrast, sections of knots induced by the rubB, pqiB, and PMP mutans only showed green fluorescent spots colonizing the periphery of the knot tissue; however, internal open cavities were not observed in these knots. Furthermore, sections of knots induced by the secG and hrpR mutants showed a completely different pattern of green fluorescence that was restricted to the vicinity of the main vascular cylinder. Internal open cavities were not observed in these knots (Fig. 6). Together, these results suggest that lysis of host cells induced by P. savastanoi pv. savastanoi requires both the Sec pathway and the T3SS to be functional. In addition, this process seems to depend on detoxification of ROS mediated by RubB and PqiB, as well as on a PMP, whose function is unknown.

Figure 6.

Spatial distribution of GFP-tagged Pseudomonas savastanoi fitness-attenuated mutant (FAM) strains inside olive tissues and histological structure of knots. Epifluorescence microscopy images of transverse sections of olive knots induced by P. savastanoi pv. savastanoi NCPPB 3335 (wild type (WT)) and FAM strains at 28 d post-inoculation (dpi) are shown. FAM strains are indicated by the name of the gene interrupted by the miniTn5Km2 transposon (Table 3). PMP, putative membrane protein-coding gene. Bars, 500 μm.

Discussion

Comparative genomics, based on an ever-increasing number of complete genome sequences, can be used to reveal numerous insights into host–pathogen interactions. However, hypotheses obtained using these strategies should be experimentally demonstrated by functional approaches. The application of STM to a range of microbial pathogens has resulted in the identification of novel fitness-related genes and virulence determinants in each screen performed to date (Autret & Charbit, 2005). To our knowledge, the only STM study involving a bacterial phytopathogen identified 19 Erwinia amylovora genes required during the infection process of shoots of apple trees (Malus domestica), most of which were metabolic-related genes (Wang & Beer, 2006). However, our application of this technique to the interaction of P. savastanoi pv. savastanoi with olive plants represents the first STM analysis of a bacterial strain belonging to the P. syringae complex. Screening for virulence factors based on STM relies on the premise that virulence of a given mutant strain within a complex mixed infection reflects its virulence in an individual infection (Hensel et al., 1995), a phenomenon dependent on the dose of inoculation, including plant infection by P. syringae (Macho et al., 2007). In relation to P. savastanoi pv. savastanoi infection of olive plants, optimization of the inoculum dose for STM analysis has been previously reported (Rodríguez-Moreno et al., 2008). Complete coverage of the NCPPB 3335 genome was not achieved in this study, as the number of open reading frames (ORFs) predicted in the genome sequence of this strain (5232 ORFs; Rodríguez-Palenzuela et al., 2010) is slightly higher than the total number of STM mutants screened (4741 strains). Considering the frequency of multiple Tn5 insertions into the genome of NCPPB 3335 (Pérez-Martínez et al., 2007), as well as the number of siblings and independent insertions in the same gene identified in this study (Table S3), the total number of STM mutants tested in olive plants would represent a genome coverage of c. 67%. Nevertheless, STM libraries composed of lower numbers of mutants have been successfully applied for the identification of virulence factors in several bacterial human pathogens (Saenz & Dehio, 2005; Wang & Beer, 2006).

Metabolic pathways required by P. savastanoi pv. savastanoi in olive plants

Plant-pathogenic P. syringae are nutritionally specialized to use a limited set of nutrients that are abundant in the plant apoplast, in leaf exudates, and on the leaf surfaces (Rico & Preston, 2008). Metabolic-related genes interrupted by the transposon in FAM strains (Fig. 2, Table 2) are, in general, not associated with virulence per se but provide information about the nutritional limitations of P. savastanoi inside the host and the metabolic pathways that are relevant for infection of olive plants by this pathogen. As expected for the screening method used in this study, over 50% of the FAM strains selected were metabolically deficient mutants; together, they had disruptions in the biosynthetic pathways of nine amino acids and three vitamins (Fig. 2, Table 2), indicating that the concentrations of these compounds are limiting for bacterial growth in olive plants but are not restrictive for growth in LB medium. Although it cannot be ruled out that mutants with disruptions in the remaining amino acid biosynthetic pathways either were not represented in our library or were discarded, these results could also indicate that some of these amino acids could be nonlimiting compounds in the apoplast. This would mean that mutants for their biosynthetic pathways would not be selected through our screening. Although no data related to the amino acid content of the olive apoplast are currently available, a recent study has revealed that P. syringae pv. tomato DC3000 uses amino acids that are abundant in the plant apoplast (Rico & Preston, 2008). Interestingly, and with the exception of glutamate, a key molecule in cellular metabolism, the amino acid biosynthetic pathways identified in this study were those corresponding to amino acids less abundant in the tomato (Solanum lycopersicum) apoplast. In addition, our screening identified FAM strains with a disrupted citrate (citN) or glutamate transporter (gltP) (Fig. 2, Table 2), indicating that these mutants are defective in the acquisition of these compounds, which are abundant in the tomato apoplast (Rico & Preston, 2008).

