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Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain
Correspondence
Francisco Javier Ollero
fjom{at}us.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Statistical significance of the differences observed using the Mann–Whitney non-parametric test; LCO, lipochitin oligosaccharide; Nops, nodulation outer proteins; T3SS, type III secretion systemThe GenBank/EMBL/DDBJ accession number for the ttsI sequence of Sinorhizobium fredii HH103 is AY184383.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The nodule organogenesis process requires the exchange of symbiotic signals between both members of the symbiosis. Flavonoids exuded by legume roots are recognized by the rhizobial protein NodD, which in turn binds to specific and conserved sequences, called nod boxes, and activates the transcription of the nod genes. These genes encode enzymes responsible for the biosynthesis and secretion of the lipochitin oligosaccharides (LCOs), also called Nod factors. These molecules are recognized by the plant and play an important role in triggering the initiation of nodule organogenesis.
Bacterial surface structures such as exopolysaccharides, lipopolysaccharides and capsular polysaccharides, or type III-secreted proteins, are also important for nodulation and host-range determination (Broughton et al., 2000
; Perret et al., 2000).
Some Gram-negative bacterial strains possess a specialized apparatus for protein secretion called the type III secretion system (T3SS). The type III secretion apparatus is formed by about 20 proteins, many of them homologous to proteins involved in the biosynthesis of the flagellum. Symbiotic and pathogenic bacteria use the T3SS to deliver proteins into the eukaryotic host cell (Pallen et al., 2003).
Plant-pathogenic bacteria such as Pseudomonas and Xanthomonas secrete harpins and avirulence proteins through the T3SS. Harpins are probably translocated to the extracellular space, inducing disease in susceptible plants. Avirulence proteins are translocated into the host cytoplasm and are involved in the hypersensitive response (HR) in resistant plants (Galan & Collmer, 1999). Some of the genes involved in the biosynthesis of the machinery of the type III secretion apparatus are well conserved among plant-pathogenic bacteria, and have been named hrp for hypersensitive response and pathogenicity. Genes homologous to hrp genes have been found in some rhizobia, such as Rhizobium sp. NGR234 (Viprey et al., 1998), Sinorhizobium fredii HH103 (de Lyra et al., 2006), S. fredii USDA257 (Krishnan et al., 2003), Mesorhizobium loti MAFF303999 (Kaneko et al., 2000) and Bradyrhizobium japonicum USDA110 (Göttfert et al., 2001). Genes responsible for the biosynthesis of the rhizobial T3SS are organized in the tts region, which also contains genes that encode secreted proteins collectively known as nodulation outer proteins (Nops). To date, eight secreted proteins have been identified: NopA, NopB, NopC, NopD, NopL, NopM, NopP and NopX (Ausmees et al., 2004; Bartsev et al., 2003; Deakin et al., 2005; Krishnan, 2002; Lorio et al., 2004; Rodrigues et al., 2007; Saad et al., 2005; Skorpil et al., 2005). Recently, eight proteins have been found to be type III-secreted in B. japonicum; one of them, GunA2, has been identified as an endoglucanase (Süss et al., 2006).
Despite type III-dependent secretion requiring the presence of flavonoids and the NodD protein, only one gene of the tts region, the ttsI gene, is preceded by a nod box. In Rhizobium sp. NGR234, the expression of ttsI is induced by flavonoids and depends on the transcriptional activator NodD1 (Marie et al., 2004). In the case of B. japonicum, the presence of NodW is also needed (Krause et al., 2002). Amino acid sequence analysis shows that TtsI shares characteristics of two-component response regulators (Marie et al., 2004), and it has been proposed as an intermediary in the regulatory cascade between NodD1 and T3SS-related genes (Viprey et al., 1998). TtsI seems to regulate the expression of some genes by binding to specific promoter sequences called tts boxes (Krause et al., 2002).
Inactivation of ttsI in Rhizobium sp. NGR234 abolishes Nops secretion and affects the symbiosis in a host-dependent manner (Marie et al., 2004). A similar phenotype has been reported in mutant strains of S. fredii USDA257 and HH103 that contain a non-functional secretion machinery (Bellato et al., 1997; de Lyra et al., 2006; Meinhardt et al., 1993).
