Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system.

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence. Previous studies showed that the D. dadantii T2SS secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins possess a signal peptide and are first addressed to the periplasm before their recruitment by Out. T2SS-specific secretion signals remain unknown which prevents in silico identification of T2SS substrates. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced and encoding proteins with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

Introduction

Soft rot Pectobacteriaceae (SRP), Dickeya and Pectobacterium, are plant pathogenic bacteria that can provoke disease on more than 35% of angiosperm plant orders, including both monocot and dicot plants [1]. Among those, there is a wide range of plants of agronomic interest such as potato, rice, chicory, cabbage or ornementals on which they can cause severe losses. Symptoms are usually soft rot but these bacteria can provoke blackleg or wilting on aerial parts of potato. Recently, diseases on woody plants caused by Dickeya have been reported [2]. There is no efficient way to fight these bacterial diseases. There are actually twelve species of Dickeya described, isolated either from infected plants (type strain of D. chrysanthemi isolated from Chrysanthemum morifolium, D. dadantii subsp. dadantii from Pelargonium capitum, D. dadantii subsp. diffenbachiae from Dieffenbachia sp., D. dianthicola from Dianthus caryophillus, D. zeae and D. parazeae from Zea mays, D. oryzae from Oryza sativa, D. solani from Solanum tuberosum, D. fangzhongdai from Pyrus pyrifolia, D. poaceiphila from Saccharum officinarum) [3-8] or from river of lake waters (D. aquatica, D. lacustris and D. undicola) [9-11]. Recently D. paradisiaca isolated from Musa paradisiaca, was renamed Musicola paradisiaca [12]. The role of protein secretion systems on the onset of the disease provoked by these bacteria has been recognized long ago [13]. In contrast to many plant pathogenic bacteria, the type three Hrp secretion system is not the main determinant for SRP virulence [14]. The main virulence factor for these bacteria is a type 2 secretion system (T2SS) named Out. It allows the secretion of enzymes that degrade the components of the plant cell wall, leading to the soft rot symptom distinctive of the disease. The first Out-secreted proteins to be identified were a set of pectinases and a cellulase which are easily detectable by simple enzymatic tests [13,15].The pectinolytic T2SS secretome of the model strain D. dadantii 3937 has been studied in detail by cloning the genes of these easily detectable enzymes. D. dadantii secretes by the Out machinery nine pectate lyases, one pectin methylesterase, one pectin acetylesterase and one rhamnogalacturonate lyase [16]. A proteomic analysis of the secreted proteins by 2D gel electrophoresis allowed the identification of two other secreted proteins, the feruloyl esterase FaeD and a protein with homology to a Xanthomonas campestris avirulence protein AvrL [17]. A search in D. dadantii of homologues of proteins secreted by the Out T2SS of Pectobacterium atrosepticum [18] recently led to the characterization of the metal binding protein IbpS [19]. There is no strict host specificity for Dickeya species, however some of them show a loose association for some plant species. Since all the pectinolytic enzymes studied in D. dadantii are present in most of other Dickeya species these enzymes are probably not responsible for the host preference observed for these bacteria [20]. We hypothesized that additional T2SS-secreted proteins specific for some species might exist and play a role in the host preference. To identify such proteins, we analyzed previously published D. dadantii transcriptome data, looking for genes induced in plant infection conditions and encoding proteins with a signal sequence. We identified several proteins secreted by the Out machinery and showed that two proteins of the DUF4879 family, SvfA and SvfB are D. dadantii virulence factors.

