- Research article
- Open Access
Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants
- Melissa G. Castillo-Lizardo†1, 2,
- Isabel M. Aragón†1,
- Vivian Carvajal3,
- Isabel M. Matas1, 4,
- María Luisa Pérez-Bueno3,
- María-Trinidad Gallegos3,
- Matilde Barón3 and
- Cayo Ramos1Email authorView ORCID ID profile
© Castillo-Lizardo et al. 2015
- Received: 7 May 2015
- Accepted: 6 August 2015
- Published: 19 August 2015
The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.
A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.
Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.
- Tomato Plant
- matE Gene
- Tomato Leave
- Lysogeny Broth
- Competition Index
The ability to produce the auxin phytohormone indole-3-acetic acid (IAA) is widespread among soil and plant-associated bacteria. As more bacterial species have been analyzed, the roles of auxins in bacterial interactions with plants appear to be diverse, varying from pathogenesis to phytostimulation . The best-characterized IAA biosynthetic pathway in phytopathogenic bacteria is the indole-3-acetamide pathway. In this pathway, the genetic determinants involved in the conversion of L-tryptophan (Trp) to IAA are Trp monooxygenase (encoded by the iaaM gene), which converts Trp to indole-3-acetamide (IAM), and IAM hydrolase (encoded by the iaaH gene), which catalyzes the transformation of IAM to IAA. These two genes have been cloned and characterized for phytopathogenic bacteria such as Agrobacterium spp. and Pseudomonas savastanoi [2, 3]. P. savastanoi pv. nerii, the causal agent of oleander (Nerium oleander) knot disease, also converts IAA to indole-acetyl-ε-L-lysine (IAA-Lys), a less biologically active compound as estimated in a coleoptile elongation assay [4–6]. This conversion involves the enzyme IAA-Lys synthase, encoded by the iaaL gene [7–9]. Although most P. syringae pathovars produce detectable amounts of IAA in the presence of Trp , IAA synthesis usually involves different genes than iaaM and iaaH [10–12]. In contrast, iaaL is widespread in P. syringae pathovars and is often found in plasmids [7, 10, 13]. Inactivation of the iaaL gene by transposon mutagenesis in P. savastanoi pv. nerii resulted in the accumulation of IAA in the culture medium; however, this mutant did not cause typical knot symptoms, probably due to its inability to multiply within host tissues .
P. syringae pv. tomato (Pto) DC3000, which causes bacterial specks on tomatoes and can infect the model plants Arabidopsis thaliana and Nicotiana benthamiana, is an important model for the study of plant-pathogen interactions [14–16]. The ability of DC3000 to infect host plants depends on numerous genes expressed by the HrpL alternative sigma factor. The Pto DC3000 HrpL regulon includes genes involved in the hypersensitive response and others encoding the pathogenicity (Hrp) type III secretion system (T3SS) machinery, a repertoire of type III effector proteins [15, 17–19], as well as genes encoding factors unrelated to the T3SS, such as iaaL (PSPTO_0371) and the genes for coronatine synthesis [17–21]. A recent analysis of the role of the Pto DC3000 iaaL gene in the infection of N. benthamiana plants concluded that an iaaL deletion mutant did not exhibit phenotypic differences in terms of in planta growth, virulence, or hypersensitive response (HR) compared to the wild type strain . Conversely, although the genome of Pto DC3000 encodes two coding sequences (CDS) that are likely involved in auxin production [14, 22], the roles of these genes in IAA biosynthesis have not been demonstrated yet . Thus, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.
A reporter transposon screen for HrpL-activated genes in Pto DC3000 identified several genes, including iaaL, that were linked to novel variations of the canonical sequence for Hrp boxes  found in HrpL-dependent promoters . Further analysis of the transposon mutants revealed that the upstream promoter-proximal ORF encoded a putative MATE (multidrug and toxic compound extrusion) family transporter gene (PSPTO_0370) . Recently, overexpression of HrpL in Pto DC3000 was shown to induce the expression of this putative MATE transporter gene, suggesting that it is part of the Pto DC3000 HrpL regulon . Efflux pumps associated with multidrug resistance (MDR) contribute to bacterial survival in plant tissues via the removal of antimicrobial secondary metabolites, such as flavonoids, isoprenoids, and alkaloids, which are present in healthy plant tissues or synthesized de novo in response to pathogen attack [24, 25]. MDR efflux pumps have been shown to contribute to the colonization of host plants by bacterial phytopathogens, including P. syringae strains [26, 27]; however, little evidence has been found for the contribution of MATE transporters to the virulence of bacterial phytopathogens .
