The Sinorhizobium fredii HH103 Lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules

The Sinorhizobium fredii HH103 Lipopolysaccharide Is Not Only Relevant at Early Soybean Nodulation Stages but Also for Symbiosome Stability in Mature Nodules Isabel Margaret 1 , M. Mercedes Lucas 2 , Sebastia ´n Acosta-Jurado 1 , Ana M. Buendı ´a-Claverı ´a 1 , Elena Fedorova 2¤ ,A ´ngeles Hidalgo 1 , Miguel A. Rodrı ´guez-Carvajal 3 , Dulce N. Rodriguez-Navarro 4 , Jose ´E. Ruiz-Sainz 1 , Jose ´M. Vinardell 1 * 1Departamento de Microbiologı ´a, Facultad de Biologı ´a, Universidad de Sevilla. Sevilla, Spain, 2Instituto de Ciencias Agrarias, CSIC, Madrid, Spain, 3Departamento de Quı ´mica Orga ´nica, Facultad de Quı ´mica, Universidad de Sevilla, Sevilla, Spain, 4IFAPA, Centro Las Torres-Tomejil, Alcala ´del Rı ´o (Sevilla), Spain Abstract In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA,lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA,lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean. Citation: Margaret I, Lucas MM, Acosta-Jurado S, Buendı ´a-Claverı ´a AM, Fedorova E, et al. (2013) The Sinorhizobium fredii HH103 Lipopolysaccharide Is Not Only Relevant at Early Soybean Nodulation Stages but Also for Symbiosome Stability in Mature Nodules. PLoS ONE 8(10): e74717. doi:10.1371/journal.pone.0074717 Editor: Biao Ding, The Ohio State University, United States of America Received May 28, 2013; Accepted August 4, 2013; Published October 1, 2013 Copyright: ß2013 Margaret et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Andalusia Government (P07-CVI-02506), the Spanish Ministry of Science and Innovation (BIO2011-30229- C02-01), and the Comunidad de Madrid (Programa Microambiente S2009/AMB-1511). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Department of Molecular Biology, Wageningen University, Wageningen, The Netherlands Introduction The lipopolysaccharide (LPS) is a complex glycolipid component present in the outer leaflet of the Gram-negative bacteria, including rhizobia [1–3]. The LPS of rhizobia shows the same general architecture as that from LPS of enteric and animal Gramnegative pathogens. It can be conceptually divided into three different structural regions: an inner acylated saccharide known as lipid A that is linked to a central core oligosaccharide which is attached to an outer O-chain polysaccharide [4]. A single preparation of rhizobial LPS contains a mixture of LPS molecules. This mixture is usually composed of molecules that carry various lengths of the O-chain polysaccharide, core oligosaccharide, and lipid A, but also contains molecules which are devoid of the O- chain polysaccharide [2]. LPS molecules with O-chain polysaccharide are often referred to as ‘‘smooth’’ LPS (or S-LPS), and those without O-chain as rough LPS, or R-LPS. LPS molecules carrying truncated (shorter) O-chains (‘‘semi-rough’’ or ‘‘semismooth’’ LPS) can also be observed, particularly in some mutants [5]. LPS may play significant roles at different stages of the (Brady/ Sino/Meso)Rhizobium-legume symbioses, such as initial recognition and infection, invasion of root cortical cells, bacterial penetration into plant cells, formation and persistence of functional symbiosomes and nodule senescence. LPS may act as inhibitors of plant defence responses, as bacterial signals, as a physical passive barrier, or as antigens mimicking a plant-like interface [2,6,7]. O-chain rhizobial mutants are defective in indeterminatenodule forming legumes (such as Pisum sativum or Medicago sativa) in the endocytotic invasion of the root nodule cells [8–11]. The presence of an intact LPS is also necessary for the formation of effective determinate nodules. Thus, Rhizobium etli,Bradyrhizobium japonicum, and B. elkanii mutants that lacked the O-chain are defective in their symbioses with determinate-nodule forming legumes, such as Phaseolus vulgaris or Glycine max. In some cases, nodules are not formed while in others the small white nodules PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e74717 formed (pseudonodules) contain aborted infection threads, show severe structural aberrations, and are devoid of bacteria and leghaemoglobin [12–14]. The symbiotic phenotypes reported for rhizobial LPS mutants clearly indicate that the O-chain polysaccharide is necessary to form a normal nitrogen-fixing symbiosis, and that structural changes can mainly occur to the O-chain and to the Lipid A portion of the LPS during the transition from a vegetative bacterium to the functional bacteroid [1]. Sinorhizobium fredii HH103 is a fast growing rhizobial strain that nodulates Glycine max (soybean) and many other determinate- and indeterminate-nodule forming legumes [15]. The genome sequence of S. fredii HH103 has been nearly completed and is available in