Results

Identification of new out-secreted proteins

To have a more complete knowledge of the proteins secreted by the D. dadantii Out T2SS that could be involved in the pathogenicity process, we searched for candidate genes in recently published transcriptome data [21,22]. We selected genes strongly induced during plant infection and coding for proteins possessing a signal sequence which is a prerequisite to be secreted by a T2SS. We retained the genes Dda3937_01687, Dda3937_00585 (thereafter named SvfA and SvfB, respectively) and Dda3937_00081 (also named yoaJ). We also retained VirK, a protein of unknown function with a signal sequence identified among the genes controlled by the transcriptional regulator PecS of many virulence factors [23]. Each protein was tagged with a C-terminal His-tag and its secretion was analyzed in the D. dadantii wild type strain, an outD mutant in which the Out machinery is not functional and this strain complemented with a outD-carrying plasmid. The proteins SvfA, SvfB, YoaJ and VirK were detected in the supernatant of the wild type but not of the mutant strain, demonstrating their secretion by the Out machinery (Fig 1). Complementation of mutant strain with the outD plasmid restored secretion of these proteins. Only about 50% of these proteins was secreted, probably because of their production from a multicopy plasmid or interference of the 6-His tag with the protein recruitment. In addition to the full-length SvfA and SvfB, a band of about 10 kDa reacting with the anti-His antibody can be seen in the supernatant of the wild type strain expressing SvfA and SvfB but not in that of the protease deficient strain A350. It has been shown that the pectate lyase PelI is cleaved by proteases in the supernatant of wild type strain to give a protein with HR-inducing property [24] but that it remains intact in strain A350. SvfA and SvfB are probably also N-terminally processed by these proteases. It is interesting to note that the processed proteins have the size of SvfC and YolA from B. subtilis (see below).

Fig 1

Identification of new secreted proteins by D. dadantii.

Wild-type, A6533 outD mutant complemented or not by outD and strain A350 containing plasmid bearing the gene of the protein to test were grown overnight in LB medium. 15 μl of supernatant (S) and cellular (C) fractions were separated by SDS-PAGE. After blotting, the proteins were detected with anti-6His antibody. * indicates the processed form of SvfA and SvfB.

YoaJ is a PecS-regulated gene [23] and it was found among the most induced genes during Arabidopsis infection or culture in the presence of plant extracts [21]. It encodes a protein with homology to expansins. These proteins are able to non-enzymatically loosen cell wall cellulose. They are found in all plants where they have a role in cell wall extension and in many plant pathogenic microorganisms [25]. The D. dadantii expansin YolA could play a similar role. VirK is a protein of unknown function that has homologues in several plant pathogenic bacteria such as R. solanacearum, Agrobacterium tumefaciens, Lonsdalea and Xanthomonas. No symptom for the D. dadantii virK mutant was observed whatever the plant tested [23].

SvfA and SvfB are virulence factors

svfA and svfB are among the most induced D. dadantii genes during Arabidopsis infection or during culture of the bacteria in the presence of plant extracts [21]. They are also strongly expressed during maceration of potato tubers by D. dianthicola and D. solani [26]. The two D. dadantii proteins share 43% identity and 58% similarity in amino acid composition (S1 Fig). SvfA is 187 amino acid long (165 for the mature form, 17.5 kDa) and SvfB is 198 amino acid long (177 for the mature form, 18.9 kDa). These proteins belong to the DUF 4879 family of proteins. Proteins of this family have no known function. YolA, a protein of the DUF 4879 family showing low homology with SvfB, is among the most highly secreted protein of Bacillus subtilis [27]. YolA is also present in B. cereus and in the insect pathogen B. thuringiensis. B. subtilis YolA is shorter than SvfA and SvfB, missing the more variable N-terminal part (S1 Fig). An additional gene of the DUF 4879 family located next to svfA and probably resulting from a duplication is found in D. fangzhongdai and D. undicola. It was named svfC and has 53% homology with D. dadantii SvfA and 34% with D. dadantii SvfB. SvfC is shorter that SvfA and SvfB (126 amino acid for the mature protein, 13.2 kDa) and has the same size as B. cereus YolA (S1 Fig) It possesses a signal sequence, indicating that it could also be secreted by the Out system.

