The halotolerant endophytic fungal strain used in the present study for expression analysis of PPIase genes was identified as P. oxalicum. Microscopic observations revealed fungal hyphae to be highly branched with long brush-like branched conidiophores producing phialides with a short narrow neck. Conidia were smooth-walled, cylindrical to ellipsoidal and produced in chains in long parallel columns (Fig. 1a). Though this strain was able to grow in the presence of up to 15% NaCl, the growth was substantially higher in the medium lacking salt (Fig. 1b) since the colony diameter after ten days of incubation at 30ºC was higher (5.1 cm) compared to salt stress (1.2 cm). Further, relative to the unamended medium, the colonies obtained in the presence of salt were compact and depicted reduced sporulation. The mycelial fresh and dry weights of the culture were affected differently by salt stress. While the fresh weight was higher in the medium lacking salt, the mycelial dry weight was significantly greater in the presence of NaCl (Fig. 1c), which is in agreement with similar observations reported earlier for other fungi44. However, ultrastructure studies and the estimation of compatible solutes are required to understand the mechanism responsible for the salt-induced increase in the mycelial biomass of this strain.
In silico analysis revealed 237 full-length CLD-containing putative cyclophilin proteins in different species of Penicillium (Table 1). The number of cyclophilins in different Penicillium spp. range between 7–11, with the P. oxalicum genome encoding ten cyclophilins (Supplementary Table S1). Based on homology, the Penicillium cyclophilins were clustered into 12 different orthogroups viz., PenCYP01-PenCYP12 (Table 2, Supplementary Table S2a-l), which was also validated by their phylogenetic clustering (Fig. 2). Genes encoding different cyclophilins of the same orthogroup depicted conservation in their intron-exon architecture (Supplementary Fig. S1). While the genes for PenCYP09 cyclophilins (Group C) showed the absence of introns, the genes of orthogroup PenCYP06 (Group H) depicted up to seven introns (Supplementary Fig. S1). The PenCYP01, PenCYP05, PenCYP06 and PenCYP11 members were observed in all Penicillium spp., suggesting their essential role (Table 2). The PenCYP12 orthogroup comprises of only two cyclophilins, PcoCYP121 (121.93 kDa) and PgrCYP121 (121.9 kDa), that were observed only in P. coprophilum and P. griseofulvum, respectively (Supplementary Table S2l). Variable homology was noticed among cyclophilins of different orthogroups, with the interspecific divergence being higher in the members of PenCYP06 and PenCYP10, that showed a minimum similarity of 51.6% and 41.3%, respectively (Supplementary Table S3f, j). On the contrary, the orthogroups PenCYP01, PenCYP11 and PenCYP12 demonstrated greater conservation, with the minimum similarity being 90.7%, 84.2 % and 97.1%, respectively (Supplementary Table S3a, k, l).
The predicted molecular weights (MWs) and pIs of the Penicillium cyclophilins range between 17.63 kDa (P. steckii) to 126.82 kDa (P. arizonense), and 4.56 (P. expansum) to 9.32 (P. steckii), respectively (Table 1). The cyclophilins in P. oxalicum also showed divergence in their MWs and pIs, with the values ranging between 17.79 kDa (PoxCYP17) to 69.82 kDa (PoxCYP69), and 5.93 (PoxCYP41) to 8.87 (PoxCYP18), respectively (Supplementary Table S1). Though predominantly cytosolic, the cyclophilins in Penicillium were also predicted to localize to the nucleus, endoplasmic reticulum (ER) and mitochondria, highlighting functional divergence of these proteins (Table 1; Supplementary Table S2a-l). Besides cytosolic, the ER-localized (PoxCYP23) and nuclear PPIases (PoxCYP54, PoxCYP62-1, PoxCYP62-2 and PoxCYP69) were also observed in P. oxalicum (Supplementary Table S1). Except for PenCYP07 cyclophilins, in which the CLD ranges between 128-179 AAs, this domain's length is similar in cyclophilins of all other orthogroups (Supplementary Table S2a-l). The secondary structure of CLD, comprising of a typical β-barrel of eight antiparallel β-sheets with the two ends closed by α-helices and represented as βⅠβⅡαⅠβⅢβⅣβⅤβⅥαⅡβⅦαⅢβⅧ in hCYPA (Supplementary Fig. S2)24, showed conservation in cyclophilins of all orthogroups except PenCYP03, PenCYP05, PenCYP11 and PenCYP12 which either lack or contain a partial β1 region. Based on the presence of domains other than CLD, the cyclophilins were further classified as single domain (SD) or multidomain (MD) proteins (Table 3). While seven orthogroups (PenCYP01-PenCYP06 and PenCYP09) consist of SD cyclophilins, five orthogroups (PenCYP07, PenCYP08, PenCYP10-PenCYP12) comprise of MD proteins that contain additional domains such as TPR, RRM, U-box, WD, PP2C, and GIT_SDH (Supplementary Table S2a-l). Both SD (6) and MD