Role of the type VI secretion systems during disease interactions of Erwinia amylovora with its plant host
© The Author(s). 2017
Received: 16 January 2017
Accepted: 2 August 2017
Published: 17 August 2017
Type VI secretion systems (T6SS) are widespread among Gram-negative bacteria and have a potential role as essential virulence factors or to maintain symbiotic interactions. Three T6SS gene clusters were identified in the genome of E. amylovora CFBP 1430, of which T6SS-1 and T6SS-3 represent complete T6SS machineries, while T6SS-2 is reduced in its gene content.
To assess the contribution of T6SSs to virulence and potential transcriptomic changes of E. amylovora CFBP 1430, single and double mutants in two structural genes were generated for T6SS-1 and T6SS-3. Plant assays showed that mutants in T6SS-3 were slightly more virulent in apple shoots while inducing less disease symptoms on apple flowers, indicating that T6SSs have only a minor effect on virulence of E. amylovora CFBP 1430. The mutations led under in vitro conditions to the differential expression of type III secretion systems, iron acquisition, chemotaxis, flagellar, and fimbrial genes. Comparison of the in planta and in vitro transcriptome data sets revealed a common differential expression of three processes and a set of chemotaxis and motility genes. Additional experiments proved that T6SS mutants are impaired in their motility.
These results suggest that the deletion of T6SSs alters metabolic and motility processes. Nevertheless, the difference in lesion development in apple shoots and flower necrosis of T6SS mutants was indicative that T6SSs influences the disease progression and the establishment of the pathogen on host plants.
Type VI secretion systems (T6SS) were discovered in ecologically-diverse pathogenic and non-pathogenic Gram-negative bacteria, including bacteria associated with eukaryotic cells maintaining pathogenic or symbiotic interactions [1–3]. T6SS gene clusters characteristically consist of a set of 13 core genes and a varying number of accessory genes  including Hcp and VgrG that were frequently identified in culture supernatants [5–7]. In some bacterial species Hcp and VgrG along with additional effectors were discovered to have antibacterial properties that are targeted to recipient cells and contribute to the competitiveness and indirectly to virulence [8–13]. The T6SS are implied in various functions in different bacterial species, e.g., biofilm formation, inter-bacterial pathogenicity, host-cell invasion and survival within macrophages [8, 14–19]. The contribution of T6SS to virulence was demonstrated for certain animal and human pathogens [20–24], whereas only few reports about functions in plant associated bacteria exist [25–29]. In Rhizobium leguminosarum, the T6SS was described as a nodulation impairment locus imp  and later identified as a secretion system . First indications that T6SS might contribute to virulence in plant pathogenic bacteria came from microarray profiling and secretome analysis in Pectobacterium atrosepticum SCRI1043 and Agrobacterium tumefaciens C58 [6, 7, 28]. In A. tumefaciens C58, it was shown that the T6SS is involved in virulence and interbacterial competition [7, 8]. A contribution to virulence was also demonstrated for Ralstonia solanacearum where mutation of a T6SS gene led to an attenuation of virulence on tomato plants and additionally to a reduction of motility and biofilm formation . The mutational analysis of the T6SSs in Pantoea ananatis showed that one of the clusters is essential for pathogenicity on onion host plants and is involved in bacterial competition .
Erwinia amylovora is a Gram-negative, enterobacterial phytopathogen causing the fire blight disease . The pathogen has a broad host-range affecting various Rosaceae (primarily Spiraeoideae), including ecologically and economically important species (e.g., apple and pear). The pathogenicity of E. amylovora is strictly dependent on a functional type III secretion system (T3SS) and the production of the exopolysaccharide amylovoran . We identified three T6SS (T6SS-1-T6SS-3) in E. amylovora CFBP 1430, of which two include the 13 core genes, whereas the third cluster is only rudimentary . T6SS-1 and T6SS-2 were also identified in other Erwinia species, whereas the third cluster is not present in all of them . Recently, it was demonstrated that the T6SSs of E. amylovora are involved in bacterial competition, exopolysaccharide production, and virulence on immature pear fruits . Bacterial competition was influenced by all T6SS mutants tested, whereas the effects on exopolysaccharide production and virulence varied between the different gene knock-out variants . In this study, the aim was to analyze a potential contribution of the T6SSs of E. amylovora in virulence to plants, antibacterial properties, and to use RNA-seq to analyze transcriptomic changes. The performed plant assays indicated a minor contribution of T6SSs to the virulence E. amylovora CFBP 1430. Additionally, competition assays showed that deletion of the T6SSs slightly affected the antibacterial properties of E. amylovora CFBP 1430. Differential expression of motility and chemotaxis genes were observed, and further experiments showed that the T6SSs have an effect on the motility of E. amylovora CFBP 1430.