svfA and svfB mutants have been constructed and their pathogenicity has been tested on potato. The svfA mutant was significantly less aggressive than the wild type strain while the svfB mutant was not significantly affected (Fig 2A). Virulence of the svfA mutant could be restored by introduction of a plasmid bearing the wild type svfA gene (Fig 2B). Virulence of the double svfA svfB mutant was further reduced showing that the role of SvfB is additive to that of SvfA (Fig 2A). However, virulence of the double svfA svfB mutant was not as reduced as in an outD mutant, confirming that D. dadantii virulence is multifactorial. Introduction in the double mutant of a plasmid bearing svfA or svfB restored partially virulence (Fig 2A). Thus, genes Dda3937_01687 and Dda3937_00585 were named svfA and svfB for secreted virulence factor A and B.

Fig 2

Virulence of svfA and svfB mutants.

A. Potatoes (n = 9) were infected with the wild type strain, and the svfA, the svfB, the svfA svfB, the outD mutant and the outD complemented strain. Rotten tissue was weighed after 48 h. Statistical tests were performed using the Wilcoxon-Mann-Whitney test. The p-value were compared with an alpha risk of 4%. There is significant difference (p<0.04) between A, B, C and D. B. Complementation of the svfA and the svfA svfB mutants. Potatoes (n = 9) were infected with the wild type strain, the svfA or the svfA svfB mutants containing the empty plasmid pBBR-MCS3 or the plasmid bearing svfA or svfB. Rotten tissue was weighed after 48h. Statistical tests were performed using the Wilcoxon-Mann-Whitney test. The p-value were compared with an alpha risk of 4%. p < 0.001 = ***, p < 0.005 = **, p < 0.01 = *.

All our attempts to overproduce the proteins SvfA and SvfB in order to purify them and to study more precisely their function were unsuccessful because their production was toxic to the bacterial cells engineered to overproduce them.

Expression of svfA and svfB

In an attempt to identify the function of SvfA and SvfB, we analyzed the conditions in which their genes are expressed. We tested the effect of galacturonate and polygalacturonate, two compounds that are inducers of the expression of the main virulence factors, the pectate lyases, and of glucose, which represses it. We also analyzed the effect of mutations in genes controlling several aspects of D. dadantii virulence. KdgR represses the pectinase, pectin catabolism and out genes [28]. Its inducer is 2-keto-3-deoxygluconate, a polygalacturonate and galacturonate catabolic derivative. PecS controls genes encoding the pectinases, diverse secreted protein, the Out machinery and proteins involved in resistance to oxidative stress [29]. PecT is a regulator of the pectate lyase, motility and exopolysaccharide synthesis genes [30]. Pir regulates hyperinduction of pectate lyses in response to plant extracts [31]. GacA, the regulator of the two-component regulatory system GacA-GacS, is a global regulator required for disease expression in response to the metabolic status of the bacteria [32]. Expression of svfA was slightly induced by polygalacturonate but not by galacturonate (Fig 3A). However, expression of this gene was not modified in a kdgR background indicating that induction by polygalacturonate is not mediated by KdgR. Growth in the presence of chicory chunks strongly induced svfA expression as expected from transcriptomic data showing induction in the presence of plant extract. A high concentration of glucose led to a strong induction of svfA expression (Fig 3A). This regulation is mediated by the catabolite repressor protein CRP since a mutation in the crp gene derepressed svfA expression. Thus, Crp is a repressor of svfA. Although it had not been previously identified as a PecS-regulated gene [23], svfA expression is increased in a pecS background. A pir mutation provoked a weak derepression of svfA expression. Neither PecT nor GacA significantly regulate svfA expression (Fig 3A).

Fig 3

Expression of svfA and svfB in various growth conditions.

