FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2013256472> ?p ?o ?g. }

• W2013256472 endingPage "5090" @default.
• W2013256472 startingPage "5079" @default.
• W2013256472 abstract "Article17 September 2001free access A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii Elisabetta Soragni Elisabetta Soragni Present address: Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0634 USA Search for more papers by this author Angelo Bolchi Angelo Bolchi Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Raffaella Balestrini Raffaella Balestrini Centro di Studio sulla Micologia del Terreno (CNR) and Dipartimento di Biologia Vegetale, Università di Torino, Vialle Mattioli 25, I-10125 Torino, Italy Search for more papers by this author Claudio Gambaretto Claudio Gambaretto Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Riccardo Percudani Riccardo Percudani Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Paola Bonfante Paola Bonfante Centro di Studio sulla Micologia del Terreno (CNR) and Dipartimento di Biologia Vegetale, Università di Torino, Vialle Mattioli 25, I-10125 Torino, Italy Search for more papers by this author Simone Ottonello Corresponding Author Simone Ottonello Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Elisabetta Soragni Elisabetta Soragni Present address: Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0634 USA Search for more papers by this author Angelo Bolchi Angelo Bolchi Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Raffaella Balestrini Raffaella Balestrini Centro di Studio sulla Micologia del Terreno (CNR) and Dipartimento di Biologia Vegetale, Università di Torino, Vialle Mattioli 25, I-10125 Torino, Italy Search for more papers by this author Claudio Gambaretto Claudio Gambaretto Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Riccardo Percudani Riccardo Percudani Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Paola Bonfante Paola Bonfante Centro di Studio sulla Micologia del Terreno (CNR) and Dipartimento di Biologia Vegetale, Università di Torino, Vialle Mattioli 25, I-10125 Torino, Italy Search for more papers by this author Simone Ottonello Corresponding Author Simone Ottonello Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma Search for more papers by this author Author Information Elisabetta Soragni2, Angelo Bolchi1, Raffaella Balestrini3, Claudio Gambaretto1, Riccardo Percudani1, Paola Bonfante3 and Simone Ottonello 1 1Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parco Area delle Scienze 23/A, I-43100 Parma 2Present address: Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0634 USA 3Centro di Studio sulla Micologia del Terreno (CNR) and Dipartimento di Biologia Vegetale, Università di Torino, Vialle Mattioli 25, I-10125 Torino, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5079-5090https://doi.org/10.1093/emboj/20.18.5079 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Important morphogenetic transitions in fungi are triggered by starvation-induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up-regulation of TbSP1, a surface-associated, Ca2+-dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A2 to be described in fungi and identifies a novel class of phospholipid-hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre-protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free-living and symbiosis-engaged hyphae. It thus appears that a dual localization phospholipase A2 is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1-related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non-filamentous microorganisms, points to a general role for TbSP1 phospholipases A2 in the organization of multicellular filamentous structures in bacteria and fungi. Introduction More than 5000 species of ascomycete and basidiomycete fungi form symbiotic associations, called ectomycorrhizas, with the fine roots of most shrubs and trees found in temperate and boreal forests (Molina et al., 1992). By promoting nutrient exchange between the two partners, mycorrhizas exert a positive influence on plant survival under a variety of unfavorable environmental conditions (Read, 1999). Ectomycorrhizal fungi, however, are not obligate symbionts, and some of