Fungi have three tetraspanin families with distinct functions

Abstract

Background: Tetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea, Botrytis cinerea and Colletotrichum lindemuthianum, and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor, and also their functional analysis in M. grisea.

Results: The exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2, which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor. A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea, suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Deltatsp3 and Deltatpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Deltapls1 mutants, which are completely non-pathogenic on barley and rice.

Conclusion: A new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea. The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi.

Figure 1

Schematic structure of Pls1 tetraspanins. Tetraspanins contain four transmembrane domains (TM1 to TM4) with conserved polar/charged residues (pink circles), a small extracellular loop (ECL1), a small intracellular loop (ICL) and a large extracellular loop (ECL2). ECL2 contains a CCG motif and further two conserved cysteine residues (yellow circles). The N-terminal and C-terminal tails are intracellular. These cysteine residues allow formation of two disulphide bridges (red lines), crucial for the folding of ECL2. One putative palmitoylation site is located proximal to TM4 (green circle).

Figure 2

Structural comparison of fungal tetraspanins (Pls1, Tsp2, Tsp3) and tetraspanin like proteins (Tpl1). The Pls1, Tsp2 and Tsp3 tetraspanin families display the typical structure of tetraspanin: four transmembrane domains (TM1, TM2, TM3, TM4, yellow boxes) and a small extracellular loop (ECL1, gray line), a small intracellular loop (ICL, purple line), a large extracellular loop (ECL2, orange line), intracellular N-terminal (black line) and C-terminal (brown line) tails. ECL2 contains a CCG motif and further two conserved cysteine residues (red triangles). A. The Pls1 tetraspanin family contains conserved polar/charged residues in TM1, TM2, TM3 and TM4 (blue lines) and one putative palmitoylation site is proximal to TM4 (green triangle). B. The Tsp2 tetraspanin family displays large intracellular N-terminal and C-terminal tails. One putative conserved palmitoylation site is located at the end of TM4 (green triangle). C. The Tsp3 tetraspanin family has a short ECL1 and a large C-terminal tail. D. The Tpl1 tetraspanin-like proteins lack the typical cysteine-based pattern in their ECL2 (CCG motif and further two conserved cysteine residues). Nevertheless, their ECL2 regions contain two conserved cysteine residues close to TM3 and TM4.

Figure 3

Alignment of Tsp3 tetraspanins. Sequences were aligned using ClustalX 1.8. Conserved amino acids are indicated in black (>80%), dark gray (>60%) and light gray (>40%). The transmembrane domains (TM) are circled in black, the small extracellular loop (ECL1), the small intracellular loop (ICL), the large extracellular loop (ECL2) are shown in gray, purple and orange lines, respectively. ECL2 contains a CCG motif and further two conserved cysteine residues (red). These cysteine residues allow formation of two disulphide bridges crucial for the folding of ECL2. Mg: Magnaporthe grisea, Pa: Podospora anserina, Nc: Neurospora crassa, Bc: Botrytis cinerea, Ss: Sclerotinia sclerotiorum, Tr: Trichoderma reesei, Gz: Gibberella zea, An: Aspergillus niger, Ur:Uncinocarpus reesii. For a tetraspanin structural model, see Figure 1.

Figure 4

Alignment and phylogenetic tree of Tpl1 tetraspanins like. A. Alignment of Tpl1 tetraspanins. Sequences were aligned using ClustalX 1.8. Conserved amino acids are indicated in black (>80%), dark gray (>60%) and light gray (>40%). The transmembrane domains (TM) are circled in black, the small extracellular loop (ECL1), the small intracellular loop (ICL), the large extracellular loop (ECL2) are shown in gray, purple and orange lines, respectively. ECL2 contains two conserved cysteine residues (red). Mg: Magnaporthe grisea, Cg: Chaetomium globosum, Nc: Neurospora crassa, Pa: Podospora anserina, Sn:Stagonospora nodorum, An: Aspergillus nidulans, Ac: Aspergillus clavatus. (For a structural model, see Figure 1). B. Phylogenetic tree of fungal Tpl1 proteins. Aligned sequences were analyzed using maximum likelihood from PHYML and XP_501745 from Yarrowia lipolytica and Nce2 (YPR149W) from Saccharomyces cerevisiae as outgroups. Bootstraps values are expressed as percentage of 100 replicates.

