A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528
© Studholme et al; licensee BioMed Central Ltd. 2009
Received: 4 March 2009
Accepted: 24 August 2009
Published: 24 August 2009
Pseudomonas syringae is a widespread bacterial pathogen that causes disease on a broad range of economically important plant species. Pathogenicity of P. syringae strains is dependent on the type III secretion system, which secretes a suite of up to about thirty virulence 'effector' proteins into the host cytoplasm where they subvert the eukaryotic cell physiology and disrupt host defences. P. syringae pathovar tabaci naturally causes disease on wild tobacco, the model member of the Solanaceae, a family that includes many crop species as well as on soybean.
We used the 'next-generation' Illumina sequencing platform and the Velvet short-read assembly program to generate a 145X deep 6,077,921 nucleotide draft genome sequence for P. syringae pathovar tabaci strain 11528. From our draft assembly, we predicted 5,300 potential genes encoding proteins of at least 100 amino acids long, of which 303 (5.72%) had no significant sequence similarity to those encoded by the three previously fully sequenced P. syringae genomes. Of the core set of Hrp Outer Proteins that are conserved in three previously fully sequenced P. syringae strains, most were also conserved in strain 11528, including AvrE1, HopAH2, HopAJ2, HopAK1, HopAN1, HopI, HopJ1, HopX1, HrpK1 and HrpW1. However, the hrpZ1 gene is partially deleted and hopAF1 is completely absent in 11528. The draft genome of strain 11528 also encodes close homologues of HopO1, HopT1, HopAH1, HopR1, HopV1, HopAG1, HopAS1, HopAE1, HopAR1, HopF1, and HopW1 and a degenerate HopM1'. Using a functional screen, we confirmed that hopO1, hopT1, hopAH1, hopM1', hopAE1, hopAR1, and hopAI1' are part of the virulence-associated HrpL regulon, though the hopAI1' and hopM1' sequences were degenerate with premature stop codons. We also discovered two additional HrpL-regulated effector candidates and an HrpL-regulated distant homologue of avrPto1.
The draft genome sequence facilitates the continued development of P. syringae pathovar tabaci on wild tobacco as an attractive model system for studying bacterial disease on plants. The catalogue of effectors sheds further light on the evolution of pathogenicity and host-specificity as well as providing a set of molecular tools for the study of plant defence mechanisms. We also discovered several large genomic regions in Pta 11528 that do not share detectable nucleotide sequence similarity with previously sequenced Pseudomonas genomes. These regions may include horizontally acquired islands that possibly contribute to pathogenicity or epiphytic fitness of Pta 11528.
Pseudomonas syringae is a widespread bacterial pathogen that causes disease on a broad range of economically important plant species. The species P. syringae is sub-divided into about 50 pathovars, each exhibiting characteristic disease symptoms and distinct host-specificities. P. syringae pathovar tabaci (Pta) causes wild-fire disease in soybean and tobacco plants [1, 2], characterised by chlorotic halos surrounding necrotic spots on the leaves of infected plants. Formation of halos is dependent on the beta-lactam tabtoxin, which causes ammonia accumulation in the host cell by inhibition of glutamine synthetase . However, whether tabtoxin is an essential component of the disease process is unclear [4, 5].