In this work, we report a complete analysis of the transcriptional regulation of ttsI and other S. fredii HH103 genes that belong to the tts region. We also show how the inactivation of the ttsI gene and its nod box blocks Nops secretion and alters, positively or negatively, the capacity of S. fredii HH103 to nodulate some of its host legumes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Sinorhizobium strains were grown at 28 °C on tryptone yeast (TY) medium (Beringer, 1974) or yeast extract mannitol (YM) medium (Vincent, 1970). Escherichia coli strains were cultured on Luria–Bertani (LB) medium (Sambrook et al. 1989) at 37 °C. When required, the media were supplemented with the appropriate antibiotics as described by Lamrabet et al. (1999). Flavonoids were dissolved in ethanol and used at 1 µg ml–1, which gave final concentrations between 3.0 µM (quercetin) and 6.2 µM (umbelliferone). Plasmids were transferred from E. coli to Sinorhizobium strains by conjugation, as described by Simon (1984), using plasmid pRK2013 as helper.
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. Bacterial cultures at OD660 0.8–1.0 were diluted 100-fold before the addition of flavonoids to ensure that bacterial cultures had an OD660 in the range 0.15–0.30 when β-galactosidase activity was measured (16 h after induction). When necessary, the pH of the YM medium was buffered to pH 6 with MES (20 mM), or to pH 7 or 8 with HEPES (20 mM).
Recombinant DNA techniques were performed according to the general protocols of Sambrook et al. (1989). For hybridization, DNA was blotted to Hybond-N nylon membranes (Amersham), and the DigDNA method of Roche was employed according to the manufacturer's instructions. PCR amplifications were performed as described previously (Vinardell et al., 2004a). Primer pairs used for amplification of the S. fredii HH103 ttsI, nodD2 and nopA genes were, respectively: y4xiF (5'-TAATCAGCCTGGCTGACA) and y4xiR (5'-AACAGAACGAGCGCGTAGA); D2d (5'-CTAACCAAGCCGGAGGA) and D2r (5'-CCGAAGCCGTGTACCA); fy1secF (5'-CCAGGGAGTCCAGATCGTGCA) and fy1secR (5'-GAGGCGTGGTTTACCGATCGA). The NCBI BLAST program was used for homology searches. Plasmids pMUS741 and pMUS746 were obtained, respectively, by cloning a 1.4 kb PCR fragment containing the ttsI gene and its nod box and a 1.4 kb PCR fragment containing the nodD2 gene into the broad-host-range vector pMP92.
The strategy used to generate the different mutant strains described in Table 1
is shown in Fig. 1. Plasmid pK18mob, which is a suicide plasmid in rhizobia, was used for the homogenotization of the mutated versions of the ttsI nod box and the ttsI, nodD2 and nopA genes in S. fredii HH103 RifR.
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Purification and analysis of secreted proteins.
Extracellular proteins from S. fredii strains were recovered from 50 ml of YM bacterial cultures grown on an orbital shaker (180 r.p.m.) for 40 h (
109 bacteria ml–1). Cultures were centrifuged for 20 min at 10 000 g at 4 °C. The supernatants were mixed with three volumes of cold acetone and maintained at –20 °C for 24 h. The mixtures were centrifuged for 45 min at 22 000 g at 4 °C. Dried pellets were resuspended in 300 µl sample buffer [62.5 mM Tris/HCl, pH 6.8, 2 % (w/v) SDS, 10 % (w/v) glycerol, 5 % (w/v) β-mercaptoethanol and 0.001 % (w/v) Bromophenol Blue]. Extracellular proteins were separated by SDS-PAGE using the discontinuous buffer system of Laemmli (1970). Electrophoresis was performed on 15 % (w/v) SDS-polyacrylamide gels and proteins were visualized by silver staining.