them, including the ascomycete truffle Tuber borchii studied in the present work, can grow in pure mycelial culture by exploiting their limited saprotrophic capabilities. The mycorrhizal transition is triggered by as yet largely unidentified, environmental and plant-generated signals that are sensed by the free-living mycelium and transduced into a multifaceted morphogenetic program (Martin and Tagu, 1999; Buscot et al., 2000). Ectomycorrhiza formation is generally considered as a very soft colonization process, not causing any irreversible damage, nor any drastic defense response in the host plant. However, various cytological and biochemical evidence clearly points to the existence, at least in early infection stages, of some basic functional similarities between mycorrhiza formation and host invasion by pathogenic fungi (Hebe et al., 1999; Martin and Tagu, 1999). As revealed by the fungal cell wall alterations that take place during symbiosis development (Bonfante et al., 1998), a central aspect of mycorrhiza formation is an extensive remodeling of the surface and aggregation state of the hyphae. Fungal molecular components that are thought to be involved in such events (albeit not exclusively associated with mycorrhiza development) have been identified recently by both large-scale (Voiblet et al., 2001) and conventional gene search approaches. These include a protein involved in vesicular transport and autophagocytosis (Kim et al., 1999), a transporter for monosaccharide movement from the plant to the mycobiont (Nehls et al., 1998), hydrophobins (Tagu et al., 1996) and the adhesin-like, symbiosis-regulated acidic polypeptides (SRAPs) of Pisolithus tinctorius (Laurent et al., 1999). The latter two are structural proteins that have been found extracellularly as well as surface associated in both free-living and symbiosis-engaged hyphae. The mRNAs for both proteins are up-regulated concomitantly with symbiosis formation, but the signals triggering such a response are still largely unknown. As revealed by the results obtained in some multicellular non-mycorrhizal fungi, many important morphogenetic transitions, such as aerial hyphae (Wosten et al., 1999) and conidiophore (Lauter et al., 1992) formation, and the development of invading appressoria in phytopathogenic fungi (Talbot et al., 1993), similarly are accompanied by the overexpression of secretable surface proteins. Many of these responses, e.g. hydrophobin mRNA up-regulation in the rice pathogen Magnaporthe grisea (Talbot et al., 1993) and in the cellulolytic filamentous fungus Trichoderma reesei (Nakari-Setala et al., 1997), can be mimicked in vitro by growth under conditions of nutrient deprivation. This link between nutritional status and surface protein expression has been interpreted as a sort of adaptive response, in which starvation for essential nutrients is perceived by specific receptors and transduced into morphogenetic changes aimed at inducing either a metabolically quiescent state (e.g. conidia) or a state of improved nutrient acquisition (e.g. invasive hyphal structures) (Madhani and Fink, 1998; Lengeler et al., 2000). It thus appears that nutritional factors, especially the conditions of inorganic nitrogen shortage often found in the root microenvironment of ectosymbiosis-susceptible plants (Keeney, 1980; Read, 1991), may also trigger adaptive changes in surface protein expression (as well as morphogenetic transitions) in mycorrhizal fungi. Here, we report on the isolation of a novel phospholipase, named TbSP1, that is strongly and reversibly up-regulated by nutrient deprivation and accumulates in the inner cell wall layer of free-living mycelia, fruitbodies and mycorrhizas of the symbiotic fungus T.borchii. TbSP1, which is also released extracellularly and contains the adhesin-like motif RGD, is reminiscent of similarly localized phospholipid-hydrolyzing enzymes involved in host invasion by pathogenic fungi (Nespoulous et al., 1999; Ghannoum, 2000; Cox et al., 2001). However, at variance with such enzymes, all of which are (lyso)phospholipases B unable to discriminate between the sn-1 and the sn-2 ester bond of glycerophospholipids, TbSP1 is absolutely specific for the