the EMBL Nucleotide Sequence Database (EMBL- Bank) under accession numbers HE616890 to HE616899 [15,16]. S. fredii HH103 produces at least five different surface polysaccharides: exopolysaccharides (EPS), lipopolysaccharides (LPS), capsular polysaccharides (KPS [K-antigen polysaccharides]), and cyclic glucans (CG). Two different types of KPS are constitutively produced by S. fredii HH103. One of them, called poly-PseAc, is a homopolymer of a derivative of the pseudaminic acid [17], while the other is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) [18]. The chemical structure of the S. fredii HH103 CG has been determined [19]. S. fredii HH103 mutants affected in the production of KPS, CG or EPS have been already constructed and described. HH103 mutants unable to produce EPS are fully effective with soybeans [20], while those unable to produce CG only form small knot-like structures (pseudonodules) that do not fix nitrogen and are devoid of rhizobial cells [19]. The HH103 poly-PseAc (hereafter called ‘‘KPS’’) plays an important role in the S. fredii-soybean symbiosis, since mutants affected in genes of the rkp-1 and rkp-3 regions are symbiotically impaired with soybean [20–23]. All these studies led to the conclusion that KPS and CG, but not EPS, are relevant for the capacity of S. fredii HH103 to nodulate soybean. Sinorhizobium meliloti and S. fredii LPS core regions are closely related [2]. The core oligosaccharide of the S. fredii USDA257 LPS is composed of Kdo, glucose, galactose, glucuronic acid, and galacturonic acid [24]. This carbohydrate composition is similar to that described for the LPS core of S. meliloti 1021 [9]. To our knowledge, neither the structure of the S. fredii HH103 LPS nor the symbiotic capacity of any lps mutant has ever been studied. In this study we constructed S. fredii HH103 mutants affected in two genes (lpsB and lpsE) coding for glycosyl transferases involved in LPS biosynthesis, and in the greA gene, which is located close to lpsB and codes for an elongation transcriptional factor. The greA lpsB lpsCDE cluster is well conserved in the genus Sinorhizobium as well as in Bradyrhizobium japonicum [11,25]. Although S. meliloti 2011 greA,lpsB,lpsE,lpsD, and lpsC mutants show all altered LPS profiles, only the greA mutants are symbiotically impaired with Medicago sativa, an indeterminate nodule-forming legume [11]. In NGR234, a S. fredii strain that does not nodulate soybeans, mutation of the lpsB gene not only alters the LPS profile but also eliminates (an average of less than one nodule per plant) the bacterial nodulation capacity with Vigna unguiculata [25]. Up to now, the effects of greA,lpsB or lpsE mutations on the bacterial symbiotic capacity had not been investigated in rhizobia able to nodulate soybean. Here we show that mutations in the S. fredii HH103 greA,lpsB, and lpsE genes also provoke alterations in the LPS structure. In contrast to the situation described for the S. meliloti-alfalfa symbiotic interaction, all these mutations impaired the S. fredii HH103-soybean symbiosis. Light and transmission electron microscopy (TEM) studies of soybean nodules displaying normal external morphology clearly showed the onset of early nodule senescence. Results Isolation of the S. fredii HH103 greA lpsB lpsCDE Genes Before the HH103 genome sequence became available [16], we employed a previously described PCR-based screening of an HH103 genomic library [19] to isolate cosmid pMUS908 and sequenced a 5646-bp DNA fragment containing the HH103 greA lpsB lpsCDE genes (accession number JX170205). This fragment matches to nucleotides 1578624 to 1572979 of the HH103 chromosome sequence (HE616890). In silico analysis of the sequenced fragment revealed that the S. fredii HH103 greA lpsB lpsCDE genes show the same genetic organization that in S. meliloti 1021 (Figure 1). The putative proteins encoded by these ORFs have 158, 351, 270, 343, and 340 residues and are 98, 83, 82, 80, and 80% identical, respectively, to the corresponding orthologues of S. meliloti 1021. The greA gene, located upstream of lpsB, encodes a transcription elongation factor. The lpsCDE genes are transcribed in the opposite direction that greA and lpsB. The products of lpsB lpsCDE are glycosyl transferases putatively involved in LPS core biosynthesis. In order to investigate the relevance of the lpsB lpsCDE genes of S. fredii HH103 in different bacterial characteristics, such as LPS synthesis and symbiotic capacity, different mutants (Figure 1 and Table 1) were generated as described in Material and Methods: SVQ613 (lpsB::V), SVQ615 (lpsB::lacZDp-Gm R ,RR), SVQ642 (lpsE::lacZDp-Gm R ,RR), and SVQ647 (lpsE::lacZDp-Gm R , Rr). In some mutants the mutated gene and the lacZDp-Gm R cassette had the same transcriptional orientation (RR), while in others was opposite (Rr).We were also interested in investigating whether greA could have an effect on lpsB expression and LPS production. For this purpose, we also generated two independent mutants in the greA gene by using the lacZDp-Gm R cassette; these two mutants differ in the orientation of the lacZ cassette: SVQ655 (greA::lacZDp-Gm R ,RR) and SVQ656 (greA::lacZDp-Gm R ,Rr). S. fredii HH103 greA, lpsB, and lpsE Mutants are Affected in LPS but not in KPS, CG or EPS Production The lipopolysaccharide (LPS) profiles of the greA,lpsB, and lpsE mutants were analyzed by PAGE experiments performed in the presence of SDS (Figure 2A) as well as by immunostaining experiments using a monoclonal antibody, NB6-228.22, that recognizes the LPS of HH103 (Figure 2B). The S-LPS of HH103 Rif R is usually composed of 5 bands [22,23], although the number of visible bands may vary from one preparation to another (Figure 2, panel A, lanes 1 and 8). The LPS electrophoretic profiles of mutants SVQ613 (lpsB::V; lane 2), SVQ615 (lpsB::lacZDp-Gm R ,RR; lane 3), SVQ642 (lpsE::lacZDp- Gm R ,RR; lane 4), SVQ647 (lpsE::lacZDp-Gm R ,Rr; lane 6), and SVQ656 (greA::lacZDp-Gm R ,Rr; lanes 11) were altered in comparison with that of HH103 Rif R (lanes 1 and 8). The LPS electrophoretic profile of mutant SVQ655 (greA::lacZDp-Gm R , RR; lane 9) was not apparently altered. The alteration of the profiles of lpsB,lpsE, and one of the greA (SVQ656) mutants consisted of an increase in the electrophoretic mobility of both S- LPS and R-LPS bands. The mobility of the S-LPS in the lpsB mutants (SVQ613 and SVQ615, lanes 2 and 3) was faster than that observed for mutants SVQ642, SVQ647, and SVQ656 (lanes 4, 6, and 11, respectively). The monoclonal antibody NB6-228.22 recognized the same pattern of LPS bands in S. fredii HH103 Rif R (Figure 2, panel B, lanes 1 and 8) and in its greA mutant derivative SVQ655 (lane 9). However, this antibody only recognized a subset of the S-LPS and R-LPS silver-stained bands of the greA mutant derivative SVQ656 (panel B, lane 11), and failed in the recognition of the fastest silver- S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 2 October 2013 | Volume 8 | Issue 10 | e74717 stained bands that respectively appear in both SVQ656 LPS regions (compare lane 11 of panels A and B) These results indicate that both greA mutants produce LPS immunorelated with those of HH103 but SVQ656 also produces prominent new LPS bands without immunoreactivity. In addition, NB6-228.22 failed in the recognition of the altered LPS produced by mutants SVQ613 Figure 1. Genetic organization of the greA-lpsB-lpsCDE region of S. fredii HH103 and a comparison to that of Sinorhizobium meliloti 1021. Black triangles indicate the position at which the lacZDp-Gm R cassette (in mutants SVQ655, SVQ656, SVQ615, and SVQ642) or the Omega interposon (in mutant SVQ613) were inserted to generate S. fredii HH103 mutants. The predicted effect of the cassette on transcription is showed as a white arrow (indicating direction of transcription from the aacC1 gene belonging to lacZDp-Gm R ) or a black cross (indicating both transcriptional and translational blocking due to V). doi:10.1371/journal.pone.0074717.g001 Table 1. Bacterial strains and plasmids used in this study. Strain or plasmid Derivation and relevant properties Source or reference S. fredii HH103-Rif R ( = SVQ269) Spontaneous Rif R derivative of HH103 [51] SVQ613 HH103-Rif R lpsB::VThis work SVQ615 HH103-Rif R lpsB:: lacZDp-Gm R , same transcriptional direction in lacZ and lpsB This work SVQ642 HH103-Rif R lpsE:: lacZDp-Gm R , same transcriptional direction in lacZ and lpsE This work SVQ647 HH103-Rif R lpsE:: lacZDp-Gm R , opposite transcriptional direction in lacZ and lpsE This work SVQ655 HH103-Rif R greA:: lacZDp-Gm R , Same transcriptional direction in lacZ and greA This work SVQ656 HH103-Rif R greA:: lacZDp-Gm R , opposite transcriptional direction in lacZ and greA This work E. coli DH5asupE44 DlacU169 hsdR17 racA1 endA1 gyr96 thi-1 relA1, Nx R Stratagene Plasmids pAB2001 Ap R vector containing the lacZDp-Gm R cassette [45] pBluescript II SK+Cloning and sequencing vector, Ap R Stratagene pHP45VAp R vector containing the Vinterposon (Spc R Str R )[46] pK18mob Cloning vector, Km R [44] pCPP46 Cloning vector, Tc R [52] pMUS908 Cosmid pLAFR1 carrying the greA and lpsBEDC genes of S. fredii HH103 This work pMUS981 pK18mob +1.5-kb NarI fragment of pMUS908 containing lpsB This work pMUS982 pK18mob containing lpsB::lacZDp-Gm R RR This work pMUS990 pK18mob containing lpsB::VThis work pMUS1005 pK18mob +2.5-kb EcoRI-SphI fragment of pMUS908 containing lpsE This work pMUS1008 pK18mob containing lpsE::lacZDp-Gm R RR This work pMUS1020 pK18mob containing lpsE::lacZDp-Gm R Rr This work pMUS1022 pK18mob