Pathogenicity of P. syringae strains is dependent on the type III secretion system (T3SS). The T3SS secretes a suite of virulence 'effector' proteins into the host cytoplasm where they subvert the eukaryotic cell physiology and disrupt host defences [6–14]. Mutants lacking the T3SS do not secrete effectors, and as a consequence do not infect plants or induce disease symptoms. Thus, understanding effector action is central to understanding bacterial pathogenesis. A single P. syringae strain typically encodes about 30 different effectors . However, different P. syringae strains have different complements of effector genes. The emerging view is that of a core of common effectors encoded by most strains, augmented by a variable set. Individual effectors appear to act redundantly with each other and are individually dispensable with a small or no loss to pathogen virulence . Effectors are also thought to play an important role in determining host range. This is most clearly true when infections are restricted by host defences. Some plants have evolved specific mechanisms to recognise certain effectors; such recognition induces strong host defences which curtail infection. For example, expression of the T3SS effector HopQ1-1 from P. syringae pathovar tomato (Pto) DC3000 was sufficient to render Pta 11528 avirulent on Nicotiana benthamiana . The opposite situation, in which acquisition of a novel effector gene confers the ability to infect new host plants, has not been demonstrated and remains speculative. However, heterologous expression of the effector gene avrPtoB conferred a plasmid-cured strain of P. syringae pathovar phaseolicola (Pph) with increased virulence . We hope that further identification and characterisation of effector repertoires of particular strains will shine new light on their roles in determining host range. Finally, bacterial virulence is also likely to be influenced by other non-T3SS-dependent virulence factors such as toxins which are often co-regulated with the T3SS .
Complete genome sequences are available for strains representing three P. syringae pathovars: Pto, pathovar phaseolicola (Pph) and pathovar syringae (Psy) [18–20]. Comparisons of these have led to the identification of core effector gene sets and to explain some of the differences in host-specificity between pathovars. However, these three sequenced strains are representatives of three distinct phylogroups within the species P. syringae, and as such are phylogenetically quite distant [21, 22]. According to DNA-DNA hybridisation studies and ribotyping , P. syringae can be divided into 9 discrete genomospecies. Representative strains of Psy, Pph and Pto fell into genomospecies one, two and three respectively . Recently, a strain of pathovar oryzae (genomospecies four) was sequenced . A draft genome sequence was also published for Pto T1 , a strain closely related to Pto DC3000 but restricted to tomato hosts, whereas Pto DC3000 is able to cause disease on Arabidopsis. In the current study, we explore genetic differences at an intermediate phylogenetic resolution; that is, we compared the genome sequences of Pta 11528 to that of P. phaseolicola (Pph) 1448A, which resides within the same phylogroup but possesses a distinct host range and causes different disease symptoms.
Pto DC3000 was the first plant-pathogenic pseudomonad to have its genome sequenced, helping to establish the Arabidopsis-Pto system as the primary model for plant-microbe interactions. However, Arabidopsis is not a natural host of Pto, and it is important to develop alternative systems given the genetic variability of P. syringae strains, particularly in regard to effectors. We work on the interaction between Pta and the wild tobacco plant N. benthamiana, which offers certain advantages over Arabidopsis. Firstly, N. benthamiana is an important model for the Solanaceae, which includes many important crop species. The Pta-N. benthamiana interaction is a natural pathosystem. Lastly, N. benthamiana is an important model plant that is more amenable to biochemistry-based approaches and facile manipulation of gene expression such as virus-induced gene silencing (VIGS). Thus N. benthamiana provides experimental options for understanding plant-bacterial interactions. Strains of Pta can cause disease on N. benthamiana, but relatively few genetic sequence data are available for this pathovar.
In this study we generated a draft complete genome sequence of Pta 11528 and used a functional screen for HrpL-dependent genes to infer its repertoire of T3SS effectors and associated Hrp Outer Proteins (Hops), which differs significantly from that of its closest relative whose complete genome has previously been published (Pph 1448A). Pta 11528 does not encode functional homologues of HopAF1 or HrpZ1. This was surprising since HopAF1 was conserved in the three previously sequenced pathovars [18–20]. HrpZ1 is conserved in most strains of P. syringae that have been investigated, albeit with differences in amino acid sequence . However, Pta strain 6605 and several other isolates from Japan, were previously shown to carry a major deletion leading to truncated HrpZ protein product . Pta 11528 encodes several novel potential T3SS effectors for which no close orthologues have been reported. We also discovered several large genomic regions in Pta 11528 that do not share detectable nucleotide sequence similarity with previously sequenced Pseudomonas genomes. These regions may be horizontally acquired islands that possibly contribute to pathogenicity or epiphytic fitness of Pta 11528.
