Pls1 tetraspanins are widespread in ascomycetes and basidiomycetes
Proteins homologous to Pls1 [see Additional file 1] and [Additional file 2] were identified using Blastp in all available sequenced genomes of ascomycetes (Sclerotinia sclerotiorum, Leptosphaeria maculans, Podospora anserina, Chaetomium globosum, Trichoderma reesei, Fusarium verticilloides, Nectria haematococca, Stagonospora nodorum, Coccidioides posadasii) and basidiomycetes (Laccaria bicolor), except Ustilago maydis, Cryptoccocus neoformans, Aspergillus species, Mycosphaerella graminicola, and all Saccharomycotina species (hemi-ascomycetes). Another strategy based on the use of the tetraspanin HMM profile (PF00335) revealed a single Pls1 tetraspanin in the same fungal genomes. SsPLS1, LmPLS1, PaPLS1, GzPLS1, CpPLS1 cDNAs were obtained either from cDNA libraries or reconstructed from ESTs and used to define introns and start and stop codons in the corresponding genes.
The sequence conservation between the different Pls1 tetraspanins is essentially in the TM domains, some regions of the ECL2 including its cysteine-based pattern (CCGY-x(13)-C-x(11/19)-C-x(14)-TM4) and the C-terminal tail (Figure 2, [Additional file 1] and [Additional file 2]). The ECL1 sequences are more variable, as those in basidiomycete Pls1 proteins (17 aa) are shorter than in ascomycetes (26 aa). Similarly, the conserved motif in ICL from Pls1 proteins of basidiomycetes (QRNHVTLGLV) is very different from that found in Pls1 from ascomycetes (RGWLK). Three TM domains contain charged/polar amino acids (D in TM1, S/T in TM2, T in TM4) conserved among all Pls1 proteins. Some Pls1 proteins from ascomycetes have additional charged/polar amino acids (N in TM2, S/T in TM3 and D in TM4; see Figure 1 and [Additional file 1]). In animal tetraspanins, these conserved polar/charged amino acids are thought to stabilize interactions between transmembrane domains [10]. A putative palmitoylation site (cysteine residue) is located at the end of TM4 in most Pls1 tetraspanins, except those from the Leotiomycetes, Botrytis cinerea and Sclerotinia sclerotiorum (Figures 1 and 2, [Additional file 1]). In animals, the palmitoylation of several conserved juxtamembrane cysteine residues influences the formation of tetraspanin-enriched membrane microdomains [15–17]. A variable region is located in the ECL2 loop of fungal Pls1 tetraspanins (Figures 1 and 2, [Additional file 1]), between the last two cysteine residues whereas, it is located between the second and the last cysteine residues of the ECL2 in animal tetraspanins. Interestingly, in animals this variable region contains sites involved in specific tetraspanin protein-protein interactions [10]. The cysteine-based pattern of fungal Pls1 tetraspanins (CCGY-x(13)-C-x(11/19)-C-x(14)-TM4) contains a conserved tyrosine residue located after the CCG motif that is not in the cysteine-based pattern of animal tetraspanins (Figures 1 and 2, [Additional file 1] and [Additional file 2]). The cysteine-based pattern of ascomycete Pls1 EC2 (CCGY-x(13)-C-x(10)-GC-x(14)-TM4) is slightly different from that found in Pls1 from basidiomycetes (CCGY-x(13)-C-x(19)-C-x(14)-TM4, [Additional file 1] and [Additional file 2]) suggesting their ECL2 tertiary structure slightly differs from those of ascomycetes. The amino acid sequences of the C-terminal tail of fungal Pls1 tetraspanins are strongly conserved and are rich in charged/polar amino acids (average 70%). Indeed, these C-terminal tails display a conserved RxExERF/YxxIDxK motif found in Pls1 from ascomycetes as well as from basidiomycetes [see Additional file 1]. This conservation suggests that the C-terminal tail of fungal Pls1 tetraspanins plays a functional role as does that of animal tetraspanins [18, 19].
