The Pisolithus tinctorius symbiosis related protein expressed sequence tag (EST PtSRP) was previously identified in the first hours of the interaction between the fungus Pisolithus tinctorius and sweet chestnut Castanea sativa, and partially characterized as a fungal marker gene of ectomycorrhizal symbiosis formation. We used the 5’ rapid amplification of cDNA ends (RACE) to obtain the PtSRP mRNA 5’ region, and together with our previously reported 3’ mRNA region, the full mRNA sequence was assembled by use of bioinformatics tools and deposited to GenBank (Accession: GU733439). The full-length mRNA sequence (636 bp) revealed the locations of the 5’ and 3’ untranslated regions (UTRs) and contained the Kozak sequence (ccc aag ATG A) in the 5’ UTR. The in silico translated PtSRP open reading frame (ORF) codes for a 127 amino acid protein and contained four putative post-translational modification sites (two N-glycosylation and two phosphorylation). The protein secondary structure is postulated to be composed of one N-terminal hydrophobic transmembrane alpha helix and at least six hydrophilic beta-strands spread across the protein. Sub-cellular localization prediction suggests that the protein is involved in cellular secretory pathway, supported by the presence of a cleavage site motif close to the membrane anchor. The data presented herein indicate the role of PtSRP as a fungal membrane secreted protein involved in early stages of ectomycorrhizal formation, with application as a possible marker for nascent ectomy-corrhiza fungal development.
The establishment of ectomycorrhiza involves controlled, intense gene expression in both partners that leads to drastic morphological and physiological changes, crucial to the development of mutualism and symbiotic harmony [1-3] . The comparison of protein extracts from mycorrhizal and non-mycorrhizal mycelia in previous studies has shown differences that suggest specific gene activation during the symbiosis process [4-6] . These findings highlighted a new class of biomolecules thought to control the ectomycorrhiza symbiosis process: the ectomycorrhizins [
Among the ectomycorrhizins, SRAPs (Symbiosis Related Acid Proteins) and hydrophobins are the most investigated and discussed classes of proteins. However, these proteins were generally isolated from fully established mycorrhiza or those developing associations after several days of interaction [8,9] . The identification of new early stage ectomycorrhizal molecules could bring new insight to the molecular and functional understanding of the ectomycorrhiza formation process. The fungal PtSRP mRNA (previously called Pisolithus tinctorius symbiosis related receptor 1, accession number EL563703) was isolated [
The 5’ RACE technique generated a partial 355 bp fragment corresponding to the 5’ portion of PtSRP mature mRNA (deposited at NCBI as nucleotide record GU733- 439). The complete sequence, assembled by contig construction between the 5’ RACE fragment and a 3’ previously reported sequence (EL563703, [
In silico analysis of PtSRP primary structure indicated theoretical molecular weight of 13,969 kDa and an iso-
electric point of 3.92. Further, four targets of post-translation modifications were predicted: two N-glycosylation sites (residues 118 to 121 (NFSQ) and 122 to 125 (NFTI)) and two casein kinase II phosphorylation sites (residues 65 to 68 (TNSE) and 99 to 102 (TVPD),
The predicted secondary structure was an N-terminal hydrophobic transmembrane alpha-helix, from residues 10 to 20, followed by six beta-sheets interspersed by short loops (
GLOBE prediction indicated that PtSRP is not a globular protein.
The studies of unknown ectomycorrhizal genes need to be carried out in several stages, as there are as yet none or few elements for comparison. Hydrophobins are fungal proteins usually found during the early stages of P. tinctorius-E. globulus interaction [
The Kozak consensus sequence was found in the 5’ UTR of the PtSRP mRNA. This regulatory element plays an important role during early processes of gene translation [
ences are common, as variation between species [18-20] . In Saccharomyces cerevisiae, for example, adenine is commonly observed to precede the initial methionine codon [
The computationally translated protein ORF showed significant local sequence alignment with two previously reported proteins. The first (48% identity and 65% similarity) was isolated from the mycelia of the ectomycorrhizal Laccaria bicolor (accession XP_001876100.1). This protein is associated with a small-secreted protein (SSP) of unknown function, up regulated in symbiotic tissues [
The presence of the integrin binding RGD domain (arginine-glycine-aspartic acid) in a protein has been related to cell adhesion [
The PtSRP post-translation modification sites previously indicated [
The fungal strain, culturing and ectomycorrhizal induction were done as described in [
Total fungal RNA was extracted using the PureLinkTM Micro-to-Midi Total RNA Purification System (Invitrogen). The 5’ PtSRP was obtained using 5’ RACE technology (GeneRacer—full-length, RNA ligase-mediated rapid amplification of 5’ cDNA ends—RLM-RACE, Invitrogen) according to the manufacturer’s instructions. The 5’ phosphate free ends were linked to GeneRacerTM RNA Oligo and the cDNA was synthesised (Superscript II—Invitrogen). PCR reactions were preformed using the primers supplied in the kit, aiming at the GeneRacerTM RNA Oligo combined with gene specific primers targeted to the 3’ sequence (PT-1440 REV: 5’-AAATCGTTCAGAGAGATAAAGTTG-3’ and PCR 1R REV: 5’- CGTCCGGTACTGTGACCATC-3’). Cloning of the largest RACE fragment was performed using the pGEM-T Easy Vector System (Promega) and the insertion was confirmed by PCR using Promega’s specific primers (SP6 and T7) directed to the cloning vector.
Cloned plasmids of recombinant bacteria (TG1) were extracted using Mini-prep. ABI PRISM BigDyeTM Terminator v3.1 Cycle sequencing Ready Reaction kit (Applied Biosystems) was used for sequencing the 5’ RACE fragment in an ABI PRISM 3100 Genetic Analyzer system (Applied Biosystems). Full-length mRNA was obtained by contig formation using SeqMan NGen v1.2 (DNASTAR Lasergene V8.0, Madison-US) after manual edition of sequences using ApE v1.15 (University of California-US) and Chromas Lite v2.01 (Technelysium Pty Ltd., Australia) software. This sequence will be referred in the text as 636 bp.
To confirm that the above contig sequence was correctly constructed, a primer pair (PtSRP FW: 5’-CCTCTCTCTCGAACACCTCCAC-3’ and PtSRP REV: 5’-ACGTACAGCAGAATGCGAAAG-3’), directed to the flanking regions of the gene ORF were designed (by use of ApE v1.15) for the direct PCR amplification of the gene from cDNA samples. The amplicons were cloned using the CloneJETTM PCR Cloning Kit (Fermentas). Cloned plasmids were extracted from recombinant DH10B using QIAprep® spin Miniprep kit and sequenced on an ABI PRISM 3100 as described above. Twelve experimental sequences were aligned giving a 604 bp consensus gene sequence which was further aligned to the 636 bp sequence described above.
The most probable ORF definition of the 636 bp was achieved using the “Find ORF” routine in the ApE v1.15, with identification of initial methionine and termination codons. Untranslated regions (UTRs) of possible ORFs were compared to the original Kozak sequence [
Structural analysis of the putative ORF was carried out using the Predict Protein Web-server (http://www.predictprotein.org) [
This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brazil) and the Post-Graduation Program on Fungal Biology (PPG-BF, Federal University of Pernambuco—Brazil).