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UMR 1090 Génomique Développement et Pouvoir Pathogène, INRA, Université de Bordeaux 2, Centre INRA de Bordeaux, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon Cedex, France
Correspondence
Joël Renaudin
renaudin{at}bordeaux.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Plant-pathogenic mollicutes, spiroplasmas and phytoplasmas cause many diseases affecting a large variety of crop plants worldwide (Firrao et al., 2005; Lee et al., 2000; Seemüller et al., 2002). The bacteria inhabit the phloem sieve elements and are transmitted from plant to plant by phloem sap-feeding leafhopper insects, which therefore are responsible for the spread of the diseases. The relationships of plant mollicutes with their hosts have been recently reviewed (Bové et al., 2003; Christensen et al., 2005).
S. citri is the aetiological agent of stubborn disease of citrus (Saglio et al., 1971, 1973) and brittle root disease of horseradish (Fletcher et al., 1981). It is transmitted, in a persistent manner, by the leafhoppers Circulifer haematoceps (Fos et al., 1986) and Circulifer tenellus (Liu et al., 1983a). Various studies have addressed the movement of S. citri into the intestines and salivary glands of its leafhopper vector (Kwon et al., 1999; Liu et al., 1983b). For transmission to occur, the ingested spiroplasmas must infect the gut epithelial cells before crossing into the haemolymph, where they continue to multiply and then migrate to other organs and eventually invade the salivary glands, from which they are injected into the phloem through the salivary duct during feeding. Thus, successful transmission of S. citri by its leafhopper vector relies on the ability of the spiroplasmas to cross two physical barriers, the gut epithelium and the salivary gland membrane. To explain how spiroplasmas pass through these barriers, a model has been proposed (Fletcher et al., 1998), in which spiroplasmas adhere to receptors on the apical membrane of the gut epithelium cell, before being taken into the cell by endocytosis. After migrating within the cell, spiroplasmas are released by exocytosis and reach the haemocoel through the basal lamina. A similar mechanism has been proposed for migration of the spiroplasmas from the haemocoel into the salivary duct through the salivary gland cells. However, the genetic determinants and the molecular mechanisms that govern the interactions between spiroplasmas and insect cells are still to be discovered.
Several surface proteins have been implicated in the transmission of S. citri by its leafhopper vector. In S. citri BR3, protein SARP1 is needed, in vitro, for adherence of spiroplasmas to insect cells (Berg et al., 2001; Yu et al., 2000), suggesting that this protein might be involved in the interaction of the spiroplasmas with insect cells. However, genetic evidence of its involvement is lacking. Using a reverse genetic approach, it has been shown that inactivation of a gene encoding a lipoprotein showing limited homology with the solute-binding protein of an ABC transporter resulted in a dramatic decrease of transmission efficiency, indicating that this protein might play a role in the transmission process (Boutareaud et al., 2004). Similarly, a S. citri mutant devoid of spiralin, the major lipoprotein at the cell surface, was poorly transmitted as compared to the wild-type strain GII-3 (Duret et al., 2003). In this case, studying interactions between spiroplasma and insect proteins revealed that, in vitro, spiralin acted as a lectin, binding glycoproteins of the vector insect (Killiny et al., 2005). However, in spite of the absence of spiralin, the spiralin-less mutant, though at low efficiency, was still transmitted, indicating that insect transmission certainly involved several proteins other than spiralin. The fact that both transmissible and non-transmissible strains possess equal amounts of spiralin supports this idea. Recently, we showed that non-transmissible strains of S. citri lacked plasmids encoding adhesin-like proteins, whereas these plasmids were detected in all transmissible strains tested (Berho et al., 2006).