Bioinformatics analysis of the genome of P. savastanoi pv. savastanoi NCPPB 3335 revealed the existence of various genes encoding candidate enzymes involved in the complete degradation of aromatic compounds to intermediates of the Krebs cycle (Rodríguez-Palenzuela et al., 2010). Related to this battery of genes, the catJ mutant (Fig. S2) showed a drastic reduction in both fitness (Fig. 1) and virulence in planta (Figs 3, 5). Together, these results suggest that P. savastanoi pv. savastanoi could be adapted to use or detoxify aromatic compounds present at high concentrations in the tissues of woody plants. In fact, the production of phenolic compounds is greatly increased in olive tree knots upon P. savastanoi pv. savastanoi attack (Cayuela et al., 2006), which strongly suggests that bacterial resistance to phenols could be of paramount importance in the pathogenicity of this bacterium. However, all these hypotheses remain to be tested.

Fitness and virulence genes not previously identified in P. savastanoi pv. savastanoi

Our STM screening identified mutations affecting previously reported virulence and pathogenicity factors in P. savastanoi, such as the biosynthesis of IAA (iaaH gene, strain FAM-110) and the T3SS (hrpR gene, strain FAM-117), which validates the use of young micropropagated olive plants for the identification of virulence genes in this pathogen. In relation to the T3SS, the virulence phenotype, the cellular localization in olive tissue and the structure of the tumors observed for the hrpR mutant (Figs 3, 5, 6) were in agreement with our previous results (Pérez-Martínez et al., 2010).

Fitness and virulence genes of P. savastanoi pv. savastanoi identified in this study included those related to the Sec pathway, which is essential in other bacteria for peptide translocation across or into the bacterial membrane and for the functional assembly of the T3SS and T4SS (Chen et al., 2000; Kimbrough & Miller, 2002). Peptides translocated through the transmembrane SecYEG complex are then correctly folded by the periplasmic chaperone PpiD (Antonoaea et al., 2008). Transposon insertions into either secG or ppiD resulted in a significant fitness reduction in planta (Fig. 1), which in the case of the secG mutant was clearly reflected by a reduction of the GFP fluorescence observed in tumors induced by this strain (Fig. 5). In addition, the spatial distribution of pathogen cells and the internal structure of the tumors induced by this strain were similar to those visualized for the hrpR mutant (Fig. 6), indicating that the P. savastanoi pv. savastanoi Sec pathway is also essential for host cell lysis and for the spatial distribution of the pathogen inside olive tissues.

Type IV secretion systems are multiprotein complexes that mediate translocation of macromolecules (proteins, DNA or DNA–protein complexes) across the bacterial cell envelope into recipient cells (Álvarez-Martínez & Christie, 2009). T4SSs have been subgrouped into type IVA (vir genes) and type IVB (tra genes) (Christie et al., 2005). Although the role of the T4SSs in virulence is still not well understood in Pseudomonas, in other animal pathogenic bacteria, such as Bartonella (Schroder et al., 2011), Helicobacter (Backert & Clyne, 2011) and Brucella (de Jong & Tsolis, 2011), this system has an important role in pathogenesis. Our screening identified transposon insertions in both type IVA (virB4 gene) and type IVB (traY gene). These mutants showed a significant virulence reduction in adult olive plants, indicating that these systems are also relevant for P. savastanoi pathogenicity.