containing greA::lacZDp-Gm R Rr This work pMUS1023 pK18mob containing greA::lacZDp-Gm R RR This work pMUS1139 pCPP46 containing lpsB This work pMUS1144 pK18mob containing lpsB This work doi:10.1371/journal.pone.0074717.t001 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 3 October 2013 | Volume 8 | Issue 10 | e74717 (lpsB::V, lane 2), SVQ615 (lpsB::lacZDp-Gm R , lane 3), and SVQ642 and SVQ647 (lpsE::lacZDp-Gm R , lanes 4 and 6). Introduction of cosmid pMUS908, which contains the greA lpsB lpsCDE genes, into S. fredii greA mutants restored the wild type LPS electrophoretic and NB6-228.22 recognition patterns (Figure 2A, B). This approach failed to complement both lpsE and lpsB mutants. S. fredii lpsE mutants could be complemented by the introduction in cis of a copy of the wild type lpsE gene (see Material and Methods), leading to the construction of strains SVQ642C1 and SVQ647C2. All our attempts to complement the lpsB mutations were unsuccessful. In S. fredii HH103 there are several examples of mutations that simultaneously affect the production of various surface polysaccharides [19,21,23]. For this reason, we have analysed the production of other surface polysaccharides in the HH103 lps and greA mutants. PAGE and/or NMR studies showed that K- antigen polysaccharides (KPS) and cyclic glucans (CG) produced by SVQ613, SVQ642, SVQ655, and SVQ656 appear to be identical to that produced by the parental strain HH103 Rif R (Figures S1 and S2). All the greA, lpsB, and lpsE mutants formed colonies in TY and YMA media that were equally mucous to those formed by HH103 Rif R (data not shown), suggesting that EPS production was not altered in these mutants. The S. fredii HH103 greA Mutants Show Reduced Mobility and their Auto-agglutination Capacity can also be Altered Surface polysaccharide alterations can affect some bacterial traits such as motility, auto-agglutination in liquid medium and biofilm formation [26,27]. Because of this, S. fredii HH103 mutants affected in greA,lpsB, and lpsE were investigated for these capacities. Inactivation of lpsB or lpsE did not affect any of these traits (Figure 3A, Figure S3, and data not shown). Interestingly, inactivation of greA did not affect biofilm formation capacity either (Figure S3), but significantly diminished bacterial motility in soft (0.24% agar) TY medium (Figure 3A). In addition, the intensity of auto-agglutination of SVQ656 (greA::lacZDp-Gm R ,Rr) was clearly higher than that shown by HH103 Rif R , while that of SVQ655 (greA::lacZDp-Gm R ,RR) was not apparently affected (Figure 3, panel B). Introduction of cosmid pMUS908 into HH103 greA mutants increased motility and diminished auto-agglutination up to wild type levels (Figure 3A and data not shown). Mutants producing altered LPS forms are frequently more sensitive to detergents [28]. S. fredii HH103 and its mutant derivatives lpsB (SVQ613), lpsE (SVQ642) and greA (SVQ655 and SVQ656) were tested for their sensitivity to different chemical compounds that cause oxidative stress (H 2 O 2 and paraquat) and/ or are present in soybean roots, such as vanillic, ferulic, and Figure 2. S. fredii HH103 lpsB and lpsE mutants and the greA mutant SVQ656 are affected in LPS production. A, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver-staining or B, immuno-staining using the monoclonal antibody NB6-228.22 of lipopolysaccharides (LPS) crude extracts from Sinorhizobium fredii HH103 and its lpsB (SVQ613, SVQ615), lpsE (SVQ642, SVQ647), and greA (SVQ655, SVQ656) mutant derivatives. Lanes 1 and 8, HH103 Rif R ; lane 2, SVQ613; lane 3, SVQ615; lane 4, SVQ642; lane 5, SVQ642 C1; lane 6, SVQ647; lane 7, SVQ647 C2; lane 9, SVQ655; lane 10, SVQ655 carrying pMUS908; lane 11, SVQ656; lane 12, SVQ656 carrying pMUS908. The rough and smooth forms of the wild-type LPS are indicated as R-LPS and S-LPS, respectively. The relevant characteristics (gene mutated, cassette employed and, when necessary, orientation of the cassette) are indicated on the top of each lane. Compl. = complemented. doi:10.1371/journal.pone.0074717.g002 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 4 October 2013 | Volume 8 | Issue 10 | e74717 salicylic acids and the isoflavone genistein [29]. The detergent SDS and ethanol were also included in these assays. S. fredii HH103 Rif R and its mutant derivatives were not significantly inhibited by the presence in the disks of 5 ml of the following stock solutions: 1 M paraquat, 100 mM salicylic acid, 100 mM ferulic acid, 3.7 mM genistein, 100% ethanol, 6.9 mM SDS or 50 mM H 2 O 2 . Only mutant SVQ613 (lpsB) was inhibited by 100 mM vanillic acid (inhibition halo of about 5 mm around the paper disk). S. fredii HH103 lpsB Expression is not Affected by pH or the Presence of Flavonoids It is known that various culture