Results and discussion
Sequencing and assembly of the Pta 11528 genome
The Illumina sequencing platform provides a cost-effective and rapid means to generate nucleotide sequence data [27–29]. Although this method generates very short sequence reads, several recent studies have demonstrated that it is possible to assemble these short reads into good quality draft genome sequences [30–41].
RefSeq accession number
G+C content (%)
Nucleic acid sequence identity to P. syringae pv tabaci 11528 draft assembly (%)
P. syringae pv. tabaci 11528 draft genome assembly
P. syringae pv. phaseolicola 1448A, chromosome
P. syringae pv. phaseolicola 1448A large plasmid
P. syringae pv. tomato str. DC3000 plasmid pDC3000B
P. syringa e pv. syringae B728a, chromosome
P. syringae pv. tomato str. DC3000 plasmid pDC3000A
P. syringae pv. phaseolicola 1448A small plasmid
P. syringae pv. tomato str. DC3000
We aligned the 71 Pta supercontigs against published complete Pseudomonas genome sequences using MUMMER . The Pta 11528 genome was most similar to that of Pph 1448A, with 97.02% nucleotide sequence identity over the alignable portions. The next most similar genome was that of Pto DC3000, with less than 90% identity (Table 1). This pattern of sequence similarity is consistent with phylogenetic studies that placed strains of Pta in the same phylogroup as Pph and revealed a relatively distant relationship to Pto [21, 22].
Comparison of the protein complement of Pta 11528 versus Pph 1448A and other pseudomonads
Using the FgenesB annotation pipeline http://www.softberry.com, we identified 6,057 potential protein-coding genes, of which 5,300 were predicted to encode proteins of at least 100 amino acids long. Of 5,300 predicted Pta 11528 proteins, 575 (10.8%) had no detectable homology with Pph 1448A proteins (based on our criterion of an E-value less than 1e-10 using BLASTP). Of these 575 sequences, 303 had no detectable homologues in Psy B728a nor Pto DC3000. These 303 Pta-specific sequences had a median length of 198 amino acids whereas the median length of the 5,300 sequences was 216 amino acids. Automated gene prediction is not infallible and inevitably a subset of the predictions will be incorrect. The reliability of gene predictions is poorer for short sequences than for longer ones. This slight enrichment for very short sequences among the Pta-specific gene predictions might be explained by the inclusion of some open reading frames that are not functional genes among those 303. However, many of the predicted proteins showed significant similarity to other proteins in the NCBI NR databases (See Additional file 1: Table S1), confirming that these are likely to be genuine conserved genes.
Conservation of the T3SS apparatus and T3SS-dependent effectors
Homologues of known hop genes in Pta 11528. Homologues were detected by searching the Pta 11528 FgenesB-predicted protein sequences against HopDB http://www.pseudomonas-syringae.org using BLASTP
Gene in Pta 11528 genome (location)
Hrp-box HMM score (bioinformatic evidence)
HrpL-dependent (functional screen)
Homologue in Pph 1448A
(PSPPH is a truncated HopW1 homologue)
Besides the core conserved hop genes, the Pta 11528 genome assembly contains full-length orthologues of hopR1, hopAS1, hopAE1 and hopV1, which are also found in Pph 1448A but are absent from Psy B728a and/or Pto DC3000.
The hrpZ1 gene encodes a harpin, which is not classified as a type III effector because it is not injected directly into host cells. Harpins are characteristically acidic, heat-stable and enriched for glycine, lack cysteine residues  and can induce defences in both host and non-host plants [45, 46]. HrpZ1 forms pores in the host membrane  suggesting a role in translocation of effectors across the host membrane. It also shows sequence-specific protein binding activity . HrpZ1 can induce defences in both host and non-host plants and tobacco has been extensively used as the non-host plant species [45, 46]. The inactivation of hrpZ1 in Pta 11528 and other strains of Pta  may be an adaptive strategy and have been an important process in the stepwise progression towards compatibility, allowing Pta 11528 to avoid detection by the tobacco host plant. This is reminiscent of the "black holes" and other processes that inactivate genes whose expressed products are detrimental to a pathogenic lifestyle [49, 50]. One excellent example is the inactivation of cadA in genomes of Shigella species as compared to the genome of their closely related but non-pathogenic Escherichia coli strain [51, 52].