A. The D. dadantii strain A6418 containing the svfA-uidA fusion and its derivative strains containing an additional regulatory mutation were grown in M63 medium in the presence of the indicated compounds (Y = glycerol, G = glucose, A = galacturonate, PGA = polygalacturonate, E = chicory chunks). Strains with additional mutations were grown with glycerol as a carbon source except the crp mutant that was grown with 0.2% glucose. ß-glucuronidase activity was measured with p-nitrophenyl-ß-D-glucuronate. B. Similar experiment for the D. dadantii strain A6467 containing the svfB-uidA fusion and its derivative strains containing an additional regulatory mutation. Activities are expressed in μmoles of p-nitrophenol produced per minute and per milligram of bacterial dry weight ± standard deviation. Data are expressed as the mean (n  =  6) from six independent experiments. Statistical tests were performed using the Wilcoxon- Mann-Whitney test. The p-value were compared with an alpha risk of 4%. p < 0.001 = ***, p < 0.005 = **, p < 0.01 = *.

Regulation of svfB shows some similarity to that of svfA: it was not induced by galacturonate, polygalacturonate or regulated by KdgR, it was induced by glucose and repressed by Crp, and it was repressed by PecS (Fig 3B). However, a few differences can be noted: in contrast to what is observed with svfA, no induction by plant pieces was observed for svfB and PecT was a repressor of svfB expression while Pir did not seem to control it (Fig 3B).

Occurrence of the new secreted proteins in other Dickeya species and soft rot Pectobacteriaceae

Presence of svfA, svfB, virK and yoaJ was searched in the genome of all the Dickeya type strains, and in a few soft rot Pectobacteriaceae strains (Table 1). Presence and number of proteins of the DUF 4879 family is variable among Dickeya species. The gene svfA is present in all strains except D. zeae, D. chrysanthemi and D. poaceiphila. The gene svfB is present in most species but is absent in D. chrysanthemi, D. poaceiphila, D. undicola and D. aquatica. The gene svfC is found in D. fangzhongdai and D. undicola. Thus, the number of genes of the DUF 4879 family in Dickeya strains varies from 0 to 3. Homologues of the svf genes can also be found in some Pectobacterium strains (Table 1). For example, two copies are present in P. carotovorum subsp carotovorum. However, even in a given species, the gene may be present or not (presence of a homologue of svfB in 10 out of the 23 P. brasiliense strains present in the ASAP data bank (https://asap.ahabs.wisc.edu/asap/home.php). svf genes are absent from M. paradisiaca. Outside Pectobacteriaceae, homologues of svfB can be found in a few Gammaproteobacteriaceae, i.d. in some Photorhabdus, Luteibacter and Pseudoalteromonas strains. yoaJ is present in all Dickeya and Pectobacterium strains except D. poaceiphila. virK is present in all Dickeya strains except in D. aquatica and absent in all Pectobacterium strains tested.

Table 1

Presence of Out-secreted proteins in various Dickeya, Musicola and Pectobacterium strains.

Strain	svfA	svfB	svfC	YoaJ	VirK	IbpS	NipE	XynA	AvrL
D. dadantii	1	1	0	1	1	1	1	1	2
D. diffenbachiae	1	1	0	1	1	1	1	0	2
D. fangzhongdai	1	1	1	1	1	1	1	1	2
D. solani	1	1	0	1	1	1	1	1	2
D. zeae	0	1	0	1	1	1	1	1	1
D. oryzae	1	1	0	1	1	1	1	1	0
D. parazeae	0	1	0	1	1	1	1	1	1
D. dianthicola	0	1	0	1	1	1	1	0	1
D. undicola	1	0	1	1	1	1	1	0	1
D. lacustris	1	0	0	0	0	1	1	0	2
D. aquatica	1	0	0	1	0	1	1	0	2
D. chrysanthemi	0	0	0	1	1	1	1	0	1
D. poaceiphila	0	0	0	0	1	1	0	1	0
M. paradisiaca	0	0	0	0	1	0	0	0	0
P. atrosepticum	0	0	0	1	0	1	1	0	1
P. carotovorum	1	1	0	1	0	1	1	0	1
P. parmentieri	0	0	0	1	0	1	1	0	1
P. polaris	0	0	0	1	0	1	1	0	1