conditions, such as the presence of flavonoids or the medium pH, can affect the expression of genes involved in surface polysaccharide production [19,25,30]. Strain SVQ615 (lpsB::lacZDp-Gm R ) was used for investigating the possible effect of different culture conditions on the expression of the lpsB gene. One of the conditions tested was the presence of genistein, a soybean secreted flavonoid able to induce the transcription of S. fredii HH103 nod genes [31]. b-galactosidase assays of SVQ615 cultures grown in the presence and absence of genistein showed similar LacZ activities (298.8612.1 and 326.265.3 Miller units respectively). Mutant SVQ615 was also cultured in acidic- (pH 6.0), neutral- (pH7.0), and alkalinebuffered (pH 8.0) liquid YM (YMB) media. Non-buffered YMB media, with a pH value of 7.0 at the time of inoculation, was also included. b-galactosidase activity of SVQ615 cultures was similar in all the conditions tested (data not shown). The S. fredii HH103 greA and lpsB Genes are not Cotranscribed but Mutations in greA Reduce the Transcription of lpsB The S. fredii HH103 greA and lpsB genes are adjacent and transcribed in the same orientation. The intergenic greA-lpsB region is composed of 134 bp. The fact that the LPS electrophoretic profile of SVQ656 (in which lacZ and greA are oppositely transcribed) showed clear alterations suggests that greA could influence the transcription of lpsB. To investigate whether greA and lpsB are cotranscribed, we used RT-PCR for searching putative HH103 mRNAs covering the 39-end of greA and the 59-end of lpsB by using primers greAlpsB-F and greAlpsB-R (Figure 4, panels A and B; Table S1). As a positive control, primer pairs greAint-F/greAint- R and rtlpsB-F/rtlpsB-R, which allow the amplification of internal fragments of the greA and lpsB genes respectively, were employed. In addition, we used primers lpsBE-F/lpsBE-R as a negative control because they would lead to the amplification of a DNA fragment covering the 39-end of lpsB and the 39-end of lpsE that cannot be originated by retrotranscription of the total mRNA population since lpsB and lpsE are transcribed in opposite directions. All these primer pairs allowed the amplification of fragments of the expected size when HH103 gDNA was used as template (Figure 4, panel B, lanes 10 to 13). However, when HH103 cDNA was employed, only the internal fragments of greA (lane 6) and lpsB (lane 8) were amplified. All these results indicate that, in S. fredii HH103, greA and lpsB do not belong to the same transcriptional unit. Although the S. fredii HH103 greA and lpsB genes are independently transcribed, the fact that the LPS of mutant SVQ656 showed an altered electrophoretic profile prompted us to investigate whether the transcription of lpsB was affected in a greA- mutant background (Figure 4, panel C). qPCR experiments showed that lpsB expression is about 30-fold reduced in mutant SVQ656 (0.03360.007) when compared to HH103 (assigned the value of 1). The fact that mutant SVQ655 only presents a slight reduction of lpsB expression (0.56960.215) could be due to a polar effect of the lacZDp-Gm R cassette, which, in this mutant strain, has the same transcriptional orientation that lpsB. Symbiotic Properties of S. fredii HH103 greA (SVQ656), lpsB (SVQ613), and lpsE (SVQ642) Mutants The symbiotic phenotype of S. fredii HH103 greA (SVQ655 and SVQ656), lpsB (SVQ613 and SVQ615), and lpsE (SVQ642 and SVQ647) mutants was investigated with G. max cv. Williams (Table 2; Figure S4). Soybean plants inoculated with any of these mutants showed severe symptoms of nitrogen starvation. All the parameters analyzed (number and fresh-weight of nodules and plant-top dry-weight) were significantly decreased in soybean plants inoculated with the greA,lpsB or lpsE mutants in comparison to those of plants inoculated with the wild-type parental strain HH103 Rif R (Table 2). Nitrogen-fixation activity (assessed by acetylene reduction assay [ARA]) was detected in nodules showing normal external morphology (data not shown). In addition to a reduced number of Fix + nodules, the greA,lpsB or lpsE mutants also Figure 3. Inactivation of the S. fredii HH103 greA gene affects swimming capacity and auto-agglutination. A, swimming capacity of (a) S. fredii HH103 Rif R and its (b) lpsB (SVQ613), (c) lpsE (SVQ642), (d,e) greA (SVQ655 and SVQ656) mutant derivatives, and (f) SVQ656 carrying cosmid pMUS908. B, auto-agglutination assays of S. fredii HH103 Rif R and its greA (SVQ655 and SVQ656) mutant derivatives. The optical density of the cultures was monitored at 0, 6, 22, 30, 45, and 69 h of incubation at static conditions. doi:10.1371/journal.pone.0074717.g003 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 5 October 2013 | Volume 8 | Issue 10 | e74717 induced the formation