Bacterial strains, media, and growth conditions
Bacterial strains and plasmids used in this study
Strain or plasmid
E. amylovora CFBP 1430
Wild type strain
E. amylovora T6-d1
ΔtssB-1/tssC-1T6SS cluster 1 deletion mutant, CmR
E. amylovora T6-d3
ΔtssB-2/tssC-2 T6SS cluster 3 deletion mutant, CmR
E. amylovora T6-d1d3
ΔtssB/tssC T6SS cluster 1 and 3 deletion mutant, CmR
E. amylovora ΔhrpL
ΔhrpL Deletion mutant, CmR
E. coli DH5α
Φ80 lacZ M15, Δ(lacZYA-argF) U169, recA1, endA1,thi-1
Red recombinase expressing plasmid, ApR
Antibiotic resistance cassette template, ApR, CmR
FLP encoding recombinase plasmid, ApR, CmR
Construction of T6SS mutants
The lambda red recombination system  was used to replace consecutive genes EAMY_3020–3021 and EAMY_3227–3228 (coding for TssB1/TssC1 and TssB2/TssC2) in both T6SS clusters. The resulting single mutants were named T6-d1, T6-d3, and the double mutant T6-d1d3. Plasmids used are listed in Table 1. Plasmid pKD3 was used as template to amplify the chloramphenicol resistance cassette with the primer pairs listed in Additional file 1: Table S1. PCR products were introduced by electroporation into competent E. amylovora CFBP 1430 cells carrying plasmid pKD46. The single mutants were constructed by deletion of the complete reading frames of tssB/tssC in each cluster. To create the double mutant, the plasmid pKD46 was removed from the T6-d1 by overnight incubation on LB plates at 37 °C and colonies were screened for loss of antibiotic resistance. Plasmid pCP20 was introduced to eliminate the chloramphenicol resistance cassette and mutation of tssB/tssC in T6SS-3 was performed as described above. All knock-out mutants were confirmed by PCR and sequencing.
Immature pear assay
E. amylovora WT, T6-d1, T6-d3, T6-d1d3, and a ΔhrpL mutant were grown overnight in KB liquid cultures, collected by centrifugation, adjusted to an OD600 = 0.1 (i.e., approximately 108 cfu ml−1) and diluted to cell-densities of approximately 103, 105, and 107 cfu ml−1. The pears (cultivar ‘Conférence’) were surface sterilized using 10% NaOCl, pierced with a needle and subsequently inoculated with 5 μl bacterial suspension or as a control with water [39, 40]. The fruits were incubated in a humidity chamber at 28 °C for 8 days. Fruits were assayed in triplicates for each treatment and the experiment was repeated twice.
Shoot inoculation assays
Shoots of potted one-year old grafted apple (Malus × domestica cv. ‘Golden Delicious’) and pear plants (Pyrus communis cv. ‘Conférence’) were used for inoculation experiments. The plants were kept at 20 °C during the day and 18 °C at night with a relative humidity of 80%. Shoots were inoculated using a syringe with a bacterial suspension containing approximately 108 cfu ml−1. The plants were assayed in pentaplicates with five repetitions. The disease progress was recorded weekly over four weeks and calculated as the percentage of total shoot length with observable lesion formation .
Newly opened flowers of two-year-old grafted apple (Malus × domestica cv. ‘Golden Delicious’) trees were spray-inoculated (200 μl/spray dose) using an atomizer. The bacterial suspensions contained approximately 107 cfu ml−1. To determine bacterial population sizes, the petals were removed and the remaining flower parts were washed in water. Bacteria were plated immediately after inoculation, 1 DPI (day post inoculation) and 2 DPI post inoculation. Twenty flowers per tree were assessed to determine the population sizes. Flower necrosis was rated at 10 DPI and 12 DPI. The experiment was repeated twice. A total of 529 (WT), 629 (T6-d1), 543 (T6-d3) and 580 (T6-d1d3) flowers were rated.
RNA from bacteria grown to the mid-log phase in liquid media was extracted using the NucleoSpin RNA II kit (Macherey Nagel, Oensingen, Switzerland) following manufacturer instructions. Flowers were inoculated with a pipette applying to the hypanthium 5 μl of bacterial suspension containing 107 cfu ml−1. The flowers were collected 2 DPI, petals were removed and washed in water. The floral parts were removed from the solution and centrifuged. The supernatant was discarded, the pellet flash-frozen in liquid nitrogen and stored at −86 °C until RNA-extraction. Total RNA was isolated using the innuPREP Plant RNA Kit (Analytikjena, Jena, Germany) according to the manufacturer’s instructions. All total RNA samples were treated with DNase I (Thermo Scientific, Wohlen, Switzerland) and control PCRs were performed to check for absence of DNA contamination. Quality and concentration of the RNA samples were determined using the Bioanalyzer 2100 (Agilent Technologies, Stuttgart, Germany). Samples were pooled to the required amount for further processing.
The RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Reinach, Switzerland) was used to create cDNA templates for RT-PCR. Aliquots of 1 μg of RNA were reversed transcribed with random hexamers according to manufacturer instructions and used for PCR with the primer pairs listed in Additional file 1: Table S1.
RNA-seq data analysis
The cDNA libraries, derived from liquid cultures (ribosomal RNA depleted), were constructed and Illumina sequenced leading to 18,598,523 (WT) and 18,275,977 (T6-d1d3) reads. The reads were aligned with Bowtie version 0.12.7  to the E. amylovora CFBP 1430 genome sequence  with an overall alignment rate of 91.43% (WT) and 91.04% (T6-d1d3). The cDNA libraries, derived from inoculated flowers, led to 44,296,072 (WT inoculated sample) and 45,470,859 (T6-d1d3 inoculated sample) reads. Alignment of the reads to the E. amylovora CFBP 1430 genome sequence had overall alignment rate of 10.65% (WT inoculated sample) and 20.77% (T6-1d-3 inoculated sample). Analysis of differential expression levels was performed using Cufflinks version 1.3.0 . Gene expression levels were normalized using fragments per kb of exon per million mapped reads (FPKM) report values. Genes were considered as significantly differentially expressed, when their fold change was ≥1.5 or ≤ −1.5, respectively, and their p value <0.001.