For immunostaining, extracellular proteins were separated on 15 % (w/v) SDS-polyacrylamide gels and electroblotted to Immun-Blot PVDF membranes (Bio-Rad) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Membranes were blocked with Tris-buffered saline (Sambrook et al., 1989) containing 2 % (w/v) BSA and then incubated with antibodies raised against NopA (Bartsev et al., 2003; Deakin et al., 2005) diluted 1 : 3000 in the same solution. Anti-rabbit immunoglobulin antibodies (alkaline phosphatase conjugate) were used as secondary antibodies. Reaction results were visualized using nitro-blue tetrazolium chloride–5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (NBT–BCIP) [45 µl NBT solution (75 mg ml–1 in 70 %, v/v, N,N-dimethylformamide) and 35 µl BCIP solution (50 mg ml–1 in N,N-dimethylformamide) were added to 10 ml developing buffer (80 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2)].
Plant assays.
Nodulation assays on Glycine max (L.) Merrill cvs Williams, Peking, Heinong 33, Kochi and Tribune, and on Erythrina variegata (L.) were performed as described by de Lyra et al. (2006). Each Leonard jar contained two plants (one plant in the case of E. variegata). For Glycyrrhiza uralensis, nodulation assays were carried out in mini-Leonard jars (200 ml for the upper part containing vermiculite and 170 ml for the reservoir containing the plant nutritive solution) according to Vinardell et al. (2004a). Each plant was inoculated with 5x108 bacteria. Plants were grown for 42 days (90 days in the case of E. variegata) with a 16 h photoperiod at 25 °C in the light and 18 °C in the dark. Plant tops were dried at 70 °C for 48 h and weighed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The 1405 bp sequenced fragment contains one complete ORF, the ttsI gene, which extends between positions 589 and 1269 and shows 100, 99, 79 and 69 % identity to the ttsI genes of S. fredii USDA257 (AF229441), Rhizobium sp. NGR234 (U00090), M. loti MAFF303099 (BA000012) and B. japonicum USDA110 (AF322012), respectively. The deduced protein sequence of the S. fredii HH103 ttsI gene (226 aa, AAO25539) was 100, 99, 80 and 67 % identical to those of strains S. fredii USDA257 (226 aa, AAL98682), Rhizobium sp. NGR234 (226 aa, AAB91932), M. loti MAFF303099 (223 aa, BAB52643) and B. japonicum USDA110 (229 aa, BAC47108).
The nod box located upstream of the Rhizobium sp. NGR234 ttsI gene was also present in S. fredii HH103 (positions 251–295), with only one mismatch (position 254: T instead of C). Interestingly, a well-conserved NolR-binding site (TTTAGGATTGGGTAAT), extending between positions 83 and 98 (Vinardell et al., 2004a), was also found upstream of the nod box.
As described in Methods, three ttsI mutants were generated. Mutants SVQ533 and SVQ534 harbour the 
interposon and the lacZ-GmR cassette, respectively, in the ttsI coding sequence. Strain SVQ519 harbours the interposon in the nod box upstream of ttsI. These mutants showed no alteration in LCO, LPS and plasmid profiles when compared with the parental strain HH103 RifR (data not shown).
Regulation of ttsI by NodD1, NodD2 and NolR
As previously mentioned, type III secretion of proteins is a flavonoid-dependent process that requires the presence of NodD (Krause et al., 2002; Marie et al., 2004). The role of other regulatory genes, such as nodD2 and nolR, in Nops secretion has also been investigated (Krishnan et al., 1995; Marie et al., 2003; Vinardell et al., 2004a).
To elucidate the effects of the S. fredii HH103 NodD1, NodD2 and NolR proteins on Nops secretion, we first individually analysed their effect on the transcriptional regulation of ttsI. For this purpose, the ttsI : : lacZ-GmR mutation was homogenotized into nodD1, nodD2 and nolR mutant backgrounds. Thus, three double mutants were obtained: SVQ544 (HH103 RifR nodD1 : : ttsI : : lacZ-GmR), SVQ553 (HH103 RifR nolR : : ttsI : : lacZ-GmR), and SVQ545 (HH103 RifR nodD2 : : ttsI : : lacZ-GmR). In addition, plasmids pMUS296, pMUS746 and pMUS675, which harbour the nodD1, nodD2,and nolR genes, respectively, subcloned into plasmid pMP92, were transferred by conjugation to strain SVQ534 (HH103 RifR ttsI : : lacZ-GmR).