middle (sn-2) position and can thus participate in both membrane remodeling and cell signaling events. TbSP1 is the first phospholipase A2 (PLA2) thus far described in fungi and identifies a new family of phospholipases that are only present in filamentous microbes. To the best of our knowledge, the nutrient-regulated expression of a phospholipid-hydrolyzing enzyme has never been reported before. Results Isolation of the cDNA for an abundant low molecular weight protein extracellularly released by suspension cultured T.borchii mycelia We initially analyzed by SDS–PAGE the growth medium of mycelia cultured for different lengths of time under suspension culture conditions. As shown in Figure 1A (lanes 1 and 2), many soluble polypeptides could be detected even at relatively early stages of in vitro culture. However, given the extremely slow growth rate of T.borchii mycelia and the correspondingly long lag phase (∼15 days under standard culture conditions; Saltarelli et al., 1998), it is possible that many of these polypeptides are the remnants of surface proteins shed from the starting inoculum rather than the products of de novo synthesis. Indeed, the overall polypeptide pattern changed strikingly after ∼2 weeks of culture. We thus focused on 3- to 5-week-old mycelial cultures and, particularly, on a highly represented polypeptide with an apparent molecular mass of 23 kDa (p23) that showed maximal accumulation around day 28 (Figure 1A, lane 4). This polypeptide was gel purified, subjected to N-terminal sequencing and the resulting sequence (p23/20; Figure 1A) was employed for the design of degenerate oligonucleotides that were utilized as primers for a PCR amplification programmed with a cDNA library prepared from 30-day-old, liquid medium-grown mycelia (SLM-30). A single DNA fragment of 54 bp, coding for a conceptual translation product perfectly matching the inner 17 amino acids of the p23/20 peptide, was obtained from such amplification and used as a probe to screen the SLM-30 library. A phage plaque harboring a cDNA insert of 899 bp was thus identified. This cDNA, designated TbSP1 (submitted to the DDBJ/EMBL/GenBank databases under accession No. AF162269), was utilized as a probe for DNA and RNA gel blot analyses. As revealed by the DNA gel blot data reported in Figure 1B, a single-band pattern was produced by hybridization of the TbSP1 probe to genomic DNA digested with three different restriction enzymes, thus indicating that TbSP1 is encoded by a single copy gene in the T.borchii genome. As shown in Figure 1C, an mRNA of ∼900 nucleotides was identified by the TbSp1 probe in total RNA extracted from T.borchii mycelia. Such a transcript is present in free-living mycelia grown in either liquid (SLM) or solid (SSM) synthetic medium, but accumulates at higher levels (∼4-fold) in the latter condition. Figure 1.TbSP1 identification. (A) Equal amounts of SLM-released protein were subjected to SDS–PAGE and stained with Coomassie Blue R-250. Days of in vitro culture (d) and the migration positions of molecular mass markers are indicated at the top and at the left, respectively. The migration position (p23) and the N-terminal sequence (p23/20) of the TbSP1 polypeptide are shown on the right. (B) Genomic DNA digested with EcoRI (lane 1), BamHI (lane 2) or HindIII (lane 3) was probed with the TbSP1 cDNA. The migration positions of DNA size markers are indicated on the left. (C) Balanced amounts of total RNA extracted from synthetic solid (SSM) or liquid (SLM) mycelial cultures were gel fractionated and probed with the 32P-labeled TbSP1 cDNA. The migration positions and the amounts of the 28S and 18S rRNAs, utilized as internal references, are shown on the right and in the lower panel, respectively. Download figure Download PowerPoint Features of the TbSP1 polypeptide The full-length TbSP1 cDNA codes for a 211 amino acid polypeptide comprising a sequence identical to that of the previously determined p23/20 peptide between positions 32 and 51. This is preceded by an N-terminal stretch of 31 amino acids (underlined in Figure 2A) corresponding to the main hydrophobic region revealed by hydropathy analysis (not shown). The above observations, together with