WT and T6SS mutants were freshly plated from glycerol stocks kept at −86 °C and grown overnight on KB plates. The bacteria were collected and resuspended in 0.8% NaCl, washed three times with 0.8% NaCl by centrifugation. The bacterial strains were adjusted to an OD600 = 0.1 (108 cfu ml−1) and 20 μl of the suspension was spotted in the middle of soft agar plates (3 g l−1 agar, 10 g l−1 plant tryptone, 5 g l−1 NaCl). The motility agar plates were assessed after 2 days incubation at 28 °C.
All strains were grown overnight in LB, washed once with 0.8% NaCl and adjusted to OD600 = 0.900. The WT or one of the T6SS mutants was mixed in a ratio of 1:1 with Escherichia coli DH5α cells and 20 μl of suspension was spotted on LB and KB agar plates. The spots were excised after 24 h from the agar and resuspended in 2 ml NaCl, vortexed, and serial dilution was performed. The plates were incubated at 30 °C overnight and subsequently cfu were counted.
Statistical analyses were performed with the Sigmaplot software Version 10 (Systat Software, San Jose, USA) or using R version 3.2.3. The lesion length data were log transformed prior to analysis as a two-way ANOVA with mean comparisons evaluated using Fisher’s least significant difference test at a significance level of 5%. The flower necrosis data were assessed with a one-way ANOVA using the Fisher’s LSD test at a significance level of 5% to test for statistical significant differences between treatments. The competition assays were analyzed using a one-way ANOVA with a Tukey posthoc test.
Generation and characterization of T6SS mutants
The T6SS gene clusters have been described, whereas their activity and role in pathogenicity were not. Therefore, to test whether T6SS related genes were transcribed under different conditions, we analyzed the expression of a set of selected T6SS genes (T6SS-1 (hcp-1, tssF-1), T6SS-3 (hcp-2, vgrG-4, tssF-2)), by isolating RNA from E. amylovora CFBP 1430 grown in different liquid media. The qualitative assessment by RT-PCR (Additional file 1: Figure S1) showed that the T6SSs genes in E. amylovora CFBP 1430 were expressed under the tested conditions. This indicated that these genes represent an active secretion system, which can be assessed both in vitro and in vivo.
Deletion mutants were constructed in order to determine whether the T6SS-1 and T6SS-3 contribute to the virulence of E. amylovora CFBP 1430. To exclude that the mutations have an influence on the growth rates of the mutants, growth curves were determined in different liquid media. No obvious differences in the growth rates of the individual mutants compared to the wildtype strain were detected (Additional file 1: Figure S2), which allows the direct comparison of the strains both in vitro and in vivo.
In vitro transcriptional differences between E. amylovora CFBP 1430 and the T6SS double mutant T6-d1d3
The cDNA libraries for Illumina sequencing were constructed from RNA isolated from WT and T6-d1d3 mutant strains of E. amylovora CFBP 1430, grown in minimal M9 medium supplemented with sucrose. The comparison of the WT to the T6-d1d3 mutant revealed differential expression of 508 genes (Additional file 1: Table S2): of these, 391 genes have an annotation and 117 are predicted genes. The differential expressed genes were classified into groups according to their gene ontology (GO) terms (Additional file 1: Figure S3). Processes affected include the categories cellular process, metabolic process, pigmentation, stimuli response, biological regulation, localization and locomotion.
Summary table of selected significantly differentially expressed genes from the in vitro experiment comparing the WT to the T6SS double mutant
Chemotaxis and motility
Chemotaxis protein CheW
Chemotaxis protein CheA
Chemotaxis protein MotB
Flagellar motor protein MotA
Chemotaxis response regulator protein-glutamate methylesterase
Chemotaxis protein-glutamate O-methyltransferase
Chemotaxis response regulator protein-glutamate methylesterase
Chemotaxis protein-glutamate O-methyltransferase
Flagellar basal body P-ring biosynthesis protein FlgA
Flagellar basal body rod protein FlgB
Flagellar basal body rod protein FlgC
Flagellar basal body rod protein FlgG
Flagellar basal body L-ring protein
Flagellar basal body P-ring protein
Flagellar rod assembly protein/muramidase FlgJ
Anti-sigma-28 factor FlgM
Flagellar biosynthesis protein FlhB
Flagellar biosynthesis protein FlhA
Flagellar biosynthesis protein FlhE
Transcriptional activator FlhC
Flagellar transcriptional activator FlhD
Transcriptional activator FlhD
Flagellar hook protein FliD
Flagellar hook-basal body complex protein FliE
Flagellum-specific ATP synthase FliI
Flagellar assembly protein FliH
Flagellar motor switch protein FliG
Flagellar M-ring protein FliF
Flagellar basal body-associated protein FliL
Flagellar biosynthetic protein FliP
Flagellar biosynthetic protein FliO
Flagellar biosynthetic protein FliR
Flagellar export apparatus protein FliQ
Flagellar protein FliS
Flagellar biosynthesis protein FliT
Flagellar regulatory protein FliZ
Cytochrome c biogenesis protein CcmH
Type III secretion systems
EscC/YscC/HrcC family type III secretion system outer membrane ring protein
EscJ/YscJ/HrcJ family type III secretion inner membrane ring
protein Hypothetical protein
Type III secretion system protein
Hrp pili protein HrpA
Pathogenicity locus protein hrpK
Type III secretion system protein InvA
CesD/SycD/LcrH family type III secretion system chaperone
Type III secretion system protein
ATP synthase SpaL
Type III secretion system protein
EscR/YscR/HrcR family type III secretion system export apparatus protein
Type III secretion system protein SpaQ
EscU/YscU/HrcU family type III secretion system export apparatus switch protein
The effect of T6SS deletion on plant pathogenicity
The transcriptomic data indicated that there is an effect of deletion of the T6SSs on pathogenicity-related gene expression. To get a first indication if T6SS is involved in pathogenicity of E. amylovora CFBP 1430 in planta, an immature pear assay was performed. The wildtype strain CFBP 1430, the single mutants and the double mutant were assessed for altered virulence on immature pear fruits. Additionally, a ΔhrpL mutant  was used as a positive control for altered pathogenicity. The alternative sigma factor HrpL controls the expression of different hrp genes and deletion thereof renders the mutant avirulent . Strain CFBP 1430 and the T6SS mutants were applied at two different concentrations and were assessed at 8 DPI. The application of 107 and 105 cfu ml−1 led to similar tissue maceration and ooze production of all T6SS mutants compared to E. amylovora CFBP 1430 (Additional file 1: Figure S4). Symptoms could not be observed for any of the strains when 103 cfu ml−1 was applied. The ΔhrpL mutant induced no symptoms at any concentration. The inoculation with E. amylovora CFBP 1430 and its T6SS mutant derivatives showed no difference in disease and symptom development.