The results are summarized in Table 2. As expected, the activity of ttsI in the absence of NodD1 could only reach basal levels when induced with genistein, confirming that the expression of ttsI depends on NodD1. The presence of multiple copies of nodD1 significantly increased the expression of the ttsI : : lacZ-GmR fusion when compared with the control without plasmid. In marked contrast, expression of ttsI in the presence of genistein and multiple copies of nolR showed a statistically significant decrease, whereas the inactivation of the nolR gene caused a twofold increase in the expression of ttsI. Although NodD2 has been described as being necessary for Nops secretion in other rhizobial strains, the inactivation or overexpression of the nodD2 gene did not exert a clear effect on the transcription of ttsI.
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, indicate that the best flavonoid inducers for ttsI were coumestrol, genistein and daidzein (all released by roots of soybeans McCall and Peking; Pueppke et al., 1998), apigenin and morin. Coumestrol was the most potent inducer among these five flavonoids. Curiously, 7-hydroxyflavone, naringenin, hesperetin and isoliquiritigenin, which induce the expression of nodA at least sixfold in comparison with the control without flavonoids, could only induce the expression of ttsI less than twofold. Other good inducer flavonoids for nodA, such as 7,4'-dihydroxyflavone, quercetin, fisetin and chrysin, only induced the expression of ttsI twofold.
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). As expression of T3SS genes occurs in the early stages of the infection process (Perret et al., 1999), the environmental pH could be a factor affecting the activity of these genes. To elucidate how the pH could affect the expression of ttsI and nodA, β-galactosidase assays were carried out, adjusting the pH of the YM medium to the range 6–8. Results obtained indicated that the expression of both ttsI and nodA, when induced with genistein, showed its maximum value at pH 6 and then decreased progressively to pH 8 (Table 4).
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As shown in Table 5, overexpression of ttsI significantly increased the activity of all genes tested upon induction with genistein. Interestingly, their activity was also enhanced in the presence of extra copies of ttsI in the absence of an inducer, almost reaching the values obtained when induced with genistein.
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nopA : : lacZ-GmR was constructed. As shown in Table 5, addition of genistein strongly induced the transcription of nopA, but this activation dropped to basal levels in the ttsI mutant background, suggesting that expression of nopA is strictly TtsI-dependent.
Semiquantitative RT-PCR assays were also carried out to study the expression of the nopA and rhcQ genes in ttsI and nodD1 mutant backgrounds and in the absence or presence of genistein. An internal fragment of the HH103 16S rDNA was used as a control. As shown in Fig. 2, inactivation of nodD1 or ttsI led to a basal level of transcription of the rhcQ and nopA genes. However, addition of genistein to the growth media clearly enhanced both nopA and rhcQ transcript levels.
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have shown previously that HH103 secretes, in response to flavonoids, at least eight Nops with approximate molecular masses of 180 kDa (NopD), 60 kDa (NopX and NopM), 37 or 34 kDa (NopL), 31 kDa (NopP), 21 kDa (NopB), 12 kDa (NopC) and 6 kDa (NopA).
SDS-PAGE experiments showed that inactivation of ttsI or its nod box completely abolished secretion of Nops to the extracellular medium upon induction with flavonoids, indicating that the nod box preceding ttsI is essential for Nops secretion (Fig. 3). Protein secretion could not be restored when a broad-host-range plasmid carrying ttsI (pMUS741) was transferred to any of the mutant strains (data not shown). However, transfer of plasmid pMUS984, a derivative of plasmid pMP92 that carries the ttsI, rhcC2 and y4xK genes, to SVQ533 restored its capacity to secrete Nops to the extracellular medium in response to flavonoids (Fig. 3).