the presence of a Lys–Arg, KEX2-like protease cleavage site at the junction, suggest that the 31 N-terminal amino acids represent a secretion signal sequence, which is proteolytically processed to produce the mature TbSP1 protein found in the culture medium. The TbSP1 polypeptide is largely hydrophilic with a theoretical pI of 5.0, and the difference between the observed (23 kDa) and predicted (19 kDa) molecular mass of the mature protein is likely to be due to anomalous gel migration or post-translational modification (see below). Also, similarly to the adhesin-like SRAPs of P.tinctorius (Laurent et al., 1999), TbSp1 contains an RGD cell attachment motif (positions 60–62; boxed in Figure 2A). Apart from this single feature, however, there is no other similarity between TbSP1 and SRAPs. Figure 2.Alignment of TbSP1 with related polypeptides and with the active site amino acid sequences of known phospholipases A2. (A) The TbSP1 polypeptide is aligned with the amino acid sequences of Neurospora crassa and Streptomyces coelicolor homologs. The predicted secretion signal peptide is underlined and the pre-protein cleavage site is marked with an arrowhead; the tripeptide RGD motif is boxed. The region utilized for the comparison with known PLA2s is indicated by an overlying dotted line. Amino acids that are identical in all or the majority of the sequences are indicated by black or gray shading, respectively. Percentage identity values are reported in the bottom right part of the alignment. (B) The most highly conserved portion of the TbSP1 polypeptide (positions 122–153) is aligned with the active site regions of various small secreted PLA2s. The sequences of archetype PLA2s (Gelb et al., 2000; Six and Dennis, 2000) were utilized for this comparison. The indicated structural elements refer to the X-ray structure of the cobra venom PLA2IA (Fremont et al., 1993): catalytic histidine (open triangle); Ca2+-binding residues (filled circles); disulfide-bonded cysteines (open circles). Download figure Download PowerPoint Shown in Figure 2A is the alignment of TbSP1 with four homologous amino acid sequences identified by a search of the non-redundant protein database and of the unfinished whole genome sequence of Neurospora crassa. TbSP1 relatives, all corresponding to small hypothetical proteins (150–250 amino acids) with putative signal sequences at the N-terminus, were identified in Streptomyces coelicor (AL360055 and AL035654) and N.crassa (Neu_1.629 and Neu_1.351) and were used to query the protein database. Interestingly, a low sequence similarity with the conodipine-M PLA2 from the marine snail Conus magus (McIntosh et al., 1995) was revealed by the Streptomyces AL03564 sequence. Although only marginally significant (E = 7.1), this similarity mainly pertains to amino acid residues that are conserved in all TbSP1 relatives and, particularly, to a His–Asp pair within the conserved block evidenced in Figure 2A. An additional clue was provided by the identification in the patent section of DDBJ/EMBL/GenBank of an unpublished sequence from Streptomyces violaceoruber having the same nucleotide sequence as AL035654 and annotated as PLA2 (E08479, Patent: JP 1994327468-A; not included in the non-redundant set of the NCBI). Small extracellular PLA2s are divided into eight groups that share little sequence similarity except for the active site region, i.e. a stretch of ∼30 amino acids containing a helix with the His–Asp catalytic dyad and a metal-binding loop for the activating calcium cation (Gelb et al., 2000; Six and Dennis, 2000). As shown in Figure 2B, the TbSP1 protein exhibits some of the distinctive features shared by the different PLA2 groups. Two other conserved amino acids, besides the HD dyad, are a pair of cysteine residues known to form an invariant disulfide bond within the active site region. TbSP1 is a novel Ca2+-activated phospholipase A2 To verify the above prediction as to the function of TbSP1, the T.borchii polypeptide lacking the putative signal peptide was expressed in Escherichia coli as an N-terminal fusion with a metal-binding His6 tag. The resulting recombinant protein, initially identified with the use of a monoclonal anti-His