Many S. citri strains have been shown to carry natural plasmids (Gasparich et al., 1993; Ranhand et al., 1980) and some of them were mapped and/or cloned in Escherichia coli (Archer et al., 1981; Bové et al., 1989; Mouchès et al., 1983). For years, spiroplasma plasmids were designated as cryptic, since no phenotypic trait had been associated with their presence, and none of them had been sequenced until recently. S. citri GII-3 (wild-type) contains seven plasmids, pSciA and pSci1–6, which were first identified as circular contigs of 7.8–35.3 kbp during the S. citri genome sequencing project (Foissac et al., 2004). In addition to proteins involved in DNA partitioning and conjugative gene transfer, these plasmids also encode proteins that are potentially involved in the interactions of the spiroplasma with its vector insect. Plasmids pSci1–5, in particular, encode eight proteins (ScARPs) sharing high homology with the adhesion-related protein SARP1 from S. citri BR3 (Berg et al., 2001). In addition, plasmid pSci6 encodes a hydrophilic protein, P32, which has been associated with insect transmissibility (Killiny et al., 2006). A comparison of protein profiles and plasmid contents of transmissible and non-transmissible strains of S. citri revealed that the non-transmissible strains possessed no ScARPs and no P32, and also did not possess plasmids pSci1–6 (Berho et al., 2006; Killiny et al., 2006). Such a correlation between the occurrence of plasmids and the ability of the spiroplasma strains to be transmitted by the leafhopper vector suggested that plasmids pSci1–6 encoded genetic determinants that might be essential for insect-transmission. To further investigate this hypothesis, plasmids from insect-transmissible strain GII-3 were introduced into the plasmid-free, non-transmissible strain 44 by electroporation. Successful transmission of S. citri 44 transformants to the host plant via injection into the leafhopper vector strongly suggests that genetic determinants required for insect transmission of S. citri 44 are encoded by plasmid pSci6.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). By injection into the leafhopper vector, it can be experimentally transmitted to periwinkle (Catharanthus roseus) plants, in which it multiplies and induces severe symptoms (Foissac et al., 1996, 1997). S. citri strains ASP-1 (Townsend et al., 1977) and 44 (Hosseini Pour, 2000) (kindly provided by Dr Hosseini Pour, University of Kerman, Iran) were isolated from stubborn-diseased sweet orange trees in Israel and Iran, respectively. In contrast to the situation with GII-3, attempts to infect periwinkle plants by injection of S. citri ASP-1 or 44 into the leafhopper repeatedly failed. Accordingly, these strains were described as insect-non-transmissible strains.
Spiroplasmas were grown at 32 °C in SP4 medium (Whitcomb, 1983), from which fresh yeast extract was omitted. Spiroplasma cells were transformed by electroporation as previously described (Stamburski et al., 1991) using 1–2 µg purified plasmid or various ligation mixtures. Spiroplasmal transformants were selected in the presence of 2 µg tetracycline ml–1. The antibiotic concentration was progressively increased from 2 to 15 µg ml–1 during passaging of the transformants.
Construction of S. citri strain 44/4.
Extrachromosomal DNA was purified from an ASP-1 transformant carrying pSci4NT, in addition to the endogenous plasmid pIJ2000 (Archer et al., 1981). Plasmid pIJ2000 shares the same restriction map as pSciA from S. citri GII-3 (GenBank accession no. AJ966734). After digestion by XhoI, which cleaves pIJ2000 but not pSci4NT, the digested DNA was used to transform S. citri 44 by electroporation. Eight tetracycline-resistant transformants were screened by PCR for the presence of pSci4NT and absence of pIJ2000. The selected S. citri 44 transformant carrying pSci4NT was named 44/4.
DNA isolation and Southern blot hybridization.