Pta 11528 contains highly conserved homologues of hopAB2, hopW, hopO1-1, hopT1-1, hopAG1, hopAH1, hopF1 and hopAR1, which are absent in Pph 1448A. Although absent from the Pph 1448A genome, hopAR1 and hopF1 have been identified in other strains of Pph [53–57]. In Pph 1302A, hopAR1 is located on the pathogenicity island PPH GI-1, though its genomic location varies between strains [56, 57]. PPH GI-1 is absent from the Pph 1448A genome . The Pta 11528 genome (supercontig 1087) possesses a region of similarity to PTPH GI-1, but which contains a substantial number of insertions and deletions (Additional file 2: Figure S1). The Pta 11528 hopAR1 homologue (C1E_2036) is not located in the PPH GI-1 region; it falls on supercontig 672 about two kilobases upstream of a gene encoding a protein (C1E_2039) sharing 43% amino acid identity with Pto DC3000 avrPto1. In contrast to AvrPto1 from Pto DC3000, the AvrPto1 homologue (C1E_2039) from Pta 11528 is not recognised by the plant Pto/Prf system (S. Gimenez Ibanez and J. Rathjen, manuscript in preparation).
The homologues of hopAG1, hopAH1 and the degenerate hopAI1' are found within a region of the Pta 11528 genome that shares synteny with the chromosome of Psy B728a. This region is also conserved in Pto DC3000A, albeit with several deletions and insertions, suggesting that these effector genes are ancestral to the divergence of the pathovars and have been lost in Pph 1448A rather than having been laterally transferred laterally between Pta 11528 and Psy B728a. In Pto DC3000, hopAG1 (PSPTO_0901) has been disrupted by an insertion sequence (IS) element. This is consistent with a model of lineage-specific loss of certain ancestral effectors.
In plasmid pMA4326B from P. syringae pathovar maculicola (Pma), the hopW1 effector gene is immediately adjacent to a three-gene cassette comprising a resolvase, an integrase and exeA. This cassette is also found in plasmids and chromosomes of several human-pathogenic Gram-negative bacteria . We found a homologue of this cassette along with a hopW1 homologue on supercontig 955 of the Pta 11528 genome assembly. Stavrinides and Guttman  proposed that the boundaries of the cassette lay upstream of the resolvase and upstream of hopW1. The presence of this four-gene unit in a completely different location in Pta 11528 is indeed consistent with the hypothesis that it represents a discrete mobile unit.
The regions of the Pph 1448A large plasmid that are apparently conserved in Pta 11528 include genes encoding the conjugal transfer system, suggesting the presence of one or more plasmids in this strain. We found an open reading frame (C1E_3950, located on supercontig 955 coordinates 59126-60394) encoding a protein with about 97% sequence identity to the RepA proteins characteristically encoded on pT23A-family plasmids (e.g. AAW01447; reviewed in ), suggesting that this 236 kilobase supercontig might represent a plasmid.