tag antibody (not shown), was purified to near homogeneity (Figure 3A) and used for the production of polyclonal anti-TbSP1 antibodies. As shown by the immunoblot reported in Figure 3B, a single polypeptide with an apparent molecular mass of 23 kDa was recognized specifically by the anti-TbSP1 antibody in the growth medium of T.borchii suspension cultures (lane 1). The difference in size between natural (lane 1) and recombinant (lane 2) TbSP1 is all accounted for by the artificial 20 amino acid extension incorporated into the His-tagged TbSP1 protein. This indicates that the previously mentioned discrepancy between the predicted (19 kDa) and the estimated (23 kDa) molecular mass of TbSP1 is most probably due to an asymmetric protein conformation, and consequent anomalous gel migration, rather than to post-translational modification. Accordingly, no glycoprotein–periodic acid–Schiff (PAS) staining was observed with natural TbSP1 (not shown). Figure 3.Phospholipase A2 activity of purified rTbSP1. (A) SDS–PAGE of total lysates from uninduced (lane 1) or IPTG-induced (lane 2) E.coli cells and of purified mature rTbSP1 (lane 3). (B) Immunodetection of natural (lane 1) and recombinant (lane 2) TbSP1; a control reaction using rTbSP1-pre-saturated antibodies is shown in lane 3. The migration positions of molecular mass markers and of the recombinant TbSP1 protein (rTbSP1) are indicated. (C) Calcium dependence of rTbSP1-catalyzed [3H]oleic acid release from radiolabeled bacterial membranes. (D) pH dependence of TbSP1 phospholipase activity. (E) Time course of rTbSP1-catalyzed hydrolysis of 1-palmitoyl-2-[1-14C]palmitoyl, L-α-phosphatidylcholine (1-P-2-[14C]P-PC; open circles); radiolabeled hydrolysis products are indicated as follows: palmitic acid (PA; open triangles), 1-palmitoyl lysophosphatidylcholine (Lyso-PC; open squares). (F) TLC fractionation and phosphorimager visualization of radiolabeled hydrolysis products from a representative lipolytic assay conducted on 1-P-2-[14C]P-PC. The results of similar assays using double-labeled 1,2-di[1-14C]palmitoyl, L-α-phosphatidylcholine (1,2-di[14C]P-PC) as a substrate are shown in (G) and (H); see Materials and methods for details. Data reported in (C), (D), (E) and (G) are the average of at least three independent determinations which differed by no more than 15% of the mean. Download figure Download PowerPoint Escherichia coli membranes labeled in vivo with [3H]oleic acid (a phospholipid precursor that is incorporated preferentially at the sn-2 position) were utilized initially as a substrate to test the predicted phospholipase activity of TbSP1. As shown in Figure 3C and D, purified recombinant TbSP1 (rTbSP1) catalyzed the calcium-dependent release of [3H]oleic acid from bacterial membranes (half-activating Ca2+ concentration <1 μM) with maximum activity at ∼pH 6.5. Under optimized assay conditions, 300 pmol of [3H]oleic acid/min/mg of protein were released by TbSP1 and a similar (albeit ∼20-fold lower) fatty acid release was catalyzed by a commercial PLA2 preparation from S.violaceoruber that was used as a control for these experiments (not shown). As revealed by the additional data reported in Figure 3E–H, the TbSP1 phospholipase is specific for the sn-2 ester bond position and has very little (if any) lysophospholipase activity. In fact, [1-14C]palmitic acid was the only product generated by the action of rTbSP1 on 1,2-dipalmitoyl-phosphatidyl choline carrying a 14C-labeled palmitoyl residue at the sn-2 position (Figure 3E and F). Instead, radioactively labeled palmitic acid and 1-palmitoyl lysophosphatidyl choline both accumulated in parallel lipolytic assays conducted on the double-labeled phospholipid 1,2-di[1-14C]palmitoyl-phosphatidyl choline, containing radiolabeled palmitoyl moieties at positions sn-1 and sn-2 (Figure 3G and H). Localization of the TbSP1 phospholipase To gain insight into TbSP1 localization, an immunoblot analysis was conducted on various fractions generated by aqueous washing and subcellular fractionation of mycelia grown on solid medium. As shown in Figure 4A, a single polypeptide having the same electrophoretic mobility as extracellularly released TbSP1 (SLM; lane 7) was