The aim of this study was to analyze the expression of the iaaL gene and of the putative MATE family transporter gene, hereafter called matE, located just upstream of iaaL, both in wild-type Pto DC3000 and in a knock-out ΔhrpL mutant generated by gene replacement. The roles of these genes in the virulence of Pto DC3000 during infection of tomato plants was analyzed, not only in terms of the pathogen-induced symptomatology observed in tomato leaves but also prior to the development of symptoms. To analyze the pre-symptomatic responses of tomato plants to bacterial infection, we used multicolor fluorescence imaging (MCFI), a technique that allows visualization of the activation of plant secondary metabolism in response to pathogen infection.
Bacterial strains, plasmids, and culture medium
Strains and plasmids used in this work
P. syringae pv. tomato
E. coli DH5α
F-, ϕ80dlacZ M15, (lacZYA-argF) U169, deoR, recA1, endA,
hsdR17 (rk − mk −), phoA, supE44, thi-1, gyrA96, relA1.
pGEM-T Easy Vector
Ampr, ori f1, lacZ
Ampr, ori f1, lacZ
Contains Kmr from pKD4 (Ampr Kmr)
Expression Vector, Tcr
Expression Vector, Tcr
pGEM-T derivative, contains approx. 0.5 kb on each side of the iaaL gene (Ampr)
pGEM-T derivative, contains approx. 0.5 kb on each side of the hrpL gene (Ampr)
pGEM-T derivative, contains approx. 0.5 kb on each side of the mat gene (Ampr)
pGEM-T derivative, contains approx. 0.5 kb on each side of the iaaL gene interrupted by the kanamycin resistance gene nptII (Amp r , Km r )
pGEM-T derivative, contains approx. 0.5 kb on each side of the hrpL gene interrupted by the kanamycin resistance gene nptII (Ampr, Kmr)
pGEM-T derivative, contains approx. 0.5 kb on each side of the matE gene interrupted by the kanamycin resistance gene nptII (Ampr, Kmr)
pMP220 derivative, contains the lacZ gene expressed from the PmatE promoter (Tcr)
pMP220 derivative, contains the lacZ gene expressed from the PiaaL promoter (Tcr)
pJB3 derivative, contains the matE gene with RBS
pJB3 derivative, contains the iaaL gene with RBS
Preparation of total RNA
RNA was extracted from cultures grown on M9 minimal media supplemented with 5 mM mannitol [30, 31] and ferric citrate at 0.0006 %. The cells were pelleted at exponential phase, i.e., when they reached an optical density of 0.5 at 660 nm (OD660nm). The pellets were processed for RNA isolation using TriPure Isolation Reagent (Roche Applied Science; Mannheim, Germany) according to the manufacturer’s instructions with the following exceptions: the TRIPure was preheated to 65 °C, the lysis step was carried out at 65 °C and BCP (1-bromo-3-chloropropane) (Molecular Research Center; Cincinnati, OH, U.S.A.) was used instead of chloroform. The RNA concentration was determined spectrophotometrically, and its integrity was assessed by agarose gel electrophoresis. Total RNA was treated with a TURBO DNA-free™-Kit (Applied Biosystems; California, U.S.A.) as detailed by the manufacturer. Subsequently, the samples were tested for genomic contamination by PCR.
Primer extension analysis
DNA-free RNA, prepared as described above, was heat-treated at 80 °C for 5 min. Subsequently, 30 μg of RNA was annealed at 75 °C for 10 min, and then the temperature was allowed to slowly decrease to 60 °C within 30 min. Annealing was performed in a buffer containing 100 mM NaCl and 50 mM Tris-Cl at pH 7.5. The specific primers (Additional file 4: Table S1) and the φX174 DNA/HinfI dephosphorylated marker were labeled using [γ32-P]ATP (3000 Ci/mmol) (PerkinElmer; Boston, USA) and T4 polynucleotide kinase, according to the manufacturer’s instructions for the Primer Extension System-AMV Reverse Transcriptase (Promega; Madison, USA). Runoff reverse transcription reactions were performed for 1 h at 60 °C using 15 units of ThermoScript™ RNase H- (Invitrogen; California, USA) in its provided buffer (complemented with 1 mM of each dNTP and 5 mM DTT). Reactions were stopped by the addition of one volume of loading dye (Promega; Madison, USA) and were analyzed on 6 % polyacrylamide sequencing gels containing 8 M urea. The results were visualized either using X-ray films or exposure for 24 h to Imaging Plates (IP BAS-MP 2040S), which were analyzed with a Fujifilm-BAS 1500 (Fuji; Tokyo, Japan).