The strains used in this study are D. dadantii 3937, D. aquatica 174/2, D. chrysanthemi ATCC 11663, D. dadantii subsp dieffenbachiae NCPPB 2976, D. dianthicola NCPPB 453, D. fanghzongdai DSM 101947, D. lacustris S29, D. oryzae ZYY5, D. parazeae 586, D. poaceiphila NCPPB 569, D. solani IPO 2222, D. undicola 2B12, D. zeae NCPPB 2538, M. paradisiaca ATCC 33242P. atrosepticum ATCC 33260, P. carotovorum subsp. carotovorum ATCC 15713, P. parmentieri RNS08.42.1A and P. polaris NIBIO 1006. The presence and number of proteins detected by search of the corresponding gene in the genome in each strain is indicated.

We also examined the presence or absence of genes of other non-pectinolytic proteins known to be secreted by a T2SS in Dickeya or Pectobacterium: ibpS, nipE, xynA, avrL/avrM (Table 1). IbpS is a metal binding protein that prevents ROS-induced killing of bacteria [19]. NipE is a toxin that provoke plant cell death [33]. XynA is a xylanase that was identified in a corn strain of Dickeya zeae previously named Erwinia chrysanthemi [34]. AvrL is homologous to the Xanthomonas campestris avirulence protein AvrL [17]. Two very similar proteins, AvrL and AvrM, are produced by D. dadantii 3937. AvrL was named Svx in P. atrosepticum where its role in virulence has been shown [35]. However, its function in Dickeya has not been studied. IbpS is present in almost all the species, except M. paradisiaca. NipE is absent in M. paradisiaca and D. poaceiphila. Presence of XynA is variable in Dickeya strains and it is absent in Pectobacterium. A variation in the presence and number of AvrL can be observed in Dickeya strains (Table 1). Thus, the repertoire of T2SS-secreted protein known to be important for virulence is very variable from species to species.

Discussion

The T2SS of Dickeya and Pectobacterium is a major virulence factor of these bacteria. The knowledge of the repertoire of secreted proteins is necessary to better understand the precise mechanisms of virulence of these bacteria. These analyses have been undertaken with the model strain D. dadantii 3937 and partially with Pectobacterium atrosepticum [18,36]. Dickeya and Pectobacterium are characterized by their ability to degrade pectin and they are identified by this characteristic on the semi selective Crystal Violet Pectate medium. They all secrete enzymes capable of degrading pectin (pectate lyases, polygalacturonases, pectin methylesterases). However, recent works show that other proteins are secreted by the Out T2SS [17,18]. In the present work we used published transcriptome data to identify new potential substrates of the D. dadantii T2SS. The most highly induced genes in a transcriptome experiment of D. dadantii infecting A. thaliana are known virulence genes (pelI, prtA, rhiE, paeY, rhaD, ibpS, etc…) [21]. However, in this top list some genes have no known function. The presence of a signal sequence in their product suggested that these proteins could be substrates of the T2SS necessary for the infection process. We showed here that the proteins SvfA, SvfB and YoaJ produced by genes present in the top list of those induced in Arabidopsis are substrates of the Out T2SS. YoaJ belongs to the family of expansins, proteins that loosen cellulose fibers. Their role as a virulence factor has been shown in P. brasiliense and P. atrosepticum [37] and it probably has the same function in D. dadantii and other Dickeya species. No function could be predicted for SvfA and SvfB which belong to the DUF 4879 family of proteins. However, a reduction of virulence of a svfA mutant and a svfA svfB double mutant observed on potato tubers proves a role of these proteins in the bacterial pathogenicity. Although an additive effect of the mutations was observed, they could not have exactly the same function. The mutants should be tested on various hosts to detect potential differences. It can be supposed that each protein would be more active on one type or one family of plant. Presence of three DUF 4879 proteins in D. fanghzongdai could explain its wide host range, from orchid to pear trees. Presence of homologues of SvfA and SvfB in Photorhabdus and in B. thuringiensis strains, two insect pathogens, indicates that the role of these proteins is not restricted to plant virulence but may participate to a common process of bacterial pathogenicity. We also showed here that the PecS-regulated protein VirK is secreted by Out. No role on virulence had been observed for this protein with the chicory leaf model of infection [23]. Other models should be tested to find the role of this protein.