Tsp2 tetraspanins are restricted to basidiomycetes
Prior to this study, the first set of Tsp2 tetraspanins was identified in basidiomycetes such as Coprinus cinereus (CcTsp2A, CcTsp2B, CcTsp2C), Phanerochaete chrysosporium (PcTsp2) and Cryptococcus neoformans (CnTsp2, [2]). Tsp2 proteins display a long N-terminal tail (83 to 200 aa) and a long C-terminal cytoplasmic tail (66 to 101 aa). These domain lengths are characteristic of the Tsp2 family as Pls1 tetraspanins contain only short N-terminal and C-terminal tails (3–5 and 17–22 aa, respectively, Figure 2 and [Additional file 3]). Furthermore, the C-terminal tails of Tsp2 proteins are not as rich in charged amino acids (30%) as those of Pls1 proteins (60–75%). We identified four homologues of TSP2 (LbTSP2A, LbTSP2B, LbTS2C and LbTSP2D) in the basidiomycete L. bicolor. The correspondingcDNAs reconstructed from ESTs were used to define introns and start/stop codons in the corresponding genes. This family displays all the structural hallmarks of tetraspanin secondary structure with four transmembrane domains, a small extracellular loop (ECL1, 19 aa), a small intracellular loop (ICL, 4 aa), a large extracellular loop (ECL2, 72 aa) with a typical cysteine-based pattern (CCGY/F-x(12)-CY/F-x(6)-GCK-x(13)-TM4) and a conserved C-terminal cytoplasmic tail (Figure 2, [Additional file 2] and [Additional file 3]). Three TM domains contain one or two conserved charged/polar amino acids (N and Y in TM1, S and T in TM2 and Y in TM3) as observed for animal and fungal Pls1 tetraspanins [10, 11]. One putative conserved palmitoylation site (a cysteine residue at the junction between a transmembrane domain and an intracellular domain) is located at the end of TM4, proximal to the inner side of the membrane according to the predicted fungal tetraspanin topology. Additionally, LbTsp2A and PcTsp2 display another putative palmitoylation site (cysteine residue) at the start of TM1, proximal to the inner side of the membrane. We were not able to identify proteins orthologous to Tsp2 in ascomycetes using blast analysis or the tetraspanin HMM profile.
Tsp3 is a novel tetraspanin family specific to ascomycetes
We identified a novel tetraspanin in M. grisea which we named MgTsp3, using the tetraspanin HMM profile (PF00335). MgTsp3 is a modified version of MGG_13913.5 and annotation errors were corrected using the TSP3 cDNA sequence. This novel tetraspanin displays the structural hallmarks of tetraspanins including a characteristic cysteine-based pattern (CCG-x(18/22)-C-x(9)-C-x(11)-TM4) and a C-terminal tail containing a high proportion of polar/charged amino acids (70%; Figures 2 and 3). However, it differs from Pls1 of ascomycetes in the following features: a lack of charged/polar amino acid in TM domains, a smaller ECL1 (8 aa compared to 26 aa), a longer ICL (22–25 aa compared to 8 aa) and a longer C-terminal tail (70–110 aa compared to 17–22 aa) similar in size to that of Tsp2 proteins from basidiomycetes (66 to 101 aa, Figure 2).
Proteins homologous to Tsp3 were detected only in ascomycetes, including P. anserina, T. reesei, N. crassa, C. globosum, G. zeae, S. nodorum, B. cinerea, S. sclerotiorum and Uncinocarpus reesii (Figure 3). A Tsp3 homologue was identified in Aspergillus niger, this being the first report of an Aspergillus tetraspanin (Figure 3). In marked contrast, the Tsp3 protein is absent from basidiomycete genomes. The amplification of MgTSP3 cDNA obtained by RT-PCR allowed the identification of three introns at positions 37, 66 and 595 bp. Other TSP3 genes were aligned to ESTs when available [see Additional file 2], allowing prediction of their exon positions and their corresponding proteins in N. crassa and S. sclerotiorum. The number of introns varies between one and four in the Sordariaceae, with a conserved intron at position 31 bp from the start codon [see Additional file 2]. TSP3 genes from Leotiomycetes (Botrytis cinerea and S. sclerotiorum) have four introns at conserved positions [see Additional file 2]. SnTsp3 and TrTsp3 did notdisplay four transmembrane domains; however, this is probably due to a sequence gap and an incorrect intron annotation, respectively. For this reason, these genes were not included in the comparative analysis. Overall Tsp3 sequences are not as conserved as Pls1 and Tsp2 proteins from the same range of species (Figures 3). In particular, ICL and C-terminal tail are not conserved, unlike that in Pls1 proteins (Figures 3).
Tpl1 is a tetraspanin-like protein restricted to ascomycetes
In addition to the tetraspanins described above, the M. grisea genomecontains a tetraspanin-like gene, which we have named TPL1 (MGG_08113.5). This gene was identified by use of the tetraspanin HMM profile (PF00335) not only in M. grisea but also in other ascomycetes such as C. globosum, S. nodorum, N. crassa, P. anserina and A. nidulans (Figure 4). In marked contrast, the Tpl1 protein is absent from basidiomycete genomes. The MgTPL1 cDNA was reconstructed from ESTs and used to define introns and start/stop codons. MgTPL1 has two introns at position 40 and 262 bp from ATG [see Additional file 2]. Tpl1 proteins display some structural hallmarks of tetraspanins such as the presence of four transmembrane domains, a small ECL1 loop (14 aa), a short ICL loop (6–12 aa), a large ECL2 loop (56–68 aa) and a C-terminal cytoplasmic tail (9–17 aa) similar in size to that of Pls1 tetraspanins (Figures 2 and 4, [Additional file 2]). However, Tpl1 proteins markedly differ from Pls1, Tsp2 and Tsp3 tetraspanins in that they lack the typical cysteine based-pattern in the ECL2. Instead, they have two conserved cysteine residues close to the TM3 and TM4, respectively (Figures 2 and 4). For this reason, we have classified these proteins as tetraspanin-like.