present in mycelial washings (lanes 1 and 2), whereas no signal could be detected in any of the fractions generated upon disruption and subcellular fractionation of pre-washed mycelia (lanes 3–6). This indicates that a portion of TbSP1 is loosely bound to the cell surface from which it can be extracted by aqueous washing. Figure 4.Immunoblot analysis of TbSP1 localization in Tuber mycelia and in yeast. (A) Mycelial washings (W1 and W2) and particulate fractions (F1, F2 and F3) generated by a series of differential centrifugation steps carried out at increasing sedimentation velocities were probed with the anti-TbSP1 antibody; fraction F4 is the final cytosolic supernatant (see Materials and methods for details). The migration positions of molecular mass markers and of SLM-secreted TbSP1 are indicated. (B) Immunodetection of cell-associated (lane 2) and extracellularly secreted (lane 4) TbSP1 in yeast cells transformed with a ‘sense’ TbSP1 construct; the migration positions of the unprocessed pre-protein (pre-TbSP1) and of the mature polypeptide (TbSP1) are indicated on the left. The results of control experiments conducted on a yeast ‘antisense’ TbSP1 transformant are shown in lanes 1 and 3; TbSP1 secreted by SLM-grown Tuber mycelia is shown for comparison in lane 5. Download figure Download PowerPoint To investigate TbSP1 localization further, the full-length TbSP1 protein, including the N-terminal hydrophobic peptide, was expressed in Saccharomyces cerevisiae under the control of a constitutive yeast promoter. As shown in Figure 4B, a single immunopositive band having the same gel mobility as the mature polypeptide released by T.borchii mycelia (SLM; lane 5) was detected specifically in the growth medium of yeast cells transformed with a ‘sense’ TbSP1 construct (lane 4). By comparison, a doublet of immunopositive polypeptides, with the sizes expected for the immature (pre-TbSP1) and the proteolytically processed (TbSP1) forms of the protein, was found in whole-cell extracts derived from the same ‘sense’ transformant (lane 2). These data, along with the initial identification of TbSP1 in the culture medium of T.borchii mycelia, thus point to TbSP1 as a dual localization protein that is both secreted extracellularly and surface associated. Nutrient-regulated expression of TbSP1 We next wished to find out whether TbSP1 expression is influenced by nutrient starvation. That this may indeed be the case initially was suggested by the results of RNase protection assays, reported in Figure 5A, showing that TbSP1 transcripts accumulate at higher levels upon culture of T.borchii mycelia in SSM or SLM than in the corresponding rich media (SM and LM). To examine this nutrient-dependent up-regulation in more detail, TbSP1 mRNA levels were determined in mycelia cultured for 21 days in SSM and then shifted for 10 days to incomplete culture media lacking a source of either carbon, nitrogen or phosphate. Data reported in Figure 5B show that the TbSP1 messenger was strikingly up-regulated by growth in either carbon- (∼80-fold; lane 5) or nitrogen- (∼130-fold; lane 8) deficient media, whereas basal TbSP1 levels, similar to those present in mock-shifted controls (lane 2), were present in mycelia grown for the same length of time in phosphate-deficient medium (lane 11). As further shown in Figure 5B (compare lanes 5 and 8 with lanes 6 and 9, respectively), this strong up-regulatory response is fully reversible, since basal levels of the TbSP1 transcript were recovered following an additional 5 day incubation in complete SSM. Furthermore, as revealed by the results of immunological analyses conducted on nutrient-deprived mycelia, starvation-induced up-regulation of the TbSP1 mRNA was accompanied by a parallel increase in the levels of the mature TbSP1 protein (Figure 6A), with a concomitantly increased accumulation of TbSP1 on the surface of nutrient-starved hyphae (cf. Figure 6B and C). Figure 5.Nutrient-regulated expression of the TbSP1 mRNA. (A) RNase protection analysis of TbSP1 mRNA levels in mycelia grown for 35 days on rich solid (SM) or liquid (LM) medium, or in the corresponding synthetic media (SSM and SLM). (B) RNase protection assays conducted on