Spiroplasma genomic DNA was prepared from 10 ml cultures using the Wizard genomic DNA purification kit (Promega), whereas plasmid DNA was purified from 25 ml cultures with the Wizard SV minipreps DNA purification kit (Promega). Southern blot hybridization of spiroplasmal DNA with appropriate digoxigenin-dUTP-labelled probes has been described elsewhere (André et al., 2003). Probes S4, specific to scarp4a, and S235, hybridizing to all scarp genes except scarp4a, were produced by PCR amplification of genomic DNA from S. citri GII-3 with primer pairs S4F/S4R and S235F/S235R, respectively (Table 1). Gene scarp4a is carried by pSci4 whereas scarp2b, 3b and 5a are carried by pSci5, and scarp2a, 3a, 3c and 3d by pSci4, pSci1, pSci3 and pSci2, respectively (Table 1) (Berho et al., 2006). Probe P32 consisted of a 546 bp fragment of gene p32 generated by PCR amplification with primer pair P32F/P32R (Table 1). Gene p32 is carried by pSci6. Probe TetM (535 bp) was obtained by PCR amplification of pSRT2 (Lartigue et al., 2002) with primer pair Tet1/Tet2 (Table 1). The tetracycline-resistance cassette carrying the tetM gene under the control of the spiralin gene promoter was retrieved from pSRT2 as a 2.3 kbp PstI restriction fragment.
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). Monoclonal antibody 10G3 (Berho et al., 2006), specific to ScARPs, was used as the primary antibody.
Experimental transmission assay.
Uninfected leafhoppers Circulifer haematoceps were reared on stock (Matthiola incana) plants at 28 °C with a photoperiod of 17 h. Micro-injection of S. citri into C. haematoceps leafhoppers and transmission to the periwinkle host plant (Catharanthus roseus) have been described previously (Foissac et al., 1996; Duret et al., 2003). Briefly, the insects were microinjected with 0.2 µl spiroplasma culture (approx. 105 spiroplasma cells) and the injected insects were caged on young periwinkle plants (12 insects per plant, 5–10 plants per spiroplasma strain) for a 2 week transmission period. After insect removal, the plants were kept in the greenhouse at 30 °C for symptom production. Under these conditions, the plant symptoms induced by the wild-type strain GII-3 were produced within 2–4 weeks post-transmission. Alternatively, in the experiments of Table 6, injected insects were first caged on stock plants for a latent period of 10 days, during which the spiroplasmas multiplied in the insects, before being transferred to young periwinkles (five insects per plant) for a transmission period of 10 days. Culture of S. citri from plants and insects was carried out as described previously (Duret et al., 2003; Foissac et al., 1996, 1997), except when otherwise stated. Spiroplasma titres in the insects were determined at the end of the transmission period from groups of three to five living insects. Isolation of spiroplasmas from plants was carried out 6–8 weeks post-transmission.
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), except that the leafhoppers were tested individually, each insect being crushed in 1 ml SP4 medium. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To determine whether S. citri GII-3 plasmids could be transferred to strains 44 and ASP-1, the tetracycline-resistance gene tetM was introduced into the plasmids and used as the selection marker. The cloning strategy was based on the observation that plasmids pSci1–6 all possessed one NsiI (pSci1 and pSci3–5) or PstI (pSci5–6) restriction site, except pSci2, which has two NsiI sites (Fig. 1). Extrachromosomal DNA purified from S. citri GII-3 was digested with NsiI or PstI to yield linear molecules. The NsiI and PstI restriction fragments (approx. 5 µg) were separately ligated to approximately 0.5 µg of the tetracycline-resistance cassette, which was retrieved from pSRT2 (Lartigue et al., 2002) as a 2.3 kbp PstI DNA fragment (NsiI and PstI yield compatible, cohesive ends). Then the ligation mixtures were used to transform S. citri strains 44 and ASP-1 by electroporation. After plating the transformation mixtures, both strains (44 and ASP-1) yielded tetracycline-resistant colonies at frequencies ranging from 10–8 (PstI) to 5x10–7 (NsiI) transformants per c.f.u. and per µg plasmid DNA in the ligation mixture.
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). The labelling of most, if not all, of the colonies indicated that these cells did carry plasmids, from which scarp genes were expressed to proteins. Production of ScARPs in spiroplasmal transformants was further confirmed by Western immunoblotting, and proteins of the expected molecular mass were detected on the blots (data not shown).