of pseudonodules on soybean roots. The antibiotic-resistance markers of nodule isolates were equal to those of the respective strain used as inoculant. Hence, soybean nodules formed by the different mutants tested were not due to reversions caused by the loss of the cassette or interposon used. Introduction of wild type copies of the mutated gene into HH103 greA or lpsE mutants restored wild type ability to nodulate soybean (Table 2). The symbiotic capacity of mutant SVQ613 (lpsB::V) was also tested with Vigna unguiculata cv. Bisbee Red (cowpea). Although SVQ613 was still able to induce the formation of nitrogen-fixing nodules on cowpea roots, the number of nodules formed was significantly reduced (a= 5%) when compared with the parental strain HH103 Rif R (Table 2). S. fredii HH103 greA (SVQ656), lpsB (SVQ613), and lpsE (SVQ642) Symbiosomes Show Early Senescence in Soybean Nodules To understand the role of greA,lpsB, and lpsE in nodule development in soybean, nodules elicited by HH103 Rif R , SVQ656 (greA), SVQ613 (lpsB), and SVQ642 (lpsE) were examined by light microscopy and transmission electron microscopy (TEM). All the mutants tested induced the formation of pseudonodules and nodules on Glycine max cv. Williams roots. Contrary to the peripheral vascular bundles found in normal soybean nodules (Figure 5), pseudonodules displayed the vascular tissues in a central zone, which was surrounded by sclereid and some other cells (Figure S5). No bacteria were observed by microscopy or were recovered from these pseudonodules. Since nodulation of G. max roots is not a synchronous process, the nodules formed vary in age and size. Therefore, the structure of nodules of different size formed on soybean roots at 21 and 35 days post inoculation (dpi) was studied (Figures 5 and 6; Figure S6). As expected, small nodules elicited by HH103 Rif R displayed a cortex surrounding the central zone. This cortex consisted of an outer cortex and of an inner cortex separated by sclereid cells (Figure S6, panel A). Meristematic cells, recently infected cells, and more mature infected cells were present in the nodule central zone (panel C). Small nodules induced by the greA (panels B and D), lpsB, and lpsE mutants (data not shown) displayed all the nodule zones described above and the infection threads also appeared to be normal. HH103 and SVQ656 symbiosomes did not show any significant difference (panels E and F). Light microscopy of nodules of larger size occupied by any of the inoculants tested (HH103 Rif R , SVQ656, SVQ613 or SVQ642) showed the typical structure of a fully grown soybean Figure 4. The Sinorhizobium fredii greA and lpsB genes are not cotranscribed but greA affects lpsB expression. A, genetic organization of the greA-lpsB-lpsE region. Solid lines show the position of the greA (1, 284-bp) and lpsB (3, 156-bp) internal fragments that were successfully amplified (positive controls) using cDNA or gDNA as template. Dotted lines show the positions of the PCR fragments covering the intergenic greA-lpsB (2, 156- bp) and lpsB-lpsE (4, 282-bp) regions that were amplified from gDNA but not from cDNA. B, agarose gel electrophoresis of samples resulting from PCR and RT-PCR experiments. lanes 1–4, controls without DNA; lane 5, 100bp-ladder DNA marker; lanes 6–9, HH103 cDNA; lanes 10–13, HH103 gDNA. Primers used: greAintF and greAintR (lanes 1, 6, and 10), greAlpsB-F and greAlpsB-R (lanes 2, 7, and 11), rtlpsB-F and rtlpsB-R (lanes 3, 8, and 12), lpsBE-F and lpsBE-R (lanes 4, 9, and 13). DNA marker 100, 200, and 300-bp bands are indicated on the left of the figure. C, relative lpsB expression of S. fredii HH103 strains SVQ655 (greA::lacZDp-Gm R ,RR) and SVQ656 (greA::lacZDp-Gm R ,Rr) in comparison to that of wild type HH103 Rif R . RNA was extracted from early exponential phase cultures grown in YMB. Each strain was pairwise compared with HH103 Rif R by using the Mann-Whitney nonparametrical test. In both cases, the relative expression of lpsB was significantly different from that of HH103 Rif R at the levels a= 5% (indicated with a black asterisk), doi:10.1371/journal.pone.0074717.g004 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 6 October 2013 | Volume 8 | Issue 10 | e74717 nodule (Figure 5A, D, G, J). In contrast to the apparent similarity of all the 21-dpi nodules investigated, the infected cells of 35-dpi nodules occupied by the different mutants showed cytological aberrations. The symbiosomes occupied by HH103 Rif R contained several bacteroids (Fig. 5B, C), while those occupied by the mutants contained only one bacteroid, the peribacteroid space was larger, and numerous vesicles were present in the cytoplasm of host cell (Fig. 5E, F, H, I, K, L). TEM analysis of 21-dpi nodules showed structural differences between nodules