mycelia grown for 21 days on SSM (lanes 1, 4, 7 and 10) and then shifted for 10 days to the same medium (mock-shift control; lane 2) or to SSM lacking either glucose (−G, lane 5), ammonium (−N, lane 8) or phosphate (−P, lane 11). TbSP1 mRNA levels in parallel mycelial cultures starved for the above nutrients and returned for 5 days to complete SSM are shown in lanes 6, 9 and 12, respectively; the corresponding mock-shift control is shown in lane 3. A T.borchii β-tubulin (β-Tub) antisense riboprobe was included in all assays as an internal standard. The bands shown, which correspond to full-length protection products of the TbSP1 and β-Tub riboprobes were visualized by autoradiography and quantified by phosphorimaging. Relative transcript abundance values (reported below each lane) were calculated by dividing the volumes of the TbSP1 signals by the volumes of the corresponding β-Tub signals, followed by normalization with respect to TbSP1 abundance in SLM (A) or SSM (B; lane 1) cultured mycelia. Download figure Download PowerPoint Figure 6.Enhanced accumulation of the TbSP1 protein in nutrient-starved mycelia. (A) Immunoblot analysis of TbSP1 levels in mycelia grown for a total of 31 days in SSM (control; lane 1) and in parallel mycelial cultures shifted for the last 10 days to SSM lacking glucose (−G, lane 2) or ammonium (−N, lane 3). Equal amounts of total protein were loaded onto each lane and probed with the anti-TbSP1 antibody; an immunopositive TbSP1 band was also detectable in control mycelia with a longer exposure time. Samples of the same control (B) and nitrogen-starved (C) mycelia used for the experiment reported in (A) were subjected to immunofluorescence analysis using an FITC-labeled secondary antibody (bars = 18 μm); a control section of hyphae (h) from which the primary anti-TbSP1 antibody was omitted is shown in (D) (bar = 9 μm). Download figure Download PowerPoint TbSP1 accumulation in the inner cell wall layer of free-living and symbiosis-engaged hyphae The surface localization of TbSP1 and its production by hyphal structures representative of different stages of the Tuber life cycle were investigated finally by transmission electron microscopy (TEM)-immunolabeling experiments. As shown in Figure 7, cell wall-associated gold granules were detected in free-living hyphae transversally sectioned close to the subapical (Figure 7A) or the fully differentiated (Figure 7B) area. An identical surface localization was revealed by TEM-immunolabeling experiments conducted on T.borchii fruitbodies, in which TbSP1-associated gold granules were found in the cell walls of both vegetative hyphae and asci as well as on sporal envelopes (data not shown). Figure 7.Immunogold TbSP1 labeling of free-living hyphae. (A) Immunogold-TEM localization of TbSP1 in 30-day-old, SM-grown hyphae (h) transversally cut close to the subapical zone. (B) TbSP1 localization in the fully differentiated area of the same mycelium; the electron-dense material coating the external surface of the hyphae (h) is marked with" @default.
• W2013256472 created "2016-06-24" @default.
• W2013256472 creator A5018869586 @default.
• W2013256472 creator A5035350243 @default.
• W2013256472 creator A5057407298 @default.
• W2013256472 creator A5065942011 @default.
• W2013256472 creator A5068763493 @default.
• W2013256472 creator A5082752753 @default.
• W2013256472 creator A5091907746 @default.
• W2013256472 date "2001-09-17" @default.
• W2013256472 modified "2023-10-05" @default.
• W2013256472 title "A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii" @default.
• W2013256472 cites W1483806268 @default.
• W2013256472 cites W1560448629 @default.
• W2013256472 cites W1595453426 @default.
• W2013256472 cites W1647692986 @default.
• W2013256472 cites W1971250044 @default.
• W2013256472 cites W1978414262 @default.
• W2013256472 cites W1992170471 @default.
• W2013256472 cites W1994570371 @default.
• W2013256472 cites W1999563507 @default.
• W2013256472 cites W2002225116 @default.
• W2013256472 cites W2005542260 @default.
• W2013256472 cites W2018289835 @default.
• W2013256472 cites W2028519434 @default.
• W2013256472 cites W2035406968 @default.
• W2013256472 cites W2039723784 @default.