Comparison of the in planta transcriptomes of E. amylovora CFBP 1430 and the T6SS double mutant
The transcriptome data derived from flowers at 2 DPI of the susceptible Malus × domestica cv. ‘Golden Delicious’ inoculated with either the wildtype E. amylovora CFBP 1430 or double mutant T6-d1d3. Alignment of the reads to the E. amylovora CFBP 1430 genome , showed that only 10.65% (CFBP 1430 inoculated) and 20.77% (T6-d1d3 inoculated sample) were mapped to the genome, probably due to cDNA derived from the apple host plants. This was confirmed by mapping the reads to the apple genome sequence  revealing that approximately 86.92% (WT inoculated sample) and 77.28% (T6-d1d3 inoculated sample) originated from apple RNA.
The bacterial transcriptome included 3563 expressed genes. Transcripts of 141 genes were found in none of the conditions, most encoding hypothetical proteins and five with annotations (flgB3, glcG, intS1, spaR3, yeeV). Among the non-expressed genes a flagellar (flgB3), a fimbrial (EAMY_0247) and a type III secretion system (spaR3) gene were identified. These genes were expressed in vitro (only intS1 differentially). The differential expressed genes were grouped according to their GO annotations (Additional file 1: Figure S3). The biological processes include the categories cellular process, metabolic process, pigmentation, response to stimulus, biological regulation, and localization and locomotion.
Summary table of selected significantly differentially expressed genes from in vivo experiment comparing the WT to the T6SS double mutant
Chemotaxis and motility
Protein phosphatase CheZ
Chemotaxis response regulator protein-glutamate methylesterase
Chemotaxis protein-glutamate O-methyltransferase
Chemotaxis protein CheA
Flagellar motor protein MotB
Flagellar motor protein MotA
Methyl-accepting chemotaxis protein
Methyl-accepting chemotaxis protein
Phosphate transporter permease subunit PtsA
Phosphate transporter permease subunit PstC
Phosphate ABC transporter substrate-binding protein
Phage shock protein PspA
Taurine ABC transporter permease
Taurine ABC transporter ATP-binding protein
Taurine ABC transporter substrate-binding protein
Sulfite reductase subunit alpha
Sulfite reductase subunit beta
In vitro experiments
To test for antibacterial properties of the T6SSs in E. amylovora CFBP 1430, a competition assay was performed challenging the WT and the T6SS mutants with E. coli DH5α on two different media. After co-inoculation with the WT and the single mutants on KB plates similar numbers of cfus were counted for E. coli DH5α. However, co-inculation with the double mutant resulted in slightly higher, statistically significant, numbers of E. coli DH5α cfus (Additional file 1: Figure S6). The competition assay on LB plates resulted in similar numbers of E. coli DH5α cfus.
In this study, a combined approach of RNA-seq, physiological tests and plant experiments was employed to analyze the impact of T6SS deletion on virulence of E. amylovora CFBP 1430. In contrast to the well-established virulence factors like the Hrp type III secretion and amylovoran [25, 44, 49, 51, 52], deletion of the T6SS did not impair the virulence of E. amylovora CFBP 1430 very strongly. Nevertheless, an alteration in virulence could be observed in apple flowers and shoots. This effect might be attributed to the impairment on motility that was displayed by the T6SS mutants, similarly to an E. amylovora motility mutant that showed reduced blossom infections . On flowers, it is required to actively gain access to the vascular system through the nectartodes [54, 55]. Hence, the impaired T6SS mutants would produce less disease symptoms on flowers compared to the WT. When injected in the shoots, the T6SS mutants might be passively transported, as attachment structures (e.g., flagella, pili) would be less produced and therefore disease progress appeared faster. Alternatively, the identified differences on apple host plants were indicative that T6SS are involved in the control of disease progression and severity. Flagellar genes were also contained in the comparison of the in planta transcriptomes of the two strains, indicating that this trait is effectively affected in the double mutant. Motility of E. amylovora was demonstrated to be an important trait to invade non-injured apple leaves, whereas no significant effect could be detected on injured plants , and several changes in regulatory systems have an influence on motility . Similarly, a difference in the expression of flagellar genes was also observed in T6SS mutants of Citrobacter freundii . There, the deletion of the complete T6SS cluster led to a lower expression of all tested flagellar genes, whereas the deletion of clpV, hcp, and vgr showed both higher and lower expression levels dependent on the deleted gene . As a direct impact of T6SSs genes on transcriptional changes is not expected, the absence of T6SS machinery might cause a change in the membrane properties or the accumulation of T6SS related proteins might lead to a deregulation and might affect transcription of the corresponding genes.