One of the most rapid plant defense reactions to pathogen attack is the so-called oxidative burst, which constitutes the production of ROS at the site of attempted invasion (Apel & Hirt, 2004). Bacteria have developed mechanisms to survive in these hostile conditions; however, the roles of these mechanisms in the virulence of the P. syringae complex have not been studied in detail. Our screening identified three P. savastanoi pv. savastanoi mutants with disruptions in rubB, pqiB, and msrA. Rapid metabolic reduction of ROS is activated in the electron transport chain, where the protein RubB acts (Hagelueken et al., 2007). However, genes induced by high levels of inline image radicals include pqiB, whose precise function in detoxification is still unknown. Finally, MsrA contributes to the virulence of Dickeya dadantii by repairing oxidized proteins via reduction of methionine sulfoxide to methionine (García-Olmedo et al., 2001; Ezraty et al., 2005). Tumors induced by rubB or pqiB mutants also exhibited a compact tissue structure (Fig. 6), indicating that host cell lysis mediated by P. savastanoi pv. savastanoi is impaired under oxidative stress conditions. Also related to oxidative stress, P. savastanoi pv. savastanoi mutants disrupted in homologs of arsB and cioA, encoding a pump membrane protein involved in arsenite resistance (Parvatiyar et al., 2005; Patel et al., 2007) and subunit A of a cyanide-insensitive terminal oxidase involved in adaptation of Pseudomonas aeruginosa to copper limitation (Frangipani et al., 2008), respectively, were selected in our screening, although virulence reduction in olive plants was more pronounced for the cioA mutant (Fig. 3).

The cell wall or exoskeleton is a critically important structural entity in bacteria. STM mutants disrupted in peptidoglycan (PG)-related genes (pomA, mltB, and ampG mutants) and the cls gene, encoding cardiolipin synthetase, were hypovirulent in woody olive plants (Fig. 3). Interestingly, and although a GFP-tagged ampG mutant induced tumors whose sizes were similar to those induced by the wild-type strain, GFP emission by these strains did not cover the entire knot surface, suggesting that intact AmpG is required for bacterial propagation inside olive tissue.

The second messenger 3′,5′-cyclic diguanylic acid (c-di-GMP), which is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase A, is a central regulator of the prokaryote biofilm lifestyle. Recent evidence also links this molecule to virulence (Cotter & Stibitz, 2007). Over 30 different genes encoding GGDEF-domain DGCs were recently identified in the genome of P. savastanoi pv. savastanoi NCPPB 3335 (Rodríguez-Palenzuela et al., 2010), among which AER-0004088 was interrupted by the transposon in strain FAM-030. Only a subtle reduction of virulence was observed for FAM-030 in woody olive plants (Fig. 3), perhaps as a result of functional redundancy of DGC proteins in P. savastanoi pv. savastanoi NCPPB 3335.

Although many bacterial virulence determinants are thought to be host-specific, several universal virulence mechanisms are shared by bacterial pathogens of plants, insects, and mammals (Rahme et al., 2000). Further supporting this notion, several of the P. savastanoi pv. savastanoi virulence genes identified in this study (Fig. 3, Table 3) were previously selected in other STM screenings involving human and animal bacterial pathogens (Table S5). Although some of the specific virulence-associated genes identified here have orthologs in other pathogenic bacteria, for example, arsB, cioA and spmAB, the exact roles of these genes in pathogenesis remain unclear.

In summary, this novel application of STM to a bacterial phytopathogen belonging to the P. syringae complex allowed us to identify metabolic pathways required for full fitness of P. savastanoi pv. savastanoi in olive plants and, additionally, revealed novel mechanisms involved in the virulence of this pathogen, such as the type IV secretion system, a battery of genes involved in tolerance and detoxification of ROS, a set of genes required for the biosynthesis of the cell wall, and a gene that regulates c-di-GMP levels. Other features identified in this study but not analyzed in detail include a chemotaxis-related protein, several DNA-binding proteins and seven hypothetical proteins. Further functional studies of the genes identified here may promote a better understanding of the pathogenic processes of bacterial phytopathogens and, more specifically, of the interaction of P. savastanoi pv. savastanoi with olive plants.

Acknowledgements

This work was supported by the Spanish MINECO grants AGL08-05311 and AGL11-30343-C02-01, co-financed by FEDER. IMM was supported by the Ramón Areces Foundation (Spain). We thank D. Holden (Imperial College, London, UK) for kindly providing the collection of tagged miniTn5Km2 transposons. A. de Vicente and A. J. Jiménez are thanked for help with the analysis of virulence in olive plants and for the preparation and microscopic visualization of olive knot sections, respectively. C. Beuzón and J. Ruiz-Albert are thanked for valuable advice regarding STM screening. We thank M. Duarte for excellent technical assistance. We are grateful to A. Barceló and I. Imbroda for the micropropagation of olive plants.

Ancillary

Advertisement