induced by the wild type and those occupied by its mutant derivatives. Following the normal symbiosome developmental program, HH103 Rif R cells released from the infection droplets (Figure 6A) formed symbiosomes that were able to divide and fuse with other symbiosomes (Figure 6B). Mature symbiosomes contained 2–4 HH103 Rif R bacteroids (Figure 6C). The fusion of symbiosomes in multibacteroid-containing structures and the presence of ghost membranes, which are indications of nodule senescence, were only observed in old cells (Figure 6D). In contrast, nodules induced by the greA,lpsB, and lpsE mutants displayed characteristics pointing to premature termination of symbiosis (Figure 6E–J, 6N, 6P). The cytoplasm of young and mature infected cells showed a large number of endosome-like vesicles fusing with symbiosomes. Bacteroids started to be lysed inside the peribacteroid membranes. The presence of normal multi-bacteroid symbiosomes was rare. Instead, most of the symbiosomes formed by the mutants only contained a single bacteroid. Only a few fusing symbiosomes were found in some infected cells (Figure 6O). Signals of symbiosis termination by lytical clearance of bacteria, a process which is structurally similar to phagocytic elimination, were already detected in 21 dpi soybean nodules. Electron-transparent single membrane vesicles, probably provacuoles, were fusing with symbiosomes. Electrodense and irregularly shaped bacteroids were found in vacuole-like structures, as a result of the fusion of symbiosomes with these structures (Figure 6F). The infected cells of nodules occupied by the greA and lps mutants showed cell walls that were thicker than those of nodules induce by S. fredii HH103 Rif R (Figure 5). This fact was especially evident in 35-dpi nodules elicited by the greA mutant (SVQ656). Apparently, the cell wall of interstitial cells was not affected (Figure 6H). In addition, vacuoles of cells occupied by the lpsB mutant (SVQ613) showed accumulations of an osmiofilic material, which might correspond to phenolic compounds (Figure 6I). Cells occupied by the lpsE mutant (SVQ642) contained an electrondense material that was deposited over the membrane surrounding the unwalled infection droplet and its lumen (Figure 6M), and also between the membrane and the cell wall of bacteroids (Figure 6P). Discussion Rhizobial surface polysaccharides are known to play important roles in symbiosis with legumes [7,32]. Our group have previously characterized S. fredii HH103 mutants unable to produce cyclic glucans (CG) and/or capsular polysaccharides (KPS) and investigated their symbiotic properties with Glycine max and other legumes as well as different bacterial traits in free living conditions [19–23]. In this work, we have investigated the physiological and symbiotic characteristics of S. fredii mutants affected in genes that are directly or indirectly involved in LPS synthesis. For this Table 2. Plant responses to inoculation of Glycine max cv. Williams and Vigna unguiculata cv. Bisbee Red with Sinorhizobium fredii HH103 Rif R and its greA or lps derivatives a . Legume tested, inoculant b Number of nodules Nodule fresh weight (g) Plant-top dry weight (g) Glycine max HH103 Rif R 146.0675.7 1.9060.68 3.761.4 SVQ613 4.162.7** 0.0860.05** 0.960.3** SVQ615 3.963.3** 0.0860.08** 0.660.2** SVQ642 24.6613.5** 0.3760.21** 0.960.3** SVQ642 C1 117.8645.0 1.5160.52 3.161.2 SVQ647 20.668.5** 0.3560.11** 1.060.2** SVQ647 C2 103.2635.6 1.4160.37 2.960.8 SVQ655 32.2614.2** 0.4360.17** 1.060.5** SVQ655 (pMUS908) 70.2645.7 0.7560.45* 1.360.8** SVQ656 16.9612.7** 0.2460.20** 0.860.3** SVQ656 (pMUS908) 163.3656.7 1.8460.41 2.961.3 Uninoculated – – 0.960.5** Vigna unguiculata HH103 Rif R 73.1613.3 1.0460.34 2.060.9 SVQ613 45.8615.2* 0.8060.43* 1.260.6* Uninoculated – – 0.260.1 a Data represent averages of 8 soybean or 10 cowpea plants. Inoculated soybean and cowpea plants were grown for 55 and 43 days, respectively. Plants were grown in Leonard jars, each containing two soybean or cowpea plants. Soybean plants inoculated with lps and greA mutants also formed a high number of pseudonodules. Bacteria isolated from soybean and cowpea nodules of normal external morphology induced by each inoculant showed the resistance markers associated to the mutation (Spc R or Gm R ) or to the cosmid (Tc R ). In SVQ655 (pMUS908), however, only 50% of nodule isolates were Tc R . Attempts to isolate bacteria from pseudonodules were unsuccessful. b Each mutant was pairwise compared with the parental strain HH103 Rif R by using the Mann-Whitney non-parametrical test. Numbers on the same column followed by a single or a double asterisk are significantly different from those of HH103 Rif R at the levels a= 5 and 1%, respectively. doi:10.1371/journal.pone.0074717.t002 