The comparison of the transcriptomic data obtained from the in vitro cultured wild-type strain CFBP 1430 and double mutant T6-d1d3 showed that virulence associated genes, e.g., Hrp T3SS associated, amylovoran biosynthesis, as well as a set of flagellar genes were affected by the deletion of the T6SS genes (Table 2). The differential expressed amylovoran biosynthesis genes amsJ and amsK might be involved in annealing the different galactose, glucuronic acid, and pyruvyl subunits while amsL in oligosaccharide transport and assembly . The differential expression of these genes in the double mutant T6-d1d3 might lead to varying structure of the exopolysaccharide as annealing and assembly were altered. A difference in amylovoran production was observed for E. amylovora Ea1189 in a two-component signal transduction systems analysis, where deletion of the gene spk1, encoding the serine kinase corresponding to ppkA (EAMY_3004) in E. amylovora CFBP 1430 and thus part of T6SS-1 showed elevated level of the exopolysaccharide . Additionally, single gene deletions in all three T6SSs had either no effect, increased or decreased exopolysacharide production . The deletion of tssB-1 and tssC-1 increased, whereas tssB-2 and tssC-2 decreased the amylovoran production in E. amylovora NCPPB 1665 .
The genes of the Hrp T3SS, beside the potential translocator encoding hrpK, were less abundant in the double mutant T6-d1d3 including hsvA and hrpA. The latter two genes are required for full virulence and systemic infection in apple plants [43, 52, 60]. The lower expression of these genes in the double mutant in vitro indicated that mutation of T6SS might lead to an altered virulence of the pathogen. The comparison with the in vivo transcriptomic data showed that only few genes were differentially expressed under both conditions and that the pathogenicity related were not part of them. Hence the differential expression of these genes was dependent on environmental factors.
The immature pear assays showed that no phenotypic difference was obtained between wildtype strain CFBP 1430 and all mutants, indicating that the T6SS is not directly involved in virulence on immature pear. A similar result was obtained for E. amylovora Ea1189 where deletion of spk1 also did not show an effect on pathogenicity . Single deletion mutants of E. amylovora NCPPB 1665 tssB-1 and tssC-1 showed as well no effect, whereas tssB-2 and tssC-2 decreased virulence of the pathogen on immature pears . All three mutants generated in this study showed the same phenotype as the mutants in tssB-1 and tssC-1. The difference to our results in the immature pear assay might arise from deletion of the consecutive genes tssB/tssC leading to a distinct phenotype. Additionally, different varieties of pear (‘Conférence’ vs. ‘Cuiguan’) as well as E. amylovora strains (CFPB 1430 vs. NCPPB 1665) were used to test for variation in virulence. The level of disease severity is dependent on both the E. amylovora strain and the genotype of host plant used to perform such experiments [61–63]. Furthermore, the differing results in the competition assay might arise as well from the E. amylovora strains used to test for antibacterial properties. As different E. amylovora strains are defined as ‘strong’ or ‘weak’ in respect to disease severity [61, 62], strains might exist that exhibit a similar pattern for interbacterial competition. Thus, E. amylovora strain NCPPB 1665 might be a better competitor than CFPB 1430 as mutational analysis of several genes in the T6SSs showed a stronger effect on survival of E. coli DH5α .
As E. amylovora is able to elicit disease symptoms in different plant organs beside fruits, we analyzed potential effects of T6SS mutations in shoots and flowers. The mutations of T6SSs genes in E. amylovora CFBP 1430 influenced the virulence in apple shoots, whereas the mutants showed no alteration on pear shoots or on immature pears. These results indicate that the T6SSs may contribute to virulence on apple, whereas on pear, the T6SSs were not strictly required to elicit disease symptoms. Additionally, these effects were dependent on the plant organ affected and thus T6SSs might be involved in adaptation, establishment and persistence of the pathogen to different host environments. This was further supported by the transcriptomic data obtained from in vitro and in vivo experiments that showed transcriptional changes were dependent on the environment. In the plant pathogenic bacterium A. tumefaciens, similar effects were identified from the deletion of hcp, that led to a reduced tumorgenesis in plant assays, whereas in P. atrosepticum, deletion mutants showed increased or slightly reduced virulence dependent on the deleted gene [7, 28].
As no visible effect of deletion of the T6SS on virulence in pears was obtained, the T6SSs might thus contribute as a host-specificity factor to the adaptation and control of disease progression. Further, the T6SSs in E. amylovora CFBP 1430 influenced only virulence on apple host plants, whereas virulence on pear was unaffected. Thus, the T6SSs might represent a host-specificity factor required to either promote or limit disease elicitation on apple shoots and flowers. It was observed that there is genetic variation of the T6SS gene clusters detected for E. amylovora strains infecting Spiraeoideae compared to E. amylovora strains infecting Rubus , which supports the hypothesis that the T6SSs could represent a potential role in host-specificity. Additionally, the closely related pear pathogens Erwinia pyrifoliae DSM 12163T and Erwinia pyriflorinigrans CFBP 5888T and the epiphyte Erwinia tasmaniensis Et1/99 possess only the two homologous T6SS gene clusters 1 and 2, while the third cluster is absent from their genome sequences [35, 65, 66].