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To characterize the plasmid content of the S. citri 44 transformants, 57 individual colonies (48 plus 9, respectively derived from the NsiI and PstI transformation mixtures) were grown in the presence of tetracycline and genomic DNAs were analysed by Southern blot hybridization with probe TetM, and a mixture of probes S235 and P32 (Fig. 3). The diversity of the hybridization patterns indicated that the various spiroplasmal transformants carried distinct plasmid contents.
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, lanes N5, N11, N27 and P9). This was further confirmed by hybridization with probes S235 and P32. Plasmids pSci1NT and pSci5PT were detected as S235-hybridizing fragments of 763 and 556, 1585 and 3309 bp, respectively (Fig. 3b, lanes N5 and N27), whereas pSci6PT was revealed as a 1870 bp fragment hybridizing with the P32 probe (Fig. 3b, lane P9). As expected, pSci21NT did not hybridize with the S235 and P32 probes (Fig. 3b, lane N11).
Interestingly, extrachromosomal DNAs from some transformants yielded additional hybridization signals, indicating that more than one plasmid might be present in these clones. In clone N19 for example, detection of two signals of 7545 and 4417 bp with the TetM probe and four signals of 763, 556, 1585 and 3309 bp with probe S235 strongly suggested that this clone contained the two plasmids pSci1NT and pSci5NT (Fig. 3a, b, lanes N19). Similarly, in clone N21, detection of signals of 7545 and 3285 bp with the TetM probe and 8121 and 763 bp with the S235 probe suggested the presence of pSci1NT and pSci2N2T (Fig. 3a, b, lanes N21). Plasmid pSci2N2T certainly occurred from the ligation of the tetM cassette to the linear pSci2 resulting from either partial NsiI digestion or combination of the two NsiI fragments.
Unexpectedly, in addition to recombinant plasmids carrying the tetM gene, some clones were found to contain unmarked plasmids (i.e. plasmids having no tetM gene). Clone P3, carrying pSci5PT, was also found to contain unmarked pSci4 as revealed by the presence of the 2549 bp fragment hybridizing with the S235 probe and the absence of the 4174 bp fragment hybridizing with the TetM probe (Fig. 3a, b, lanes P3). Similarly, clone P10 was found to contain unmarked pSci1 and pSci4, in addition to pSci6PT. The three plasmids were detected as hybridization signals of 763, 2549 and 1870 bp, respectively (Fig. 3b, lane P10). Three plasmids, pSci1 (unmarked), pSci2N2T and pSci21NT, were also detected in clone N33 (Fig. 3a and b, lanes N33). In clones N23 and N24, containing pSci1NT and pSci4, as well as in clone N30, carrying pSci5NT, the tetM hybridization signals of 7545 bp and 4417 bp were barely detected (Fig. 3a, lanes N23, N24 and N30, respectively). In addition, a few clones yielded hybridization signals of unexpected sizes, suggesting that plasmid rearrangements might have occurred in these clones. In clone N25, for example, the presence of pSci1NT was detected as the 763 bp fragment hybridizing with the S235 probe (Fig. 3b, lane N25). However, hybridization with the TetM probe did not reveal the expected signal of 7545 bp but, instead, a fragment of approximately 5600 bp (Fig. 3a, lane N25).