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 7 October 2013 | Volume 8 | Issue 10 | e74717 purpose, different mutants in the greA,lpsB, and lpsE genes were constructed and analysed. The S. fredii HH103 greA-lpsB-lpsCDE region shows the same genetic organization than that of S. meliloti 1021 [9,11] or S. fredii NGR234 [25]. The S. fredii HH103 GreA protein, a transcription elongation factor, is nearly identical to those of S. meliloti 1021 (98% of identity) and S. fredii NGR234 (99% identity). The LpsB, LpsC, LpsD, and LpsE proteins are members of the type 1 glycosyltransferase family, a large family of proteins that transfer activated sugars to a variety of substrates, including glycogen and lipopolysaccharides [11,33]. The S. fredii HH103 LpsB, LpsC, LpsD, and LpsE glycosyl transferases are also homologous to the corresponding orthologues of S. meliloti 1021 and S. fredii NGR234, although the aminoacid identity is higher with NGR234 (92–96%) than with S. meliloti 1021 (80–83%). Figure 5. Structure of soybean nodules elicited by Sinorhizobium fredii strains HH103 Rif R (A–C), SVQ656 ( greA ) (D–F), SVQ613 ( lpsB ) (G–I), and SVQ642 ( lpsE ) (J–L). Nodules were collected from 35 dpi plants. A, section of a nodule induced by HH103 Rif R , showing a structured cortex and infected zone with infected and interstitial cells. B, detail of A; infected cells containing infection threads (white arrow) and symbiosomes. C, cells from an older zone than that shown in B. D, image of a SVQ656-induced nodule with evident cytological alterations (white arrow). E, F, images showing different grade of cell damage. The infected cells contain altered symbiosomes with single bacteroids and numerous vesicles (*). Completely collapsed infected cells are visible (**). Note the cell wall thickness of the infected cells. Interstitial cells show amyloplast with huge starch granules. G, image showing the structural features of an SVQ613-induced nodule. H, I, detail of cells showing similar symbiosome damages to those described above. J, structure of a SVQ642-induced nodule. K, infected cells showing different grade of structural damage. L; cell with numerous transparent vesicles (*) after lysis of bacteroids. Additional lettering and symbols: a, amyloplast; c, cortex zone; cw, cell wall; iz, infection zone. Bar size: A, D, G, J: 100 mm; B,C,E,F,H,I,K,L: 20 mm. doi:10.1371/journal.pone.0074717.g005 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 8 October 2013 | Volume 8 | Issue 10 | e74717 At least two different S. fredii HH103 mutants in each of the greA, lpsB and lpsE genes were generated by using the lacZDp-Gm R cassette or the omega (V) interposon. All the S. fredii HH103 lpsB (SVQ613 and SVQ615) and lpsE (SVQ642 and SVQ647) mutants Figure 6. Ultrastructure characteristics of infected cells in soybean nodules elicited by Sinorhizobium fredii strains HH103 Rif R (A–D), SVQ656 ( greA ) (E–H), SVQ613 ( lpsB ) (I–L), and SVQ642 ( lpsE ) (M–P). A, in HH103-elicited nodules, bacteria released from an infection drop (id) are surrounded by the peribacteroid membrane and forms symbiosomes (s). B, symbiosomes harboring several bacteroids after division and fusion of symbiosomes (black arrowhead). C, mature infected cell filled with symbiosomes harboring 1 to 4 bacteroids. D, older infected cell than in (C); symbiosomes display huge peribacteroid space, symbiosome fusion and ghost membranes (black arrow) can be seen. E, in SVQ656-induced nodules, freshly infected cells show numerous vesicles (v) and loss of cytoplasmic content. F, an older infected cell than (E) with an altered symbiosome, which fuses with vesicles (white arrowhead). G, image showing empty symbiosomes (*) in a cell with numerous amyloplasts. H, different cell wall thickness of an interstitial cell and of a deleterious infected cell with empty symbiosomes. I, young infected cell showing bacteroid division and vacuolar structures with osmiophilic content (black arrowhead). J, K, cells filled with single bacteroid symbiosomes. Note the presence of degenerated bacteroids and empty symbiosomes (*). L, infected cell with abundant vesicle structures containing electrodense material. M, infection drop showing osmiophilic material (white arrow). N, O, two cells with different grade of degeneration. Fusion of symbiosomes, degenerated bacteroids (black arrowhead), and empty symbiosomes (*) are shown. P, deposits of dark material on the cell wall of bacteroids (double arrows). Additional lettering and symbols: a, amyloplast; cw, cell wall; ic, interstitial cell. Bar size: A, B, C, D, E, G, I, L, M, N, O: 0.5 mm; F, H, P: 0.2 mm; J, K: 1 mm. doi:10.1371/journal.pone.0074717.g006 S. fredii HH103 LPS Relevance on Symbiosis PLOS ONE | www.plosone.org 9 October 2013 | Volume 8 | Issue 10 | e74717