Transcription initiation mapping by 5′cRACE
The transcription start site of matE was determined using the 5′cRACE method [32–34]. cDNA synthesis was performed using total DNA-free RNA, obtained as described above. One microgram of this RNA was used as a template to synthesize first-strand cDNA using a SMART™ RACE cDNA Amplification synthesis kit (Clontech; California, USA), and a gene-specific primer was designed to anneal within the coding region of the gene (Additional file 4: Table S1). The reactions proceeded for 90 min at 42 °C. Then, they were diluted 10-fold in water, and 1 μl of these dilutions were used as templates in 20-μl PCR reactions. The PCR cycles were as follows: 5 cycles for 30 s at 94 °C; 3 min at 72 °C; 5 cycles for 30 s at 94 °C; 30 s at 70 °C; 3 min at 72 °C; 25 cycles for 30 s at 94 °C; 30 s at 68 °C; 3 min at 72 °C. The amplification products were cloned into the pGEM®-T Easy Vector (Promega; Madison, USA) and sequenced.
DNA-free RNA was reverse transcribed using random hexamers included in the iScript™ cDNA synthesis kit (BioRad; California, USA). Afterwards, PCR reactions were performed with GoTaq® polymerase (Promega; Madison, USA) using 100 ng of cDNA as a template and specific primers detailed in Additional file 4: Table S1. Products were analyzed by agarose gel electrophoresis.
β-galactosidase activity assays
β-galactosidase activity was measured from DNA fragments cloned into the expression vector pMP220  using the methods developed  and modified previously . In summary, cells carrying the plasmids were cultured in LB media supplemented with 10 μg/ml tetracycline. Grown cultures were harvested by centrifugation and washed three times with 0.9 % NaCl. Finally, cultures were adjusted to an OD600nm of 0.5 and cultured in minimal media as described above [30, 31].
To measure β-galactosidase activity, 200 μl of each culture were lysed with 100 μl of chloroform and 50 μl of 0.1 % SDS and were then mixed with 800 μl of Z buffer (40 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, pH 7.0 and 50 mM β-mercaptoethanol, added just before use). After mixing, samples were incubated at room temperature for 5 min. Then, 200 μl of Z buffer containing 4 mg/ml o-nitrophenyl-β-D-galactopyranoside (ONPG) were added. The samples were then incubated at room temperature for 30–60 min until they turned yellow. Afterwards, reactions were stopped with 500 μl of 1 M Na2CO3, and the absorbance was measured at 420 and 550 nm. In addition, absorbance at 600 nm was measured in the initial culture. The enzymatic activity (in Miller units) was determined with the equation [(A420 − 1.7*A550)*1000]/(V*A600*t) , where AXXX is the absorbance at 420, 550 and 600 nm, V is the volume of the culture in ml and t is the time of the reaction in minutes.
Generation of knockout mutants
Plasmids pIAC1, pIAC2, and pIAC3 were generated to obtain ΔiaaL, ΔhprL, and ΔmatE mutant strains, respectively. DNA fragments of approximately 0.5 kb corresponding to 5′ and 3′ regions flanking the target gene were cloned into the pGEM-T Easy vector (Promega; Madison, USA). This fragment was generated with three rounds of PCR amplification using the method described by . All plasmids were sequenced to verify the absence of mutations.
Following sequencing, plasmids were labeled with the nptII kanamycin resistance gene flanked by FRT (flippase recognition target) sites. The fragment containing this gene sequence was obtained from the plasmids pGEM-T-KmFRT-BamHI or pGEM-T-KmFRT-EcoRI (Table 1). The fragments resulting from digestion were cloned into the BamHI or EcoRI sites of vectors derived from pGEM-T containing previously amplified fragments.