The transcriptomic data obtained from flower inoculation experiments additionally showed that some metabolic processes were affected, namely phosphate transport and sulfur metabolism. Under the given conditions the genes of these pathways had lower transcript abundance in the double mutant T6-d1d3 than in the wildtype indicating that the T6SSs have an influence on metabolic functions on apple flowers. Sulfur and phosphate are two essential nutrients for growth and cell functions , but the lower expression of these genes did not significantly alter flower colonization.
This work was supported by the Department of Life Sciences and Facility Management of ZHAW, the European Union FP7 KBBE project DROPSA (grant agreement 613678) and the Swiss Federal Office for Agriculture project ACHILLES and was conducted within the Swiss ProfiCrops translational research program.
Availability of data and materials
The dataset supporting the conclusions of this article was deposited at ENA and is available in the ArrayExpress database repository (http://www.ebi.ac.uk/arrayexpress/experiments/), under accession number E-MTAB-5444 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5444/).
TK, CP, BD and THMS conceived and designed the experiments; TK, JFP, CP, and FR performed the experiments; TK and JFP analyzed the data; TK, BD and THMS, interpreted the results. All of the authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.
The authors declare not to have any competing interests.
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- Ryu CM. Against friend and foe: type 6 effectors in plant-associated bacteria. J Microbiol. 2015;53:201–8.View ArticlePubMedGoogle Scholar
- Records AR. The type VI secretion system: a multipurpose delivery system with a phage-like machinery. Mol Plant-Microbe Interact. 2011;24:751–7.View ArticlePubMedGoogle Scholar
- Jani AJ, Cotter PA. Type VI secretion: not just for pathogenesis anymore. Cell Host Microbe. 2010;8:2–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I. Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics. 2009;10:104.View ArticlePubMedPubMed CentralGoogle Scholar
- Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, Heidelberg JF, Mekalanos JJ. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A. 2006;103:1528–33.View ArticlePubMedPubMed CentralGoogle Scholar
- Mattinen L, Nissinen R, Riipi T, Kalkkinen N, Pirhonen M. Host-extract induced changes in the secretome of the plant pathogenic bacterium Pectobacterium atrosepticum. Proteomics. 2007;7:3527–37.View ArticlePubMedGoogle Scholar
- Wu HY, Chung PC, Shih HW, Wen SR, Lai EM. Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens. J Bacteriol. 2008;190:2841–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Ma LS, Hachani A, Lin JS, Filloux A, Lai EM. Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe. 2014;16:94–104.View ArticlePubMedPubMed CentralGoogle Scholar
- Murdoch SL, Trunk K, English G, Fritsch MJ, Pourkarimi E, Coulthurst SJ. The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors. J Bacteriol. 2011;193:6057–69.View ArticlePubMedPubMed CentralGoogle Scholar
- LeRoux M, De Leon JA, Kuwada NJ, Russell AB, Pinto-Santini D, Hood RD, Agnello DM, Robertson SM, Wiggins PA, Mougous JD. Quantitative single-cell characterization of bacterial interactions reveals type VI secretion is a double-edged sword. Proc Natl Acad Sci U S A. 2012;109:19804–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Russell AB, LeRoux M, Hathazi K, Agnello DM, Ishikawa T, Wiggins PA, Wai SN, Mougous JD. Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature. 2013;496:508–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Zheng J, Ho B, Mekalanos JJ. Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae. PLoS One. 2011;6:e23876.View ArticlePubMedPubMed CentralGoogle Scholar
- Bondage DD, Lin J-S, Ma L-S, Kuo C-H, Lai E-M. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex. Proc Natl Acad Sci U S A. 2016;113:E3931–40.View ArticlePubMedPubMed CentralGoogle Scholar
- MacIntyre DL, Miyata ST, Kitaoka M, Pukatzki S. The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci U S A. 2010;107:19520–4.View ArticlePubMedPubMed CentralGoogle Scholar
- De Pace F, Nakazato G, Pacheco A, De Paiva JB, Sperandio V, Da Silveira WD. The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain. Infect Immun. 2010;78:4990–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Schwarz S, West TE, Boyer F, Chiang W-C, Carl MA, Hood RD, Rohmer L, Tolker-Nielsen T, Skerrett SJ, Mougous JD. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog. 2010;6:e1001068.View ArticlePubMedPubMed CentralGoogle Scholar
- Pukatzki S, Ma AT, Revel AT, Sturtevant D, Mekalanos JJ. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A. 2007;104:15508–13.View ArticlePubMedPubMed CentralGoogle Scholar
- Bernal P, Allsopp LP, Filloux A, Llamas MA. The Pseudomonas putida T6SS is a plant warden against phytopathogens. ISME J. 2017;11:972–87.View ArticlePubMedPubMed CentralGoogle Scholar
- Mulder DT, Cooper CA, Coombes BK. Type VI secretion system-associated gene clusters contribute to pathogenesis of Salmonella enterica serovar Typhimurium. Infect Immun. 2012;80:1996–2007.View ArticlePubMedPubMed CentralGoogle Scholar
- Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordoñez CL, Lory S, Walz T, Joachimiak A, Mekalanos JJ. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science. 2006;312:1526–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Blondel CJ, Yang H-J, Castro B, Chiang S, Toro CS, Zaldívar M, Contreras I, Andrews-Polymenis HL, Santiviago CA. Contribution of the type VI secretion system encoded in SPI-19 to chicken colonization by Salmonella enterica serotypes Gallinarum and Enteritidis. PLoS One. 2010;5:e11724.View ArticlePubMedPubMed CentralGoogle Scholar
- Burtnick MN, DeShazer D, Nair V, Gherardini FC, Brett PJ. Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages. Infect Immun. 2010;78:88–99.View ArticlePubMedGoogle Scholar
- Ma AT, Mekalanos JJ. In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation. Proc Natl Acad Sci U S A. 2010;107:4365–70.View ArticlePubMedPubMed CentralGoogle Scholar
- Schell MA, Ulrich RL, Ribot WJ, Brueggemann EE, Hines HB, Chen D, Lipscomb L, Kim HS, Mrázek J, Nierman WC, DeShazer D. Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol. 2007;64:1466–85.View ArticlePubMedGoogle Scholar
- Zhang L, Xu J, Xu J, Zhang H, He L, Feng J. TssB is essential for virulence and required for type VI secretion system in Ralstonia solanacearum. Microb Pathog. 2014;74:1–7.View ArticlePubMedGoogle Scholar
- Shyntum DY, Theron J, Venter SN, Moleleki LN, Toth IK, Coutinho TA. Pantoea ananatis utilizes a type VI secretion system for pathogenesis and bacterial competition. Mol Plant-Microbe Interact. 2015;28:420–31.View ArticlePubMedGoogle Scholar
- Haapalainen M, Mosorin H, Dorati F, Wu RF, Roine E, Taira S, Nissinen R, Mattinen L, Jackson R, Pirhonen M, Lin N-C. Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. Tomato DC3000, is required for fitness for competition against bacteria and yeasts. J Bacteriol. 2012;194:4810–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Mattinen L, Somervuo P, Nykyri J, Nissinen R, Kouvonen P, Corthals G, Auvinen P, Aittamaa M, Valkonen JPT, Pirhonen M. Microarray profiling of host-extract-induced genes and characterization of the type VI secretion cluster in the potato pathogen Pectobacterium atrosepticum. Microbiology. 2008;154:2387–96.View ArticlePubMedGoogle Scholar
- Barret M, Egan F, Fargier E, Morrissey JP, O’Gara F. Genomic analysis of the type VI secretion systems in Pseudomonas spp.: novel clusters and putative effectors uncovered. Microbiology. 2011;157:1726–39.View ArticlePubMedGoogle Scholar
- Roest HP, Mulders IH, Spaink HP, Wijffelman CA, Lugtenberg BJ. A Rhizobium leguminosarum biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group. Mol Plant-Microbe Interact. 1997;10:938–41.View ArticlePubMedGoogle Scholar
- Bladergroen MR, Badelt K, Spaink HP. Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant-Microbe Interact. 2003;16:53–64.View ArticlePubMedGoogle Scholar
- Malnoy M, Martens S, Norelli JL, Barny M-A, Sundin GW, Smits THM, Duffy B. Fire blight: applied genomic insights of the pathogen and host. Annu Rev Phytopathol. 2012;50:475–94.View ArticlePubMedGoogle Scholar
- Oh C-S, Beer SV. Molecular genetics of Erwinia amylovora involved in the development of fire blight. FEMS Microbiol Lett. 2005;253:185–92.View ArticlePubMedGoogle Scholar
- Smits THM, Rezzonico F, Kamber T, Blom J, Goesmann A, Frey JE, Duffy B. Complete genome sequence of the fire blight pathogen Erwinia amylovora CFBP 1430 and comparison to other Erwinia spp. Mol Plant-Microbe Interact. 2010;23:384–93.View ArticlePubMedGoogle Scholar
- De Maayer P, Venter SN, Kamber T, Duffy B, Coutinho TA, Smits THM. Comparative genomics of the type VI secretion systems of Pantoea and Erwinia species reveals the presence of putative effector islands that may be translocated by the VgrG and Hcp proteins. BMC Genomics. 2011;12:576.View ArticlePubMedPubMed CentralGoogle Scholar
- Tian Y, Zhao Y, Shi L, Cui Z, Hu B, Zhao Y. Type VI secretion systems of Erwinia amylovora contribute to bacterial competition, virulence, and exopolysaccharide production. Phytopathology. 2017;107:654-61.Google Scholar
- King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954;44:301–7.PubMedGoogle Scholar
- Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97:6640–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Rezzonico F, Duffy B. The role of luxS in the fire blight pathogen Erwinia amylovora is limited to metabolism and does not involve quorum sensing. Mol Plant-Microbe Interact. 2007;20:1284–97.View ArticlePubMedGoogle Scholar
- Kamber T, Lansdell TA, Stockwell VO, Ishimaru CA, Smits THM, Duffy B. Characterization of the biosynthetic operon for the antibacterial peptide herbicolin in Pantoea vagans biocontrol strain C9-1 and incidence in Pantoea species. Appl Environ Microbiol. 2012;78:4412–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25.View ArticlePubMedPubMed CentralGoogle Scholar
- Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Oh C-S, Kim JF, Beer SV. The Hrp pathogenicity island of Erwinia amylovora and identification of three novel genes required for systemic infection. Mol Plant Pathol. 2005;6:125–38.View ArticlePubMedGoogle Scholar