A functional screen for HrpL-regulated genes
Pta 11528 genes confirmed by the functional screen to be under the transcriptional control of HrpL
Gene in Pta 11528 gnome
Hrp-box HMM score (bioinformatic evidence)
Orthologue in Pph 1448A
Major facilitator superfamily permease
Putative M20 peptidase
Other differences in predicted proteomes of P. syringae strains
Host range and pathogenicity are likely to be further influenced by genes other than those associated with type III secretion. Virulence determinants in P. syringae include toxins as well as epiphytic fitness; that is, the ability to acquire nutrients and survive on the leaf surface . Epiphytic fitness depends on quorum-sensing , chemotaxis , osmo-protection, extracellular polysaccharides, glycosylation of extracellular structures  iron uptake  and the ability to form biofilms. Cell-wall-degrading hydrolytic enzymes play a role in virulence in at least some plant-pathogenic pseudomonads ). Secretion systems (including type I, type II, type IV, type V, type VI and twin arginine transporter) may also contribute to both virulence and epiphytic fitness , whilst multidrug efflux pumps may confer resistance to plant-derived antimicrobials .
To identify differences between Pta 11528 and the previously sequenced Pph 1448A, Psy B728a and Pto DC3000 with respect to their repertoires of virulence factors, we performed BLASTP searches between the predicted proteomes. We found no significant differences in the repertoires of secretion systems between the proteomes. However, we found that Pta 11528 lacks homologues of several Pph 1448A polysaccharide modifying enzymes (glycosyl transferase PSPPH_0951, polysaccharide lyase PSPPH_1510, glycosyl transferase PSPPH_3642). Conversely, Pta 11528 encodes two glycosyl transferases (C1E_0355 and C1E_0361) and a thermostable glycosylase (C1E_4802) that do not have homologues in any of the three fully sequenced P. syringae genomes. This may imply differences in the extracellular polysaccharide profiles. In contrast to Pph 1448A, Pta 11528 lacks homologues of RhsA insecticidal toxins (PSPPH_4042 and PSPPH_4043). However, a tabtoxin biosynthesis gene cluster is found in the Pta 11528 genome and shows a high degree of conservation with the previously sequenced Pta BR2 tabtoxin biosynthesis cluster .
Proteins encoded by the draft Pta 11528 genome that have no detectable homologues on three previously fully sequenced P. syringae genomes.
Length (amino acids)
Predicted function (FgenesB automated annotation)
Glycosyltransferase involved in cell wall biogenesis
Phosphoadenosine phosphosulfate reductase
Serine/threonine protein kinase
TetR family ranscriptional regulator. 49% amino acid sequence identity to G. bethesdensis GbCGDNIH1_1777 .
Hydrolases or acyltransferases (alpha/beta hydrolase superfamily)
Tabtoxin biosynthesis enzyme, TblA
Histone acetyltransferase HPA2
Amine oxidase, flavin-containing
Rhs family protein
Xenobiotic response element family of transcriptional regulator. 37% amino acid sequence identity to Xylella fastidiosa PD0954 .
Histone acetyltransferase HPA2
Similar to Mucin-1 precursor (MUC-1)
Site-specific recombinases, DNA invertase Pin homologs
LacI family transcriptional regulator. 57% amino acid sequence identity to Rhizobium leguminosarum plasmid-encoded pRL1201 .
Tfp pilus assembly protein, major pilin PilA. 42% amino acid sequence identity to P. aeruginosa UniProt:P17838 .
Histone acetyltransferase HPA2
NTPase (NACHT family)
Permeases of the major facilitator superfamily
Short-chain dehydrogenase/reductase SDR
ASPIC/UnbV domain-containing protein
Xenobiotic response element family of transcriptional regulator. 38% amino acid sequence identity to P. aeruginosa PACL_0260 .
Cro/CI family transcriptional regulator. 36% amino acid sequence identity to Pto DC3000 PSPTO_2855 .
Methyl-accepting chemotaxis sensory transducer (C terminus)
Methyl-accepting chemotaxis sensory transducer (N terminus)
Type II secretory pathway, component PulK
DNA-binding HTH domain-containing
Outer membrane efflux protein
Plasmid stability protein
Phage integrase family protein
Superfamily I DNA or RNA helicase
ATP-dependent DNA helicase, UvrD/Rep family
Thermostable 8-oxoguanine DNA glycosylase
PP-loop superfamily ATPase
Sugar kinase, ribokinase
ATP-dependent DNA helicase, UvrD/Rep family
ATP-dependent DNA helicase, UvrD/Rep family
Histone acetyltransferase HPA2
Phage collar protein
S-layer domain protein
Major facilitator superfamily (MFS) permease
IclR-like transcriptional regulator. 62% amino acid sequence identity to Acinetobacter baumanii ACICU_01897 .