Plasmids pIAC1, pIAC2 and pIAC3 (Table 1) were transformed by electroporation into the Pto DC3000 strain. Transformants were selected on LB media supplemented with kanamycin (15 μg/ml). Replica plates of the resulting colonies were created in plates with ampicillin (300 μg/ml) to determine whether each transformant was the result of a single recombination event (integration plasmid, AmpR) or a double recombination event (allelic exchange, AmpS). Southern Blot analysis using a sequence complementary to a target gene as a probe was used to confirm whether allelic exchange had occurred in the correct location of the genome and that only one copy of the construct was inserted.
Virulence assays and symptom quantification in tomato plants
Seeds of tomato plants (Solanum lycopersicum var. Moneymaker) were germinated and grown in a growth chamber with a 16/8 h light/dark photoperiod at 24/18 °C day/night and at 70 % relative humidity. Bacteria were grown on LB agar plates for 48 h at 28 °C and resuspended in 10 mM MgCl2 at an OD600nm of 0.5, corresponding to about 108 colony forming units (CFU) per ml. Further serial dilutions were carried out to obtain suspensions for inoculations with different doses. Four to 5-week-old plants were inoculated by infiltrating bacterial suspensions into the intracellular spaces. Infiltration was achieved by pressing the bacterial suspension against the leaf with a 2-ml syringe without the needle. The negative control plants were mock-inoculated with a 10 mM MgCl2 solution. Each plant was infiltrated in three leaflets of the same leaf, and the infiltrated (IF) area covered half of the midrib on the right side of the main vein. The other halves of those leaflets were analyzed as non-infiltrated (NIF) areas. The evolution of disease symptoms was recorded at 2, 4, 7 and 9 days post-inoculation (dpi). Bacteria were recovered from the infected leaves using a 10-mm diameter cork borer. Five disks (3.9 cm2) per plant were homogenized via mechanical disruption in 1 ml of 10 mM MgCl2, and the CFU per cm2 were counted by plating serial dilutions on LB plates with corresponding antibiotics. Quantification of necrotic lesions was performed at 9 dpi using the image analysis software Visilog 6.3 (Noesis; Courtaboeuf, France). Data were represented as the ratios of necrotic lesions per total leaf area.
Competition assays in tomato plants
Competition assays were performed by mixing cultures of mutants and wild-type strains with an OD660nm of 0.5 in a 1:1 ratio. Four to 5-week-old tomato plants were inoculated with 5 × 104 CFU/ml mixed bacterial suspensions using a 2-ml syringe without the needle. Serial dilutions of the inocula were plated in LB with and without 15 μg/ml kanamycin for the selection of mutant and wild-type strains, respectively. Four days post-inoculation, bacteria were recovered from the infected leaves by grinding the tissue with 1 ml of 10 mM MgCl2, and the CFU per cm2 were counted by plating serial dilutions on LB plates amended with the corresponding antibiotics. 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 competition indices shown are the means of three replicates showing typical results from two independent experiments, i.e. six replicates in total. Statistical analysis of CI values was carried out using Student’s t test and the hypothesis that the mean index was not significantly different from 1.0 (P-value = 0.005).
Fluorescence images of the adaxial (AD) surfaces of leaves were captured with the customized fluorescence imaging system Open FluorCam FC 800-O (Photon Systems Instruments, Brno, Czech Republic). Autofluorescence emission of the leaves was excited with a UV source (360 nm), and the F440, F520 and F690 images were acquired sequentially from identical fields of view according to . Black and white images of both measured fluorescence intensity and the calculated fluorescence ratio (F440⁄F690) are shown using a false color scale. Images and numerical data from regions of interest were processed with FluorCam software version 22.214.171.124 (PSI Systems, Brno, Czech Republic). Measurements were carried out in mock-control and bacterial-challenged plants at 2, 4, 7, and 9 dpi. Four plants per treatment were analyzed, and the experiment was carried out three times with similar results.
Genomic context analysis of the iaaL and matE loci in P. syringae and P. savastanoi
The existence of hrp box promoter sequences upstream of both the iaaL and matE genes has been reported in Pto DC3000 [17–20]. However, these two hrp box motifs were defined as non-canonical [17, 18, 20]. Although polymorphisms in hrp box sequences have been correlated with the loss of HrpL-dependent expression , the divergent hrp box promoter of the iaaL gene  actually binds HrpL and promotes HrpL-dependent transcription . Interestingly, the promoter sequence of the iaaL gene is encoded within the 3′ end of the matE gene in Pto DC3000 (Fig. 1b), suggesting that it could be constrained by the genetic code. Indeed, multi-sequence alignment of the iaaL promoter showed a conserved 5′-GGAACC-N20-TCAT-3′ motif in the genomes of all P. syringae strains analyzed, including members of genomospecies 2, 3 and 4 (Fig. 1b). Other hrp box promoters found embedded within their upstream encoded ORFs include PSPTO_2130 and its orthologs, which are also members of the non-effector genes included in the P. syringae HrpL regulon .