In summary, transformants with a wide range of plasmid contents were obtained with both NsiI and PstI ligation mixtures. Considering the tetM-marked plasmids in the S. citri 44 transformants, pSci1NT was the most frequently detected (58 %) as compared to pSci21NT (21 %), pSci2N2T (10 %) and pSci4NT and pSci5NT (4 % each) (Table 3). Depending on the transformants, pSci1NT was detected alone (20 clones) or together with other marked or unmarked plasmids (eight clones). Taking into account that cells of S. citri GII-3 contain seven plasmids, pSciA and pSci1–6, the finding that S. citri 44 transformants carried up to three plasmids within the same cell was not totally surprising. None of the transformants was found to carry either pSci22NT or pSci3NT. It is thought that, in contrast to pSci21NT, which was detected in 23 % of the transformants, pSci22NT probably did not possess the replication origin of pSci2 and therefore could not replicate in spiroplasma cells. In pSci3, the location of the NsiI site, downstream of trsE, is identical to that in pSci4, and thus cannot explain why pSci3NT, in contrast to pSci4NT, was not detected. Unmarked pSci3 was also not detected in the S. citri 44 transformants tested. In the case of transformation with the PstI mix, plasmids pSci5PT and pSci6PT were detected at similar frequencies, 44 % and 33 %, respectively. Similar data were obtained with S. citri ASP-1 (hybridization data are not shown) except that pSci4NT was detected at a higher frequency (39 % versus 4 % for strain 44) and pSci2N2T was not detected (Table 3). Considering a complete digestion of pSci2 into two NsiI fragments, the probability of combination of the two fragments together with the tetM cassette to yield pSci2N2T was expected to be very low. Taken as a whole, these results showed that plasmids pSci1, pSci2, pSci4, pSci5 and pSci6, as well as their tetM-marked derivatives, were efficiently introduced into S. citri strains 44 and ASP-1, in which they replicated.
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). In contrast, with both cultures Mix44/1256 and 44/146, four plants out of 10 developed severe symptoms, with spiroplasma titres [1.2–3.3x107 c.f.u. (g midribs)–1] similar to that [1.2x107 c.f.u. (g midribs)–1] of plants infected by S. citri GII-3.
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, the PCR data were in good agreement with the multiplication, in the insects, of the spiroplasma strains that were initially injected. For example, PCR with primer pairs S235F/S235R and P32F/P32R yielded positive amplification with all isolates from insects (Fig. 4a, c, lanes 5–8) except in the case of 44 (Fig. 4a, c, lanes 3 and 4). In agreement with the absence of pSci4 in the Mix44/1256, PCR amplification with primer pair S4F/S4R was positive with isolates GII-3 (Fig. 4b, lane 2) and 44/146 (Fig. 4b, lanes 7 and 8), but not with isolates 44 and Mix44/1256 (Fig. 4b, lanes 3–6). With DNAs from spiroplasma cultures isolated from plants, PCR with primer pair P32F/P32R yielded positive amplification with all isolates, suggesting that they all contained pSci6 carrying the p32 gene (Fig. 4c, lanes 10–13). As expected, due to the absence of pSci4, PCR amplification specific to scarp4a yielded negative results with isolates from Mix44/1256 (Fig. 4b, lanes 10 and 11). The failure to amplify scarp4a from these plants confirmed that symptom production was not due to infection by contaminating, transmissible strains such as GII-3. Interestingly, no scarp genes were detected in Mix44/1256 isolated from plant P4 (Fig. 4a, b, lanes 10), suggesting that this isolate only contained pSci6.
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). In the case of Mix44/1256, hybridization signals corresponding to pSci1, 2, 5 and 6 were detected in the insects, indicating that all four strains 44/1, 44/2, 44/5 and 44/6 did multiply in the vector insect (Fig. 5, lanes 2 and 3). Unexpectedly, a faint signal, which seemed to correspond to scarp3c of pSci3, was detected in the Mix44/1256B isolate (Fig. 5, lane 3). This suggests that one of the four cultures that were mixed prior to injection into the insects might contain a mixed population of cells, a few of them carrying pSci3 or pSci3NT. These plasmids were not detected in the S. citri 44 transformants that were initially selected (see Fig. 3). DNAs from plant isolates displayed two distinct profiles. As indicated by the 1870 bp fragment hybridizing with the P32 probe, pSci6 was the only plasmid detected in the isolate from plant P4 (Fig. 5, lane 4). This isolate was named 44/6P4. In contrast, isolate from plant P5 seemed to contain both pSci1 (as revealed by the 763 bp hybridization signal) and pSci6 (Fig. 5, lane 5), suggesting that strains 44/1 and 44/6 were transmitted to the plant whereas 44/2 and 44/5 were not.