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). In this work, the effect of the mutation of nodD1 or nodD2 on Nops secretion was analysed. As shown in Fig. 3, inactivation of nodD1 completely abolished Nops secretion, whereas the mutation of nodD2 had no effect on secretion of proteins to the extracellular medium upon induction with genistein. These results were confirmed by using specific antibodies raised against NopA. This protein was not detected in the supernatants of the ttsI or nodD1 mutant strains when induced with genistein (Fig. 3b).
Secretion of Nops did not become constitutive in the presence of multiple copies of ttsI (Fig. 4a, b) or a DNA fragment that contained the ttsI, rhcC2 and y4xK genes (data not shown). However, the nopX, rhcJ and nopA genes were expressed in the presence of multiple copies of ttsI but in the absence of flavonoids (Table 5). The absence of Nops secretion in the presence of flavonoids when multiple copies of the nolR gene were present (Vinardell et al., 2004a) was confirmed by using anti-NopA antiserum (Fig. 4b). In contrast to the results obtained by Krishnan et al. (1995), constitutive secretion of Nops could not be observed in the presence of extra copies of nodD2 (Fig. 4).
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) were determined in plant infection tests with five different soybean cultivars: Williams, Peking, Kochi, Tribune and Heinong 33, all of which are effectively nodulated by the wild-type strain S. fredii HH103. Effectors secreted by the T3SSs are usually recognized by specific plant receptors. Therefore, distinct soybean varieties could show differences in these receptors that would change their symbiotic behaviour with HH103. Two other hosts, E. variegata, a tropical legume that is poorly nodulated by HH103, and Glycyrrhiza uralensis, which forms indeterminate nodules, were also tested.
In Glycine max cultivars Williams and Peking, the number and fresh weight of nodules formed, as well as the plant-top dry weight, were significantly lower (=5 %, where is the statistical significance of the differences observed using the Mann–Whitney non-parametric test) in plants inoculated with the mutant strain SVQ519 than in those inoculated with the parental strain HH103 RifR. In soybean cultivars Kochi and Heinong 33, only the number of nodules was significantly reduced (=5 and 10 %, respectively). No differences were detected between mutant SVQ519 and the parental strain HH103 RifR in ‘Tribune’ soybean (Table 6). Complementation of the mutant SVQ533 with plasmid pMUS984 restored its capacity to nodulate soybean Williams to that of the parental strain HH103 RifR (data not shown).
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=5 %) in plants inoculated with SVQ519 than in those inoculated with the parental strain (Table 6).
In marked contrast, in E. variegata there was a significant increase (=5 %) in nodule number, fresh weight and plant-top dry weight when plants were inoculated with mutant SVQ519 in comparison with those inoculated with HH103 RifR (Table 6). These data suggest that Nops may be acting as a positive factor for a successful nodulation of S. fredii HH103 on some Glycine max cultivars and Glycyrrhiza uralensis, but could be interfering with nodulation in E. variegata.
Strains SVQ533 (HH103 RifR ttsI : : ) and SVQ534 (HH103 RifR ttsI : : lacZ-GmR) exhibited the same symbiotic phenotype shown by SV519 in Glycine max, E. variegata and Glycyrrhiza uralensis plants (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Viprey et al., 1998). TtsI has been proposed to be an intermediary in the regulatory cascade between NodD1 and T3SS-related genes, and seems to regulate the expression of the tts genes by binding to specific promoter sequences called tts boxes (Krause et al., 2002; Viprey et al., 1998). We have shown that the S. fredii HH103 ttsI gene is also activated by NodD1 and flavonoids (Table 2). As expected, inactivation of nodD1 completely abolished Nops secretion (Fig. 3). In contrast, the absence of the repressor protein NolR enhanced the expression of ttsI (Table 2) but did not cause an apparent change in the profiles of secreted proteins upon induction with genistein (Vinardell et al., 2004a; Fig. 3). However, the presence of multiple copies of nolR clearly reduced the activity of ttsI (Table 2) and repressed secretion of Nops (Vinardell et al., 2004a; Fig. 4). In S. meliloti, NolR repressed both nodD1 and nodD2, causing a general decrease in nod gene expression (Cren et al., 1995). In S. fredii HH103 the presence of multiple copies of nolR repressed the transcription not only of nod genes but also of several tts genes, including nopX and rhcJ (Vinardell et al., 2004a). In this strain, the nodD1 and ttsI coding regions are preceded by a NolR-binding site. Therefore, the negative effect of NolR on the activity of ttsI and in Nops secretion could be explained by a repression of nodD1, which is necessary for the transcription of ttsI, by a repression of ttsI mediated by direct binding of NolR to the NolR-binding site upstream of ttsI, or by both mechanisms.