In terms of the promoter region of matE, a highly conserved hrp box motif, 5′-GGAACT-N19-TCAG-3′, was found in the strains of genomospecies 3 and exhibited a slight variation in its −35 box (5′-GGAGCT-3′) in P. syringae pv. oryzae (Por) 1_6 (genomospecies 4, MLST group 4). However, although the putative −10 region of this promoter (5′-TCAG-3′) was also conserved in all strains included in genomospecies 2, a consensus −35 region was not found in the genomes analyzed (Fig. 1b), suggesting a possible loss of HrpL regulation of the matE promoter in this genomospecies. Transcription of the matE gene has been shown to be upregulated in response to HrpL overexpression both in Pto DC3000 (genomospecies 3) and Por 1_6 (genomospecies 4) , suggesting that the polymorphisms observed between these genomospecies in the −35 hrp box of matE do not seem to alter HrpL-dependent transcription of this gene. Low conservation of the −35 region has been observed for other promoters regulated by extracytoplasmic function (ECF) sigma factors, of which HrpL is an example [44, 45], and also for RpoD-dependent promoters [46, 47], However, downregulation of matE in a ΔhrpL background has not yet been reported for any P. syringae strain.
The iaaL gene is transcribed both independently and in formation in an operon with the matE gene
RNA-Seq combined with Illumina high-throughput sequencing technology have been used to identify 5′-ends of transcripts in Pto DC3000 grown in iron-limited MG medium, including those corresponding to the iaaL gene [33, 34]. Moreover, transcriptome profiling using RNA-seq coupled with the GENE-counter software package  recently expanded characterization of the HrpL regulon from six P. syringae strains, including Pto DC3000, and pointed again at the iaaL and matE genes as members of this regulon . Moreover, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and RNA-Seq was used to identify HrpL-binding sites and likely hrp promoters, including that of the iaaL gene . Despite the growing use of RNA-Seq as a high-throughput strategy to analyze the transcriptome of Pto DC3000 on a global scale, very few confirmed transcriptional start sites have been reported for this model pathogen . With the experimental aim of confirming the transcription start sites of both the iaaL and matE genes in Pto DC3000, we first used primer extension analysis and then 5′RACE. The transcription start site of iaaL was located just three nucleotides downstream of the 3′ end of its proximal hrp box promoter, at the C located 79 bp upstream of its start codon (Additional file 2: Figure S2). This position is placed two nucleotides upstream and one nucleotide downstream, respectively, of those previously determined [20, 34]. Extension of several primers complementary to the matE sequence yielded no amplification products. However, the transcription initiation site of the matE gene was identified by 5′RACE at the G located 66 bp upstream of its start codon (Additional file 2: Figure S2). This start site is identical to one of the two possible sites proposed by  and is located 17 bp downstream of the 3′ end of its proximal hrp box promoter (Fig. 1). A detailed analysis of the transcription start sites of HrpL-regulated promoters determined in Pto DC3000 by  revealed that most of them, 39 of the 49 identified, were located at a distance within 2–4 bp to the proximal hrp box promoter; the remaining distances from the start sites ranged from 0 to 75 bp (Additional file 3: Figure S3).
Transcription from the P. syringae pv. tomato DC3000 P matE promoter is HrpL-dependent
The activity of the P iaaL promoter increased after 6 h and remained moderately stable from 24 to 56 h, reaching a maximum level of approximately 200 Miller units after 72 h. In agreement with other authors , the activity of this promoter was fully dependent on HrpL, as ß-galactosidase activity levels obtained in the ΔhrpL mutant were generally similar to those of the negative control (Fig. 3b). In summary, expression from both P matE and P iaaL is dependent on HrpL and is induced in late stationary phase, although the induction levels of P matE are approximately 2.5 times higher than P iaaL under the conditions tested. Although analysis of P. syringae virulence has primarily focused on the characterization and function of the T3SS and its effector proteins, a role in virulence has also been demonstrated for other non-effector genes regulated by HrpL, e.g., the Pto DC300 corR [17, 51] and aprI [20, 52] genes and the PSPPH_A0106-A0112 operon of P. syringae pv. phaseolicola 1448A . In the light of these findings, the possible roles of the matE and iaaL genes in the survival and virulence of Pto DC3000 in tomato plants were analyzed.