TetR family transcriptional regulator. 56% amino acid sequence identity to Ralstonia solanacearum RSc0820 .
Pta protein C1E_6026 has a significant match to the sulphotransferase domain (Pfam:PF00685). Examples of this protein domain have not been found in other pseudomonads except for P. fluorescens PfO-1. Sulphotransferase proteins include flavonyl 3-sulphotransferase, aryl sulphotransferase, alcohol sulphotransferase, estrogen sulphotransferase and phenol-sulphating phenol sulphotransferase. These enzymes are responsible for the transfer of sulphate groups to specific compounds. The sulphotransferase gene (C1E_6026, 82% amino acid identity to P. fluorescens Pfl01_0157) overlaps a two kilobase Pta 11528-specific genomic island that also encodes a phage tail collar-protein encoding gene (C1E_5461, 61% amino acid identity to P. fluorescens Pfl01_0155) and an acetyltransferase (C1E_5459, 76% amino acid identity to P. fluorescens Pfl01_0148). We speculate that this region has been horizontally acquired in the Pta 11528 lineage via a bacteriophage.
An 80 kilobase region of Pta 11528 supercontig 684 contains two open reading frames (ORFs) (C1E_2584 and C1E_2585) whose respective predicted protein products show 48 and 55% amino acid identity to the C- and N-termini of a P. putida methyl-accepting chemotaxis protein (MCP) (PP_2643) and little similarity to any P. syringae protein. Since the N- and C-termini are divided into separate reading frames, this probably represents a degenerate pseudogene. Immediately downstream of these ORFs is a gene (C1E_2583) that specifies a MCP showing greatest sequence identity (70%) to PP_2643 from P. putida, whilst sharing only 65% identity to its closest homologue in P. syringae (PSPPH_4743). This region also encodes another MCP (C1E_2587) that shares only 50% amino acid identity with any previously sequenced P. syringae homologue. It remains to be tested whether these MCPs play a role in pathogenesis and/or epiphytic fitness.
Transcriptional regulators are not normally considered to be virulence factors. However, expression of virulence factors may be coordinated by and dependent on regulators. Moreover, heterologous expression of the RscS regulator was recently shown to be sufficient to transform a fish symbiont into a squid symbiont . Pta 11528 encodes several predicted transcriptional regulators that are not found in Pto DC3000, Psy B728a and Pph 1448A. These include two predicted TetR-like proteins (C1E_0901 and C1E_6027), two predicted xenobiotic response element proteins (C1E_2056 and C1E_2563), a LacI-like protein (C1E_2286), a Cro/CI family protein (C1E_2570) and an IclR family protein (C1E_5715).
Pta 11528 encodes a novel pilin (C1E_2329) not found in previously sequenced P. syringae strains but sharing significant sequence similarity with a type IV pilin from P. aeruginosa . Pilin is the major protein component of the type IV pili, which have functions in forming micro-colonies and biofilms, host-cell adhesion, signalling, phage-attachment, DNA uptake and surface motility, and have been implicated as virulence factors in animal-pathogenic bacteria . The precise function of the C1E_2329 pilin is unknown but it may be involved in epiphytic fitness or plant-pathogenesis or could even be involved in an interaction with an insect vector.