Regulation of expression of the svfA and svfB genes is atypical for a D. dadantii gene involved in pathogeny. While expression of most of the virulence factors is induced in the presence of pectin or its derivatives through the repressor KdgR and repressed by glucose, that of svfA and svfB is opposite: it is activated by glucose and not controlled by KdgR. Expression of svfA is induced in the presence of plant tissue. This pattern of regulation has been described for ibpS, which is also strongly induced in A. thaliana [19]. This could correspond to conditions encountered during the early phases of infection: pectin has not yet been degraded and glucose and sucrose are plentiful in plant tissues. svfA and ibpS could be among the earliest gene to be induced at the onset of infection, before the genes involved in pectin degradation. However, regulation of these genes by PecS and PecT shows that svfA and svfB are fully integrated in the network of regulators that controls D. dadantii virulence.

This work has extended our knowledge of the Out-dependent secretome of D. dadantii, showing that besides pectinases several other proteins are secreted. If the number of pectinolytic enzymes secreted is almost identical in the various Dickeya species, the number of additional non-pectinolytic secreted proteins varies markedly. Among the proteins analyzed (Table 1), M. paradisiaca has only one (VirK) while D. fanghzongdai has ten. All the intermediate combinations can be found in the various species. There seems to be less variations in the Pectobacterium strains surveyed. It is tempting to speculate that the presence/absence of these proteins could influence the host preference of some Dickeya species, providing additional virulence factors favorable to infect certain hosts. Works that compare Dickeya strains to understand what makes difference in their host range or aggressivity often focus only on the presence of the six known types of secretion systems without analyzing what proteins could be secreted [20,38,39]. An exhaustive analysis of the secreted proteins would be more informative.

Are there other T2SS-secreted proteins to be identified in Dickeya strains? No specific signal is present on T2SS-secreted proteins that would allow their identification. 2D gels which were used in previous studies performed on D. dadantii and P. atrosepticum to identify their secretome have a limited sensitivity [17,35]. More sensitive methods such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) can now be used [40]. However, they give many false positive results since periplasmic and cytoplasmic proteins are often found in the culture supernatant. The approach we used here allowed the identification of four new secreted proteins. However, all these methods have a drawback. They can only detect proteins in conditions where they are produced. For instance, the rhamnogalacturonate lyase RhiE could only be detected when the bacteria were cultivated in the presence of rhamnose [41]. The genes encoding YoaJ and VirK were not induced in D. dianthicola grown on potato [26]. Another problem is that a protein may not exist in the strain tested. An analysis of the secretome of several Dickeya strains grown in several conditions will be necessary to have a global view of all the additional virulence factors that can be secreted by Dickeya species and evaluate their potential role in pathogenicity.

Material and methods

Bacterial strains and growth conditions

Bacterial strains, plasmids and oligonucleotides used in this study are described in S1 Table. D. dadantii and E. coli cells were grown at 30 and 37°C respectively in LB medium or M63 minimal medium supplemented with a carbon source (0.2%, w/v unless otherwise indicated). When required antibiotics were added at the following concentrations: ampicillin, 100 mg/l, kanamycin, tetracycline, 10 mg/l and chloramphenicol, 25 mg/l. Media were solidified with 1.5% (w/v) agar. Transduction with phage ΦEC2 was performed according to Résibois et al. [42].