• W2013256472 cites W2044598948 @default.
• W2013256472 cites W2044629539 @default.
• W2013256472 cites W2050127281 @default.
• W2013256472 cites W2059347808 @default.
• W2013256472 cites W2070916151 @default.
• W2013256472 cites W2071991630 @default.
• W2013256472 cites W2078209921 @default.
• W2013256472 cites W2082572360 @default.
• W2013256472 cites W2097884283 @default.
• W2013256472 cites W2100837269 @default.
• W2013256472 cites W2104696612 @default.
• W2013256472 cites W2106675944 @default.
• W2013256472 cites W2106882534 @default.
• W2013256472 cites W2107280636 @default.
• W2013256472 cites W2107964261 @default.
• W2013256472 cites W2109312212 @default.
• W2013256472 cites W2110519006 @default.
• W2013256472 cites W2119297635 @default.
• W2013256472 cites W2126478262 @default.
• W2013256472 cites W2129629034 @default.
• W2013256472 cites W2131461400 @default.
• W2013256472 cites W2132305492 @default.
• W2013256472 cites W2143326683 @default.
• W2013256472 cites W2155866148 @default.
• W2013256472 cites W2158111073 @default.
• W2013256472 cites W2184428827 @default.
• W2013256472 cites W2405100023 @default.
• W2013256472 doi "https://doi.org/10.1093/emboj/20.18.5079" @default.
• W2013256472 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/125632" @default.
• W2013256472 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11566873" @default.
• W2013256472 hasPublicationYear "2001" @default.
• W2013256472 type Work @default.
• W2013256472 sameAs 2013256472 @default.
• W2013256472 citedByCount "74" @default.
• W2013256472 countsByYear W20132564722012 @default.
• W2013256472 countsByYear W20132564722013 @default.
• W2013256472 countsByYear W20132564722014 @default.
• W2013256472 countsByYear W20132564722015 @default.
• W2013256472 countsByYear W20132564722016 @default.
• W2013256472 countsByYear W20132564722017 @default.
• W2013256472 countsByYear W20132564722018 @default.
• W2013256472 countsByYear W20132564722019 @default.
• W2013256472 countsByYear W20132564722020 @default.
• W2013256472 countsByYear W20132564722021 @default.
• W2013256472 countsByYear W20132564722022 @default.
• W2013256472 countsByYear W20132564722023 @default.
• W2013256472 crossrefType "journal-article" @default.
• W2013256472 hasAuthorship W2013256472A5018869586 @default.
• W2013256472 hasAuthorship W2013256472A5035350243 @default.
• W2013256472 hasAuthorship W2013256472A5057407298 @default.
• W2013256472 hasAuthorship W2013256472A5065942011 @default.
• W2013256472 hasAuthorship W2013256472A5068763493 @default.
• W2013256472 hasAuthorship W2013256472A5082752753 @default.
• W2013256472 hasAuthorship W2013256472A5091907746 @default.
• W2013256472 hasBestOaLocation W20132564721 @default.
• W2013256472 hasConcept C142796444 @default.
• W2013256472 hasConcept C181199279 @default.
• W2013256472 hasConcept C18903297 @default.
• W2013256472 hasConcept C2776551241 @default.
• W2013256472 hasConcept C2779678110 @default.
• W2013256472 hasConcept C43144210 @default.
• W2013256472 hasConcept C523546767 @default.
• W2013256472 hasConcept C54355233 @default.
• W2013256472 hasConcept C55493867 @default.
• W2013256472 hasConcept C59822182 @default.
• W2013256472 hasConcept C86803240 @default.
• W2013256472 hasConcept C95444343 @default.
• W2013256472 hasConceptScore W2013256472C142796444 @default.
• W2013256472 hasConceptScore W2013256472C181199279 @default.
• W2013256472 hasConceptScore W2013256472C18903297 @default.
• W2013256472 hasConceptScore W2013256472C2776551241 @default.

• next