Pta-specific genomic islands
We identified 102 genomic regions of at least one kilobase in length which gave no BLASTN matches against previously sequenced Pseudomonas genomes (Additional file 3: Table S2). Ten of the Pta 11528-specific regions are longer than 10 kilobases, the longest being 37.7, 21.8, 18.7, 17.9 and 16.6 kilobases. The 16.6 kilobase region corresponds to the tabtoxin biosynthesis gene cluster . These regions will be good candidates for further study of the genetic basis for association of Pta with the tobacco host. For example, several of the islands encode MFS transporters and other efflux proteins that might be involved in protection from plant-derived antimicrobials (Additional file 3: Table S2).
We have generated a draft complete genome sequence for the Pta 11528 a pathogen that naturally causes disease in wild tobacco, an important model system for studying plant disease and immunity. From this sequence, combined with a functional screen, we were able to deduce the pathogen's repertoire of T3SS-associated Hop proteins. This has revealed some important differences between Pta and other pathovars with respect to the arsenal of T3SS effectors at their disposal for use against the host plant. We also revealed more than a hundred Pta-specific genomic regions that are not conserved in any other sequenced P. syringae, providing many potential leads for the further study of the Pta-tobacco disease system.
The previously published sequences of P. syringae pathovar phaseolicola 1448A , P. syringae pathovar syringae B728a , P. syringae pathovar tomato DC3000  were downloaded from the NCBI FTP site ftp://ftp.ncbi.nih.gov/genomes/Bacteria/Pseudomonas_syringae_pv_B728a. The NCBI non-redundant (NR) Proteins database was downloaded from the NCBI FTP site ftp://ftp.ncbi.nih.gov/blast/db/ on 10th December 2008.
De novo sequence assembly and annotation
Solexa sequence data were assembled using Velvet 0.7.18 . We used Softberry's FgenesB pipeline http://www.softberry.com to predict genes encoding rRNAs, tDNAs and proteins. Annotation of protein-coding genes by FgenesB was based on the NCBI NR Proteins database.
Prediction of HrpL-binding sites (Hrp boxes)
We built a profile hidden Markov model (HMM) based on a multiple sequence alignment of 26 known Hrp boxes from Pto DC3000 using hmmb from the HMMER 1.8.5 package http://hmmer.janelia.org. DNA sequence was scanned against this profile-HMM using hmmls from HMMER 1.8.5 with a bit-score cut-off of 12.0.
Functional screen for candidate type III effectors
Library preparation and the Flow cytometric-based screen for HrpL-induced genes of Pta 11528 were done according to .
Visualisation of data
Library preparation for Illumina sequencing
DNA was prepared from bacteria grown in L-medium using the Puregene Genomic DNA Purification Kit (Gentra Systems, Inc., Minneapolis, USA) according to manufacturer's instructions. A library for Illumina Paired-End sequencing was prepared from 5 mg DNA using a Paired-End DNA Sample Prep Kit (Pe-102-1001, Illumina, Inc., Cambridge, UK). DNA was fragmented by nebulisation for 6 min at a pressure of 32 psi. For end-repair and phosphorylation, sheared DNA was purified using QIAquick Nucleotide Removal Kit (Quiagen, Crawley, UK). The end repaired DNA was A-tailed and ada Pto rs were ligated according to manufacturer's instructions.
Size fractionation and purification of ligation products was performed using a 5% polyacrylamide gel run in TBE at 180V for 120 min. Gel slices were cut containing DNA in the 500 to 10 bp range. DNA was than extracted using 0.3 M sodium acetate and 2 mM EDTA [pH 8.0] followed by ethanol precipitation. Using 18 PCR cycles with primer PE1.0 and PE2.0 supplied by Illumina, 5' ada Pto r extension and enrichment of the library was performed. The library was finally purified using a QIAquick PCR Purification Kit and adjusted to a concentration of 10 nM in 0.1% Tween. The stock was kept at -20°C until used.