The presence of multiple copies of the nodD2 gene in S. fredii USDA257 makes Nops secretion constitutive (Bellato et al., 1997; Krishnan et al., 1995). However, in Rhizobium sp. NGR234 the presence of multiple copies of the nodD2 gene blocks Nops secretion (Marie et al., 2003). In contrast, in S. fredii HH103 no changes in the activity of ttsI or in Nops secretion were observed, either when the nodD2 gene was mutated or when it was overexpressed, suggesting that nodD2 is not involved in the regulation of expression of ttsI in S. fredii HH103 (Table 2, Fig. 4).
Inactivation of the S. fredii HH103 ttsI gene completely abolished secretion of Nops to the extracellular medium (Fig. 3). Moreover, inactivation of the nod box preceding ttsI gave the same protein profile as the ttsI mutant strain, confirming for the first time, to our knowledge, that this nod box is essential for the expression of the tts genes and secretion of nodulation outer proteins (Fig. 3). This is in agreement with the transcriptional regulation function assigned to TtsI. The S. fredii HH103 ttsI mutant was only complemented by a DNA fragment that, in addition to the ttsI gene, carries the rhcC2 and y4xK genes (Fig. 3a, b). This result suggests that these genes constitute an operon. If this were the case, the mutation of the ttsI gene would have a polar effect on the transcription of downstream genes, which could be required for the biosynthesis of the secretion apparatus. The rhcC2 gene encodes a secretin that is thought to be required for the biosynthesis of the type III secretion apparatus, while the amino acid sequence of Y4xK indicates that it could be a lipoprotein (Marie et al., 2004; Viprey et al., 1998). To our knowledge, this is the first time that it has been possible to complement a mutation in a rhizobial ttsI gene.
Despite the dependence of the expression of both ttsI and nodA on NodD1, clear differences in the capacity of certain flavonoids to activate these genes were found (Table 3). The mechanism by which different flavonoids can differentially modulate the expression of the nod and tts genes is currently unknown. Although clear differences in flavonoid-mediated expression were found between ttsI and nodA, the expression of both genes was much higher at acidic than at alkaline pH (Table 4). The fact that ttsI is responsive to differences in pH suggests that the ttsI gene could be sensitive to environmental changes that may occur in the rhizosphere in the early steps of the symbiotic interaction.
The HH103 ttsI gene is responsible for the transcriptional activation of at least four genes of the tts region, nopX, nopA, rhcJ and rhcQ (Table 5). Proteins NopX and NopA have been described as being associated with pilus-like surface appendages (Deakin et al., 2005; Krishnan et al., 2003), whereas RhcJ and RhcQ are components of the type III secretion machinery (Viprey et al., 1998). Nucleotide sequence analysis revealed the presence of a tts box-like element upstream of the coding region of these genes in HH103. Overexpression of ttsI and induction with genistein caused an increase in the activity of these genes when compared with the parental strain (Table 5). In addition, inactivation of ttsI completely blocked the expression of nopA and rhcQ (Table 5, Fig. 2). These results suggest that TtsI acts as a transcriptional activator of the tts genes and are in agreement with those reported by Krause et al. (2002) and Marie et al. (2004) that show that TtsI is responsible for the activation of nolU, nopP and rhcV in B. japonicum USDA110 and of the nopB operon in Rhizobium sp. NGR234. Interestingly, addition of extra copies of ttsI induced the expression of nopX, rhcJ and nopA in the absence of flavonoids, making their expression constitutive (Table 5). However, ttsI was not constitutively expressed in the presence of extra copies of nodD1, although in this case the expression of the ttsI gene upon induction with genistein was double that obtained without extra copies of nodD1 (Table 2). It is also remarkable that the constitutive expression of the tts genes due to the presence of extra copies of ttsI (Table 5) did not result in constitutive Nops secretion (Fig. 4), suggesting that extra regulatory controls could prevent the translation of the constitutively expressed tts mRNAs.