Mutant construction

To construct strain A6418 that contains a svfA-uidA fusion a 1.3 kb DNA fragment containing svfA was amplified with primers 17176H+ and 17176A. The resulting fragment was inserted into the pGEM-T plasmid (Promega). A XbaI site was created by site directed mutagenesis with the primers 17176XbaF and 17176XbaR into the svfA coding sequence and a uidA-kanR cassette was inserted into this XbaI site. To construct strain A6467 that contains a svfB-uidA-kanR fusion a 2000 bp DNA fragment containing svfB was amplified with the primers 15544L2+ and 15544L2. The resulting fragment was inserted into the pGEM-T plasmid. A XmaI site was created by site directed mutagenesis into svfB coding sequence with the primers 15544XmaF and 15544XmaR and a uidA-kanR cassette was inserted into this created unique XmaI site. To create strain A6417, a CmR cassette was introduced into the XmaI site. All the constructs were recombined into the D. dadantii chromosome according to Roeder and Collmer [41]. Recombinations were checked by PCR. His-tagged versions of the proteins SvfA, SvfB, YoaJ and VirK were constructed by amplifying the corresponding genes with the primers 17176H+ and 17176H-, 15544H+ and 15544H-, 14642H+ and 14642H-, VirKH+ and VirKH-, respectively. The resulting DNA fragments were cloned into plasmid pGEMT. For complementation experiments, the DNA fragment containing svfA was cut from plasmid pGEMT-svfA by PstI and SacII and introduced into the same site of plasmid pBBR-MCS3 and the DNA fragment containing svfB was cut from plasmid pGEMT-svfB by PstI and SacI and introduced into the same sites of plasmid pBBR-MCS3. To construct the plasmid complementing the outD mutation, the outD gene was cut from plasmid pTdB-OD (Shevchik 1997) by HindIII and SmaI and introduced into the same sites of plasmid pBAD33.

Secretion assays and western blots

D. dadantii strains containing the plasmid to test were grown overnight in LB medium in the presence of the appropriate antibiotic. 2 ml of culture were centrifuged at 10,000 g for 3 min, the supernatant was filtered at 0.45 μm, the pellet was resuspended in 2 ml of water and 15 μL and both fractions were loaded onto 12% polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were next transferred onto Immobilon P membrane (Millipore) and probed with anti 6-His antibody (Covalab, Villeurbanne).

Pathogenicity tests

Bacteria were grown overnight in LB medium, centrifuged and resuspended at OD600 1 in M63 medium. Potatoes (var. Jazzy) were surface sterilized with 70% ethanol and dried. A hole was made with a pipette tip and 10 μl of bacteria were deposited in the hole which was covered with mineral oil. Potatoes were placed over a wet paper in a tray contained in a plastic bag to maintain moisture. After 48 h at 30°C, the weight of rotten tissue was measured.

Enzymatic assays

β-glucuronidase assays were performed on toluenized extracts of cells grown to exponential phase using the method of Bardonnet et al [43] with p-nitrophenyl-β-D-glucuronate as the substrate.

Statistical analysis

For all statistical analyses, a non-parametric Wilcoxon-Mann-Whitney test was conducted with a significance level of p<0.04. Statistical analysis was performed using R (v4.1.2) with RStudio (RStudio Team (2022). RStudio: Integrated Development Environment for R. RStudio, PBC, Boston, MA URL; http://www.rstudio.com/)

Alignment of Svf proteins.

The sequences of D. dadantii SvfA (Dda3937_01687) and SvfB (Dda3937_00585), D. fanghzongdai SvfC (CVE23_15565), B. cereus WP-193674364.1 and Photorhabdus asymbiotica CAQ86327.1, without their signal sequence, were aligned with Clustal omega. Identical residues are indicated by a star and chemically equivalent residues by a double dot.

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Strains, plasmids and oligonucleotides used in this study.

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Complete blots.

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