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, lanes 6 and 7, and 8 and 9, respectively). These results indicated that strain 44/146 was transmitted to periwinkle plants by the leafhopper vector and that the three plasmids were maintained in the absence of selection pressure. Therefore, it was thought that plasmids pSci1, and/or pSci4, and/or pSci6PT could confer insect transmissibility to S. citri 44. Furthermore, the detection of pSci6PT in every spiroplasma culture isolated from plants infected by S. citri 44 transformants suggested that this particular plasmid might be involved. To further investigate the correlation between the presence of pSci6PT and the ability of S. citri 44/6 to be transmitted via injection into the leafhopper vector, experimental transmission tests were carried out with strains 44/6 (initial clone P9) and 44/6P4 (strain 44/6 isolated from plant P4 after insect transmission of Mix44/1256), both of which contained pSci6PT only (Table 5). In contrast to S. citri 44, for which none of the plants was infected, both strains 44/6 and 44/6P4 were transmitted by the leafhopper vector, as indicated by the high proportion of plants showing symptoms, 8/10 and 9/10, respectively. As expected, spiroplasmas isolated from these plants were shown to contain pSci6PT, as revealed by the restriction pattern of the purified plasmid and the detection of the 1870 bp fragment hybridizing with the P32 probe (data not shown). These results indicated that plasmid pSci6PT alone conferred insect transmissiblity to S. citri strain 44. To determine whether pSci4NT, like pSci6PT, could confer transmissibility, experimental transmission assays were conducted to compare transmissibility of strain 44/4, carrying pSci4NT, and strain 44/6, carrying pSci6PT. The results, presented in Table 6, showed that, in spite of multiplication of both strains in the insects, strain 44/6 (initial clone P9) was transmitted, as revealed by the occurrence of symptomatic plants (4/10), whereas strain 44/4 was not (0/10). In the case of strain 44/4, the detection of pSci4NT in the spiroplasmas isolated from insects at the end of the transmission period (Fig. 6, lane 6) indicated that the absence of transmission was not due to plasmid loss. However, the possibility that strain 44/4 may actually be transmissible, but at a rate lower than would be detected in our experiments, cannot be excluded. Nevertheless, these results strongly suggest that pSci6 from S. citri GII-3 encodes genetic determinants that are essential for transmission of S. citri 44 via injection into the leafhopper C. haematoceps.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Recently, sequencing data revealed that plasmids pSKU146 from Spiroplasma kunkelii, pBJS-O from S. citri BR3 and pSci1–6 from S. citri GII-3 encode proteins that are thought to be involved in the interactions of the spiroplasmas with their insect hosts (Davis et al., 2005; Foissac et al., 2004; Joshi et al., 2005).
In S. citri GII-3, plasmids pSci1–5 and pSci6, encoding respectively ScARPs and the hydrophilic protein P32, have been associated with insect transmissibility as they were detected in all insect-transmissible strains tested, but not in insect-non-transmissible ones (Berho et al., 2006; Killiny et al., 2004, 2006). Such an apparent correlation between the presence of plasmids and the ability of S. citri strains to be transmitted by the leafhopper vector is reminiscent of the situation found in S. citri ASP-1. In this particular strain, the initial culture, which possesses three plasmids of 7.8, 14.8 and 35.4 kbp, could be experimentally transmitted via injection into the leafhopper Euscelidius variegatus (Townsend et al., 1977). However, due to extensive in vitro propagation, loss of the two larger plasmids resulted in loss of insect transmissibility (Archer et al., 1981). Interestingly enough, the estimated sizes of these two plasmids (14.8 and 35.4 kbp) are similar to those of S. citri GII-3 plasmids pSci2 (14.4 kbp) and pSci6 (35.3 kbp) carrying scarp3d and p32, respectively. Taken together, these studies suppport a role of plasmid-encoded determinants in transmission of S. citri by its leafhopper vectors.