The flow cell was prepared according to manufacturer's instructions using a Paired-End Cluster Generation Kit (Pe-103-1001) and a Cluster Station. Sequencing reactions were performed on a 1G Genome Analyzer equipped with a Paired-End Module (Illumina, Inc., Cambridge, UK). 5 pM of the library were used to achieve ~20,000 to 25,000 clusters per tile. Capillary sequencing of avrE, HrpW1 and other individual genes was done on an ABI 3730. PCR products were directly sequenced after treatment with ExoI and SAP. Primer sequences are available upon request from JHC.
Verification of Illumina sequence data
Three of the core hop genes in Pta 11528 appeared to be degenerate, based on the de novo assembly of short Illumina sequence reads. The avrE1 gene appeared to have a 20-nucleotide deletion, hrpZ1 a 325-nucleotide deletion, whilst hrpW1 appeared to have three insertions of 22, 6 and 12 nucleotides. Currently, the reliability of de novo sequence assembly from short Illumina reads has not been fully characterised. In particular, repetitive and low-complexity sequence might generate artefacts in assembled supercontigs. Therefore, we checked these putative insertions and deletions by aligning the Illumina sequence reads against the relevant regions of both the Pph 1448A reference genome sequence and our Pta 11528 assembly. As an additional control, we also performed Velvet assembies on previously published Illumina short-read data from Psy B728a . We found that the B728a avreE1, hrpZ1, hrpW1 and hopAF1 were assembled intact [Additional file 4: Figure S2], indicating that there is nothing inherently 'un-assemble-able' about these gene sequences. Sequence alignment is much more robust than de novo assembly and is not subject to assembly artefacts. The alignments supported the presence of a large deletion in hrpZ1. However, the alignments were not consistent with the assembly for avrE1 and hrpW1. Therefore, we amplified the Pta 11528 avrE1 and hrpW1 genes by PCR and verified their sequences by capillary sequencing [Additional file 5: Table S3]. This confirmed that the apparent deletion in avrE1 was an artefact of the de novo assembly and that the avrE1 sequence encodes a full-length protein product. Furthermore, transient expression of avrE1 in N. benthamiana induces cell death (S. Gimenez Ibanez and J. Rathjen, unpublished). Capillary sequencing also confirmed that the de novo assembly of hrpW1 was incorrect and that Pta 11528 encodes a full-length HrpW1 protein, albeit with repetitive sequence insertions of 69 and 12 nucleotides relative to the Pph 1448A sequence.
The absence of hopAF1 from Pta 11528 is supported not only by the de novo assembly, but also by the absence of aligned (unassembled) reads. As an additional control for the degeneracy of hopAF1 and hrpZ1, we performed the same bioinformatics and sequencing protocols to Psy B728a  and recovered hopAF1 and hrpZ1 intact in the de novo assembly assembly (Additional file 4: Figure S1).
We used GenomeMatcher  for generating and visualising whole-genome alignments. For aligning short Illumina sequence reads against a reference genome, we used MAQ  and for other sequence alignments and searches we used BLAST . We used previously published complete genomes as reference sequences for comparative analyses [81–85].
List of abbreviations
conserved effector locus
exchangeable effector locus
hidden Markov model
methyl-accepting chemotaxis protein
polymerase chain reaction
- Pma Pseudomonas syringae :
- Pph Pseudomonas syringae :
- PPH GI-1 Pph:
genomic island 1
- Pta Pseudomonas syringae :
- Psy Pseudomonas syringae :
- Pto Pseudomonas syringae :
virus-induced gene silencing
We would like to thank Dr. Larry Arnold and Theresa Law for their assistance in the functional screen for candidate type III effector genes of Pta 11528, Jodie Pike for performing the Illumina sequencing, Eric Kemen for advice and help with Illumina sequencing and Kee Hoon Sohn for handling and maintaining the bacterial strains. We thank Ashley Chu and Caitlin Thireault for assistance with capillary sequencing and Dr. Jonathan Urbach for guidance on searching for hrp-boxes. We thank Robert Jackson, Gail Preston and two anonymous reviewers for very helpful comments on the manuscript. Financial support by the Gatsby Charitable Foundation is gratefully acknowledged.
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