Figure 5

Phylogenetic tree of fungal Pls1, Tsp2 and Tsp3 tetraspanins. The Pls1, Tsp2 and Tsp3 were aligned using ClustalX 1.8. Aligned sequences were analyzed by maximum likelihood using PHYML and RO3G_14268 and RO3G_02583 from R. oryzae as outgroups [see Additional files 4]. Bootstraps values are expressed as percentage of 100 replicates. Mg: Magnaporthe grisea, Cg: Chaetomium globosum, Cl: Colletotrichum lindemuthianum, Nc: Neurospora crassa, Pa: Podospora anserina, Nh: Nectria haematococca, Gz: Gibberella zeae, Fv: Fusarium verticilloides, Tr: Trichoderma reesei, Sn: Stagonospora nodorum, Lm: Leptosphaeria maculans, Cp: Coccidioides posadasii, Bc: Botrytis cinerea, Ss: Sclerotinia sclerotiorum, An: Aspergillus niger, Lb: Laccaria bicolor, Cc: Coprinus cinereus, Pc: Phanerochete chrisosporium, Cn: Cryptococcus neoformans, Ur:Uncinocarpus reesii.

Figure 6

Expression profiles of PLS1, TSP3, TPL1 in different tissues from M. grisea. PLS1, TSP3, TPL1 expressions were quantified by real-time RT-PCR using RNA extracted from mycelia (M), spores (S), 24-h old appressoria (A) from P1.2 wild type strain, perithecia (P) from crosses between P1.2 (MAT1.2) and TH12 (MAT1.1) strains and 3-days old barley leaves infected by P1.2 (I). PLS1, TSP3, TPL1 expressions were calculated relative to the transcripts levels of the constitutively expressed gene ILV5 (MGG_01808.5) according to the formulae: 2^-ΔCt = 2^{-(CtgeneX-CtILV5)}. Each data point is the average of three biological replicates. Standard deviation is indicated by error bars. PLS1 is expressed in all tissues whereas TSP3 and TPL1 are expressed in mycelia and perithecia.

Figure 7

Expression profiles of LbPLS1, LbTSP2-A, LbTSP2-B, LbTSP2-C and LbTSP2-D in different tissues from Laccaria bicolor. LbPLS1 and LbTSP2-A, LbTSP2-B, LbTSP2-C and LbTSP2-D expressions were measured by 60-mer oligoarrays (NimbleGen) using RNA extracted from mycorrhiza, mycelia and fruiting bodies (carpophores). LbPLS1, LbTSP2-A, LbTSP2-B, LbTSP2-C and LbTSP2-D expressions were calculated relative to the transcripts levels of the constitutively expressed gene EIF-5A (LACBI1_192615). Each data point is the mean between values of two biological replicates and a third value corresponding to the mean of two biological replicates. Standard deviation is indicated by error bars. PLS1 is constitutively expressed in all tissues, LbTSP2-A transcripts are barely detectable in comparison to LbTSP2-B, LbTSP2-C and LbTSP2-D transcripts that are expressed in all tissues. LbTSP2-B is strongly expressed in mycorrhiza and mycelia whereas LbTSP2-C and LbTSP2-D are up-expressed in fruiting bodies and in mycorrhiza, respectively.

Figure 8

Pathogenicity of Δtsp3 and Δtpl1 deletion mutants from M. grisea. (A) Pathogenicity on barley. Barley cv. Plaisant leaves were inoculated with droplets of conidial suspension (3 × 10⁴ conidia/ml) from pathogenic P1.2-Δku80::bar (+) and Δku80::bar/Δtsp3 and Δku80::bar/Δtpl1 mutants. Leaves were kept on water agar for 7 days under alternate day/night at 26°C and scored. Typical symptoms induced by M. grisea are visible as pale green ellipsoid lesions surrounded by a yellow halo for wild type as well as tetraspanin mutants showing that Δtsp3 and Δtpl1 mutants are still pathogenic on barley. (B) Pathogenicity on rice. Seedlings of rice cultivars Azucena and CO-39 were sprayed with conidial suspensions (2.5 × 10⁴ conidia/ml) from pathogenic P1.2-Δku80::bar (+) and Δku80::bar/Δtsp3 and Δku80::bar/Δtpl1 mutants. Seedlings were incubated for 7 days under alternate day/night at 25°C for development of disease. The deletion mutants Δku80::bar/Δtsp3 and Δku80::bar/Δtpl1 produced significantly fewer disease lesions than P1.2-Δku80::bar (reference strain).