In the present study, we transformed the non-transmissible S. citri strain 44 (having no plasmids) with plasmids from S. citri GII-3 and screened the spiroplasmal transformants for insect transmissibility. Transformation of S. citri 44 was achieved through electroporation of spiroplasma cells with a ligation mixture of the extrachromosomal DNA from S. citri GII-3 with the tetM gene, used as the selection marker. Detection of the expected recombinant plasmids pSci1–5NT and pSci5–6PT in the spiroplasmal transformants indicated that insertion of the tetM gene at the NsiI or PstI sites did not prevent plasmid replication. For clarity, we will not discriminate the natural plasmids pSci1–6 from their tetM-marked counterparts (pSci1–5NT and pSci5–6PT) in the subsequent discussion. The five plasmids pSci1–2 and pSci4–6 were each detected by Southern blot hybridization in at least one of the transformants, whereas pSci3 was not detected in the first screen of the transformants. With the NsiI mix, a large majority of transformants was found to contain pSci1, alone or together with one or two additional plasmids. The occurrence of several plasmids in a single cell indicates the absence of incompatibility between these plasmids, in agreement with the situation in S. citri GII-3, in which each plasmid (pSci1–6) was shown to occur at approximately 10 copies per cell (Foissac et al., 2004). The high proportion of S. citri 44 transformants carrying pSci1 probably resulted from the high proportion of small plasmids in the DNA preparation, due to high recovery during the isolation process. In addition, transformation experiments with isolated plasmids revealed that transformation of S. citri 44 with pSci1 was approximately ten times more efficient (0.5–1.5x105 transformants µg–1) than with pSci6 (0.5–1x104 transformants µg–1).
Experimental transmission assays via injection into the leafhopper vector C. haematoceps showed that, in contrast to S. citri 44 (wild-type), S. citri 44 transformants were transmitted to the host plant, in which they multiplied and induced symptoms. When introduced into the insects by intra-abdominal injection, both transmissible and non-transmissible strains multiplied in the leafhopper (Bové et al., 2003; Wayadande & Fletcher, 1995). Accordingly, S. citri 44 and each one of the S. citri 44 tranformants (regardless of their plasmid content), multiplied in the insects, in which they were detected at the end of the transmission period. In contrast, after feeding the infected leafhoppers on periwinkles, only spiroplasmas carrying pSci6 were isolated from every infected plant, suggesting that this plasmid could confer transmissibility to S. citri 44. Indeed, independent transmission of strains 44/4 and 44/6 clearly indicated that pSci6 alone conferred insect transmissibility to strain 44, whereas pSci4 did not. Therefore, it is likely that pSci6 encodes genetic determinants that are essential for insect transmission of S. citri 44. In agreement with these data, S. citri strain GII3-
5, having lost pSci5 but which still carried pSci6, has been shown to be transmitted at high efficiency, similarly to the wild-type strain GII-3 (Berho et al., 2006). In addition, strain GII3-135, lacking pSci1, 3 and 5, could also be experimentally transmitted (S. Duret & J. Renaudin, unpublished data).