The symbiotic role of nodulation outer proteins has been mainly investigated in Rhizobium sp. NGR234, S. fredii strains HH103 and USDA257, and B. japonicum USDA110. In Rhizobium sp. NGR234, tts mutants show different symbiotic phenotypes depending on the host plant (Viprey et al., 1998). Abolition of Nops secretion results in delayed nodulation of B. japonicum USDA110 on soybean Williams (Krause et al., 2002). In S. fredii, different strain-specific phenotypes have been observed. In strain USDA257, mutants in genes belonging to the nolXWBTUV locus gain the ability to induce nitrogen-fixing nodules on agronomically improved American soybean cultivars that are not naturally nodulated by the wild-type strain (Meinhardt et al., 1993). In contrast, in HH103, which naturally induces the formation of nitrogen-fixing nodules in both American and Asiatic soybeans, an rhcJ mutant strain is impaired in its symbiotic capacity with soybean cultivars Williams and Peking. However, it shows an improved symbiotic behaviour with E. variegata (de Lyra et al., 2006).
The inactivation of the S. fredii HH103 ttsI gene provoked similar but more dramatic symbiotic effects on Williams soybean than the inactivation of the rhcJ gene (de Lyra et al., 2006). When compared with the parental strain, the reductions in nodule number and plant-top dry weight were, respectively, 68.5 and 78.2 % for the ttsI mutant and 40.0 and 24.6 % for the rhcJ derivative. Similar results were observed in Peking soybean. In symbiosis with E. variegata, the positive effect of the inactivation of ttsI was stronger than that caused by the rhcJ mutation: increases of 372.4 versus 270.8 % in nodule number and of 664.4 versus 348.2 % in plant-top dry weight when compared with the parental strain.
Although it is difficult to compare results of different nodulation assays, the differences observed between the symbiotic capacities of the rhcJ and ttsI mutants could suggest a higher symbiotic relevance for the ttsI gene. The mutation of the rhcJ gene blocks the biosynthesis of the secretion machinery, but the lack of the transcriptional regulator TtsI could be able to affect not only the secretion of Nops but also the biosynthesis of other symbiotic signals in S. fredii HH103.
In Rhizobium sp. NGR234, ttsI activates the transcription of the rmlBDAwbg operon that is involved in the synthesis of rhamnose-rich polysaccharides (Marie et al., 2004). However, HH103 did not synthesize this polysaccharide. In fact, hybridization studies showed that the rmlB gene was not present in the genome of this S. fredii strain (data not shown). The HH103 ttsI mutant showed the same LPS and LCO profiles as the parental strain, and its production of exopolysaccharide was also not affected (data not shown). However, we cannot discount the possibility that other important symbiotic signals, such as capsular polysaccharides, cyclic glucans or even molecules still unknown, could be controlled by ttsI.
In S. fredii HH103, the inactivation of the ttsI gene completely abolished Nops secretion, and the symbiotic effect of the mutation was host-dependent. Thus, the absence of Nops was detrimental to the symbiosis with all the Glycine max cultivars tested and with Glycyrrhiza uralensis, but beneficial in the case of the tropical legume E. variegata (Table 6). Glycine max and E. variegata plants form determinate nodules, whereas Glycine uralensis forms indeterminate nodules. Therefore, the beneficial or detrimental effect exerted by S. fredii HH103 Nops does not depend on the type of nodule formed by the host legume.
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ACKNOWLEDGEMENTS |
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á (MCyT) (AGL2006-13758-C05-03) for funding the project. We also thank Dr W. J. Deakin (Département de Biologie Végétale, University of Geneva, Switzerland) for the generous gift of the NopA antibodies. Edited by: H.-M. Fischer
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Received 27 December 2007;
revised 23 February 2008;
accepted 4 March 2008.
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