Phylogeny of fungal tetraspanins reveals paralogs only in the Tsp2 family
Protein sequences from Pls1, Tsp2 and Tsp3 families were aligned using ClustalX 1.8 [see Additional file 4]. A phylogenetic analysis was then conducted using the PHYML software. The Tpl1 tetraspanin-like family was excluded from this alignment as these proteins are too divergent from Pls1, Tsp2 and Tsp3 proteins to be aligned correctly. The resulting phylogenetic tree (Figure 5) shows that Pls1, Tsp2 and Tsp3 form three distinct families. This phylogenetic tree also shows that the PLS1 genes from ascomycetes and basidiomycetes are orthologs, as the tree is congruent to the corresponding species phylogeny [20]. Similarly, TSP3 genes are orthologs (Figure 5). The TSP2 family consists of numerous paralogs, some being recent as their closest relative gene is in the same species (Tsp2B and Tsp2D from L. bicolor). The Tsp2 cluster is rooted by tetraspanins in the zygomycete, R. oryzae (RO3G_08988/RoTsp2-A and RO3G_17009/RoTsp2-B). The presence of tetraspanins in R. oryzae shows that this family of tetraspanins is ancient and predates the split between zygomycetes and higher fungi.
The number of introns in PLS1 genes varies from one to three in ascomycetes and from three to four in basidiomycetes [see Additional file 2]. One intron position is conserved in all PLS1 genes from ascomycetes (position 403, [see Additional file 2]) except in the related BcPLS1 and SsPLS1 (Leotiomycetes). This intron conservation supports the orthology found between PLS1 genes from ascomycetes. In PLS1 from basidiomycetes, three of the four intron positions are conserved [see Additional file 2] confirming a common origin for these three genes. The number of introns in genes from the TSP2 family varies from zero to four. However closely related TSP2 genes such as CcTSP2A, CcTSP2B, LbTSP2B and LbTSP2D (60% identity at the protein level) that cluster as a single clade in the phylogenetic tree (Figure 5) share the 2 among three introns, suggesting that they result from recent duplications in both C. cinereus and L. bicolor g enomes. In Sordariaceae, TSP3 genes contain three introns [see Additional file 2] except N. crassa which has four, although this may be due to an incorrect exon/intron annotation in the N. crassa gene that could not been corrected since it has no ESTs. Other TSP3 genes have three conserved intron positions (positions 31–52, 60–81 and 565–601, [see Additional file 2]) except in the related BcPLS1 and SsPLS1 (Leotiomycetes), which have an additional specific intron. This conservation of intron number and position supports the orthology between TSP3 genes.
Expression of fungal genes encoding tetraspanins
The expression patterns of PLS1, TSP2, TSP3 and TPL1 were evaluated in different fungal species in silico (absence or presence of ESTs from various libraries, [see Additional file 2]), and by quantitative PCR in M. grisea for MgPLS1, MgTSP3 and MgTPL1 (Figure 6) or by microarrays in L. bicolor for LbPLS1, LbTSP2-A, LbTSP2-B, LbTSP2-C and LbTSP2-D (Figure 7).
ESTs corresponding to genes from the PLS1 family were identified [see Additional file 2] in M. grisea (perithecia-sexual fruiting bodies), T. reesei,B. cinerea (mycelia), S. sclerotiorium (mycelia, sclerotia, and apothecia-sexual fruiting bodies), Gibberella moniliformis (mycelia), P. anserina (perithecia-sexual fruiting bodies, germinating ascospores, mycelia),C. cinerea (mycelia, sexual fruiting bodies), P. chrysosporium (tissue undetermined) and L. bicolor (free-living mycelia andmycorrhiza). In all these fungi PLS1 genes are expressed, ruling out the possibility that they correspond to pseudogenes. Indeed PLS1 cDNAs of L. maculans, S. sclerotiorium and C. posadasii were obtained by the screening cDNA libraries. qPCR expression profiling in M. grisea revealed that MgPLS1 mRNAis expressed at similar levels in mycelia and perithecia (0.9 × reference constitutive gene ILV5, Figure 6) and in spores, appressoria and infected barley leaves (0.5 × ILV5, Figure 6). In L. bicolor, PLS1 is constitutively expressed in all tissues analyzed (mycorrhizal symbiotic tissues, mycelia and fruiting bodies, Figure 7).