It is noteworthy that pSci6, which was shown to confer insect transmissibility to strain 44, encodes the hydrophilic protein P32, which has been associated with insect transmissibility of S. citri (Killiny et al., 2006). However, expression of P32 in S. citri 44 through transformation with a recombinant, oriC plasmid carrying p32 failed to restore transmissibility (Killiny et al., 2006). This result suggests that pSci6-encoded determinants other than P32 might be essential for the spiroplasmas to invade, multiply in, or be released from the salivary glands of the leafhopper vector. Plasmids pSci1–5 encode eight adhesin-like proteins closely related to each other (Foissac et al., 2004) and to SARP1 of S. citri BR3 (Berg et al., 2001). Plasmid pSci4, in particular, encodes ScARP2a and 4a. In contrast, pSci6 contains no full-length scarp gene. Instead, it possesses short scarp-like sequences that putatively encode truncated products with limited homology to ScARPs. Since no scarp sequences were detected in the genomic DNA of S. citri 44 (by Southern blot hybridization), the finding that pSci6 alone conferred transmissibility to this particular strain suggests that ScARPs are not essential for the spiroplasma cells present in the haemolymph to cross the salivary gland barrier. However, in natural transmission (in contrast to experimental transmission, in which spiroplasmas are directly injected into the haemolymph) the ingested spiroplasmas must cross the gut epithelium before reaching the haemolymph. The involvement of ScARPs at this point of the transmission process cannot be excluded. The fact that SARP1 of S. citri BR3 seems to be involved in adhesion in vitro of spiroplasmas to insect cells is in good agreement with this hypothesis (Yu et al., 2000). The finding that S. citri 44/6, which does not possess ScARPs, was poorly acquired by insects fed on 44/6-infected plants also fits the hypothesis. However, the presence of ScARPs 2a, 3a and 4a in strain 44/146 seems not to improve spiroplasma acquisition by the leafhoppers, when compared to strain 44/6. Therefore the involvement of ScARPs is still uncertain. In addition to P32, pSci6 putatively encodes many other hypothetical proteins of unknown function, some of which are specific to this plasmid. Their possible involvement in the transmission of S. citri by its leafhopper vector is being investigated.
Even though pSci6 conferred insect transmisibility to S. citri 44, transmission of S. citri 44/6 was repeatedly less efficient than that of S. citri GII-3, as indicated by the fact that the proportion of infected plants never reached 100 %. Two distinct hypotheses could explain these results. In contrast to the situation in GII-3, pSci6 could not be stably maintained in the absence of selection pressure (in the leafhopper and/or in the plant), resulting in a low proportion of cells carrying the plasmid. Indeed, whereas attempts to cure S. citri GII-3 of pSci6 were unsuccessful (Berho et al., 2006), in vitro propagation of S. citri 44/6 in the absence of selection pressure resulted in loss of plasmid pSci6 in more than 90 % of the cells after 20 successive propagations. Alternatively, genetic determinants other than those carried by pSci6 might be required for efficient transmission, and these determinants might be absent in the genome of S. citri 44, as compared to GII-3. However, equal amounts of spiralin, a protein that is required for efficient transmission of S. citri (Duret et al., 2003), were detected in S. citri strains 44 and GII-3 (Killiny et al., 2006).
In mycoplasmas, attachment to the host cells is by a specialized structure, which is formed through the recruitment of several proteins that interact between the mycoplasma and the host cell (Balish & Krause, 2005). Similar structures have been observed in cells of S. kunkelii attached to the gut epithelium of the leafhopper vector (Ammar et al., 2004; Özbek et al., 2003). However, whether particular proteins are specifically involved in these structures remains to be determined. In phytoplasmas, the immunodominant membrane protein of ‘Candidatus Phytoplasma asteris' has been shown to interact specifically with microfilaments of the leafhopper vector intestine, suggesting that this complex formation is part of the transmission mechanism (Suzuki et al., 2006). The presumed implication of plasmid-encoded proteins has also been suggested (Nishigawa et al., 2002).
While transformation of S. citri with artificial oriC plasmids has been extensively used for molecular genetic studies (Bové et al., 2003; Renaudin & Lartigue, 2005), this study is, to our knowledge, the first report of successful transformation of spiroplasma cells by natural plasmids, and opens the way to investigate the role of plasmid-encoded determinants in the biology of the spiroplasma. As an example, transformation of S. citri 44 by pSci6 from S. citri GII-3 represents the first clear-cut evidence of a link between plasmids and insect transmissibility.
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Received 21 April 2006;
revised 15 June 2006;
accepted 19 June 2006.
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