- Wei ZM, Beer SV. hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of σ factors. J Bacteriol. 1995;177:6201–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Rosen HR. The mode of penetration of pear and apple blossoms by the fire-blight pathogen. Science. 1935;81:26.View ArticlePubMedGoogle Scholar
- Zusman T, Feldman M, Halperin E, Segal G. Characterization of the icmH and icmF genes required for Legionella pneumophila intracellular growth, genes that are present in many bacteria associated with eukaryotic cells. Infect Immun. 2004;72:3398–409.View ArticlePubMedPubMed CentralGoogle Scholar
- Parsons DA, Heffron F. sciS, an icmF homolog in Salmonella enterica serovar Typhimurium, limits intracellular replication and decreases virulence. Infect Immun. 2005;73:4338–45.View ArticlePubMedPubMed CentralGoogle Scholar
- Bellemann P, Geider K. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization. J Gen Microbiol. 1992;138:931–40.View ArticlePubMedGoogle Scholar
- Kim JF, Beer SV. HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class. J Bacteriol. 1998;180:5203–10.PubMedPubMed CentralGoogle Scholar
- Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagné D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R. The genome of the domesticated apple (Malus × domestica Borkh.). Nat Genet. 2010;42:833–9.View ArticlePubMedGoogle Scholar
- Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science. 1992;257:85–8.View ArticlePubMedGoogle Scholar
- Jin Q, Hu W, Brown I, McGhee G, Hart P, Jones AL, He SY. Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae. Mol Microbiol. 2001;40:1129–39.View ArticlePubMedGoogle Scholar
- Bayot RG, Ries SM. Role of motility in apple blossom infection by Erwinia amylovora and studies of fire blight control with attractant and repellent compounds. Phytopathology. 1986;76:441–5.View ArticleGoogle Scholar
- Bubán T, Orosz-Kovács Z, Farkas Á. The nectary as the primary site of infection by Erwinia amylovora (Burr.) Winslow et al.: a mini review. Plant Syst Evol. 2003;238:183–94.View ArticleGoogle Scholar
- Wilson M, Sigee DC, Epton HAS. Erwinia amylovra infection of hawthorn blossom: III the nectary. J Phytopathol. 1990;128:62–74.View ArticleGoogle Scholar
- Cesbron S, Paulin J-P, Tharaud M, Barny M-A, Brisset M-N. The alternative sigma factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions. FEMS Microbiol Lett. 2006;257:221–7.View ArticlePubMedGoogle Scholar
- Zhao Y, Wang D, Nakka S, Sundin GW, Korban SS. Systems level analysis of two-component signal transduction systems in Erwinia amylovora: role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC Genomics. 2009;10:245.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu L, Hao S, Lan R, Wang G, Xiao D, Sun H, Xu J. The type VI secretion system modulates flagellar gene expression and secretion in Citrobacter freundii and contributes to adhesion and cytotoxicity to host cells. Infect Immun. 2015;83:2596–604.View ArticlePubMedPubMed CentralGoogle Scholar
- Benini S, Caputi L, Cianci M. Cloning, purification, crystallization and 1.57 Å resolution X-ray data analysis of AmsI, the tyrosine phosphatase controlling amylovoran biosynthesis in the plant pathogen Erwinia amylovora. Acta Crystallogr. F Struct Biol Commun. 2014;70:1693–6.View ArticleGoogle Scholar
- Kamber T, Smits THM, Rezzonico F, Duffy B. Genomics and current genetic understanding of Erwinia amylovora and the fire blight antagonist Pantoea vagans. Trees Struct. Funct. 2012;26:227–38.View ArticleGoogle Scholar
- Cabrefiga J, Montesinos E. Analysis of aggressiveness of Erwinia amylovora using disease-dose and time relationships. Phytopathology. 2005;95:1430–7.View ArticlePubMedGoogle Scholar
- Lee SA, Ngugi HK, Halbrendt NO, O'Keefe G, Lehman B, Travis JW, Sinn JP, McNellis TW. Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings. Phytopathology. 2010;100:539–50.View ArticlePubMedGoogle Scholar
- Przybyla AA, Bokszczanin KL, Schollenberger M, Gozdowski D, Madry W, Odziemkowski S. Fire blight resistance of pear genotypes from different European countries. Trees Struct Funct. 2012;26:191–7.View ArticleGoogle Scholar
- Mann RA, Smits THM, Bühlmann A, Blom J, Goesmann A, Frey JE, Plummer KM, Beer SV, Luck J, Duffy B, Rodoni B. Comparative genomics of 12 strains of Erwinia amylovora identifies a pan-genome with a large conserved core. PLoS One. 2013;8:e55644.View ArticlePubMedPubMed CentralGoogle Scholar
- Smits THM, Rezzonico F, López MM, Blom J, Goesmann A, Frey JE, Duffy B. Phylogenetic position and virulence apparatus of the pear flower necrosis pathogen Erwinia piriflorinigrans CFBP 5888T as assessed by comparative genomics. Syst Appl Microbiol. 2013;36:449–56.View ArticlePubMedGoogle Scholar
- Smits THM, Jaenicke S, Rezzonico F, Kamber T, Goesmann A, Frey JE, Duffy B. Complete genome sequence of the fire blight pathogen Erwinia pyrifoliae DSM 12163T and comparative genomic insights into plant pathogenicity. BMC Genomics. 2010;11:2.View ArticlePubMedPubMed CentralGoogle Scholar
- Madigan MT, Martinko JM, Parker J. Brock biology of microorganisms. 10th ed. Upper Saddle River: Pearson Education, Inc.; 2003.Google Scholar
- Paulin J-P, Samson R. Le feu bactérien en France. II.—caractères des souches d’Erwinia amylovora (Burril) Winslow et al. 1920, isolées du foyer franco-belge. Ann Phytopathol. 1973;5:389–97.Google Scholar