The expression patterns of the different TSP2 genes in L. bicolor were determined using genome wide long oligonucleotide microarrays (Figure 7). LbTSP2-A shows a barely detectable expression level in contrast to LbTSP2-B, LbTSP2-C and LbTSP2-D, which are expressed in all tissues of L. bicolor. LbTSP2-B is highly expressed in mycorrhiza and mycelia and at a lower level in fruiting bodies. Transcripts corresponding to LbTSP2-C are mainly found in fruiting bodies, whereas LbTSP2-D is over-expressed in mycorrhiza (Figure 7). ESTs corresponding to TSP2 were found in P. chrysosporium, C. neoformans, C. cinerea (mycelia, sexual fruiting bodies) and L. bicolor (mycelia) suggesting that these genes are expressed in the corresponding species.
ESTs corresponding to TSP3 were identified in G. zeae (mycelia, infected wheat heads and perithecia-sexual fruiting bodies), T. reesei (mycelia), Trichoderma harzianum (mycelia), B. cinerea (mycelia), S. sclerotiorium (apothecia-sexual fruiting bodies) and A. niger (mycelia). Quantitative PCR expression profiling in M. grisea showed that MgTSP3 is only weakly expressed in perithecia (0.1 × ILV5) and mycelia (0.05 × ILV5), and negligible in spore and appressoria (Figure 6). Spliced TSP3 transcripts were detected by RT-PCR using mycelial RNA (data not shown) suggesting that TSP3 is indeed expressed in mycelia, although at a very low level.
ESTs for TPL1 were only identified in M. grisea and P. anserina [see Additional file 2]. Quantitative PCR expression profiling in M. grisea revealed that MgTPL1 is over-expressed in perithecia and mycelia (0.4 × ILV5 and 0.3 × ILV5, respectively), but its expression is not detected in spores and appressoria (Figure 6).
Functional analysis of TSP3 and TPL1 in M. grisea
The M. grisea deletion mutants of TSP3 and TPL1 were obtained by targeted gene replacement in a P1.2-Δku80::bar mutant background that increases the frequency of homologous recombination [21]. Fifteen and 11 hygromycin-resistant transformants of TSP3 and TPL1, respectively, were isolated andanalyzed by PCR for the replacement of their wild-type alleles. Deletion mutants were obtained with an efficiency of 100% (15/15) and 82% (9/11) for TSP3 and TPL1, respectively. Their mycelial growth and sporulation rates were similar to those of P1.2-Δku80::bar (reference strain). These mutants were inoculated on detached barley leaves using droplets of conidial suspensions or on barley plants by spraying. Mutants Δtsp3::hyg or Δtpl1::hyg caused foliar lesions identical in number, size and aspect to those induced by the reference strain P1.2-Δku80::bar. The penetration frequencies of thesemutants on barley epidermis were similar to those of wild type, suggesting that TSP3 and TPL1 are not involved in pathogenicity on barley (Figure 8A). This behavior differs from Δpls1 mutant which is non-pathogenic on barley [3]. These mutants were spray-inoculated on two different rice cultivars compatible with M. grisea isolate P1.2, Azucena (O. sativa japonica) with an intermediate level of partial resistance to M. grisea and CO-39 (O. sativa indica) that is more susceptible to M. grisea than Azucena. Quantitative analysis of leaf blast infection revealed that the pathogenicity of Δtsp3::hyg and Δtpl1::hyg deletion mutants is significantly reduced on both cultivars. On cv. Azucena, the Δtsp3::hyg and Δtpl1::hyg mutants produced respectively less than 30% (z = 5, P = 0) and 35% (z = 4.6, P = 2.3 × 10-6) of the number of foliar lesions induced by the P1.2-Δku80::bar reference strain (Figure 8B). On cv. CO-39, the Δtsp3::hyg and Δtpl1::hyg mutants produced respectively less than 55% (t = 3.2, P = 1.2 × 10-3) and 40% (z = 4.3, P = 8.6 × 10-6), respectively, of the number of foliar lesions produced by the P1.2-Δku80::bar strain (Figure 8B).
The up-regulation of TSP3 and TPL1 in perithecia suggested involvement of these genes in mating. To address this question, the differentiation of perithecia and ascospores was examined in crosses between wild-type TH12 isolate and Δtsp3::hyg or Δtpl1::hyg mutants. A cross between the P1.2-Δku80::bar mutant (phenotypically similar to the wild-type P12 isolate) and wild-type TH12 isolate was used as control. Production of perithecia was monitored and three weeks after mating and those ascospores were observed and then allowed to germinate. No significant differences were observed in the fertility in crosses between Δtsp3::hyg, Δtpl1::hyg and wild type M. grisea strains, indicating that these genes are not essential for sexual reproduction.