The iaaL and matE genes are required for full virulence of P. syringae pv. tomato DC3000 in tomato plants
Figure 4b shows the symptoms in tomato leaves caused by wild-type Pto DC3000 and the ΔiaaL and ΔmatE mutants at 9 dpi. Moreover, image analysis of infected leaves was also performed at 9 dpi, allowing the quantification of the symptoms induced as the ratio of necrotic lesions/total leaf area (Fig. 4c). At 9 dpi, chlorosis of the tissue induced by wild-type Pto DC3000 covered the entire inoculated area, and the necrotic lesions covered approximately 6 % of the total leaf area. In contrast, leaves inoculated with the ΔmatE or ΔiaaL mutants developed late chlorosis and exhibited significantly fewer necrotic lesions than those caused by the wild-type strain (approximately 0.8 and 1.2 % of the total leaf area, respectively) (Fig. 4b and c). Therefore, both matE and iaaL are needed for growth and fitness of Pto DC3000 in tomato leaves, which also have an effect on its virulence.
It has been recently studied whether some of the non-effector genes identified as new members of the Pto DC3000 HrpL regulon contributed to pathogenicity. For that, deletion mutants were constructed (on wild-type Pto DC3000 and ΔhopQ1-1 backgrounds) for seven candidates, including the iaaL gene. Despite the fact that the annotated functions of all seven genes were elusive in terms of plant association, no phenotypic differences were observed for in planta growth, virulence, or HR for any of these mutants in Nicotiana benthamiana plants . In addition to differences in the experimental design of these two pathogenicity tests, the iaaL gene may have a relevant role in the interaction of this pathogen with tomato plants and not with N. benthamiana, a host plant for a mutant lacking the T3SS effector HopQ1-1 but not for wild-type Pto DC3000 . On the other hand, MatE most likely contributes to the intrinsic bacterial resistance to toxic compound(s) produced by tomato plants, thus promoting Pto DC3000 survival during infection. Since the matE gene was shown to be induced in planta , the contribution of MatE to bacterial resistance might be emphasized in this case.
Multicolor fluorescence imaging of infected tomato plants
After the complete genome sequencing of Pto DC3000, the application of several high-throughput experimental screens and bioinformatics approaches allowed the identification of a suite of genes regulated by the sigma factor HrpL in this model bacterial phytopathogen. In addition to the genes required for the biosynthesis of T3SS and its effectors, other non-effector regulon members with diverse functions were also identified, including the iaaL gene and the putative MATE family transporter gene here called matE. In this work, we show that the iaaL gene, which is highly conserved within the P. syringae complex, is found in synteny with the matE gene in the genome of P. syringae and P. savastanoi strains belonging to genomospecies 2, 3 and 4. We demonstrate that in Pto DC3000, both of these genes encode upstream hrp box-like promoters that are HrpL-dependent. We also present evidence for the cotranscription of iaaL and matE (matE-iaaL operon) in this pathogen. Finally, we show that deletion of either the iaaL or the matE gene in Pto DC3000 results in decreased fitness, colonization and virulence, together with a lower activation of secondary metabolism in infected tomato plants.
This research was supported by the Spanish Plan Nacional I+D+i grants AGL2011-30343-CO2-01, AGL2014-53242-C2-1-R and BIO2007-67874-C02-02 as well as by grants ref. P08-CVI-03475 and P12-AGR-0370 from the Junta de Andalucía (Spain). A. de Vicente and V. Carrión (University of Malaga, Spain) are thanked for help with the analysis of virulence in tomato plants and for assistance with 5′-RACE, respectively. We thank Manuela Vega [Servicio Centrales de Apoyo a la Investigación (SCAI), University of Malaga, Spain] for excellent assistance with the image analysis of knot volumes. We also thank M. Pineda (EEZ, CSIC, Granada, Spain) for fruitful discussions and technical help with imaging techniques.
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