cyclophilins (4) were also observed in P. oxalicum (Supplementary Table S1). This study predicted 15 different motifs within and outside the CLD (Supplementary Fig. S1), the motif composition being conserved in different cyclophilins of the same orthogroup. Comparative analysis with hCYPA revealed that all the active site residues corresponding to Arg (55), Phe (60), Met (61), Gln (63), Ala (101), Phe (113), Trp (121), Leu (122) and His (126), essential for PPIase activity and CsA interaction, are conserved in all cyclophilins of orthogroups PenCYP03, PenCYP06, PenCYP11 and PenCYP12 (Table 4, Supplementary Fig. S2). In P. oxalicum also, the PoxCYP17, PoxCYP18, PoxCYP23 and PoxCYP69 proteins showed retention of all the active site residues (Supplementary Table S4). Thus, these proteins are likely to be enzymatically active and might perform different cellular functions due to their PPIase activity. On the contrary, the cyclophilins belonging to the groups PenCYP02, PenCYP04, PenCYP05 and PenCYP07-PenCYP10 exhibited several substitutions in their active site residues, with the most common being Trp (121)/His (126) replaced with other residues (Table 4, Supplementary Fig. S2). While Trp121 in hCYPA is essential for CsA binding and changes in this residue result in decreased sensitivity to this immunosuppressant, mutations in the other active site residues are known to result in alteration in the PPIase activity45–48. The effect of alterations in the active site residues on PPIase activity of these cyclophilins needs further evaluation by cloning and characterizing these proteins.
The phylogenetic relationship among different cyclophilins was studied by constructing an unrooted tree based on proteins consisting of full-length or partial CLD sequences. This analysis divided the Penicillium cyclophilins into 11 distinct groups, A-K (Fig. 2). Interestingly, no P. oxalicum cyclophilin was observed in group G, suggesting that this gene might have been acquired by other species or lost from the P. oxalicum during the course of evolution. Similar events were implicated earlier in the evolution of plant NAC gene family also49. A noteworthy feature of Group K, comprising of PenCYP07 orthogroup, is the presence of PcaCYP7, PexCYP8, PitCYP8, PgrCYP7, and PsoCYP7 (that contain only N-terminus CLD) along with PcaCYP33, PexCYP33, PitCYP33, PgrCYP33 and PsoCYP33 (which possess only C-terminus CLD). It is likely that PcaCYP7, PexCYP8, PitCYP8, PgrCYP7 and PsoCYP7 might be the result of deletion of N-terminus region of CLD in PcaCYP33, PexCYP33, PitCYP33, PgrCYP33 and PsoCYP33, respectively. This speculation is supported by the fact that pairwise alignment of PcaCYP7, PexCYP8, PgrCYP7, PitCYP8, and PsoCYP7 with PcaCYP33, PexCYP33, PgrCYP33, PitCYP33, and PsoCYP33, respectively, corresponded to full-length cyclophilins that are homologous to other members of the same group (Supplementary Fig. S3). Though PgrCYP121 and PcoCYP121 were clustered in Group I, pairwise comparison prompted us to designate these proteins as a separate orthogroup PenCYP12 due to the presence of a large stretch of 950 AA residues that was not observed in other members of this group. The two proteins depicted 97% and 94% similarity in their GIT_SDH and CLD domains. Interestingly, GIT_SDH domain has not been reported yet in any of the Penicillium cyclophilins.
FK506-binding proteins (FKBPs)
Ninety-three putative FKBPs were identified in Penicillium spp. by basic local alignment search tool (BLAST) analysis using the human FKBP, hFKBP12, as a query. The hFKBP12 is the smallest member (12 kDa) of the FKBP family and contains the PPIase core domain50,51. Based on similarity, these proteins were categorized into four different orthogroups viz., PenFKBP01, PenFKBP02, PenFKBP03 and PenFKBP04 (Table 2). This grouping was also supported by the phylogenetic analysis, which depicted a close relationship of these proteins within a group (Fig. 3a). All Penicillium spp. except P. decumbens, P. occitanis and P. steckii depicted four different FKBPs. While P. decumbens contain only two FKBPs, both P. occitanis and P. steckii consist of three each (Table 2). Interestingly, P. antarcticum exhibited two different PenFKBP02 proteins, PanFKBP12-1 and PanFKBP13, that are 72.1% similar and appear to be paralogous (Supplementary Table S5b). The presence of PenFKBP02 and PenFKBP03 FKBPs in all Penicillium spp. underlines their essential role in the cell. The number of introns in FKBP genes varies between 1 to 5, with PsuFKBP61 of the orthogroup PenFKBP01 being the only exception with seven introns (Supplementary Fig. S4). Except for PsuFKBP61, the intron-exon architecture showed conservation in the FKBP genes of the same orthogroup. The FKBPs of orthogroup PenFKBP02 showed highest similarity with hFKBP12 (58%-71.3%), followed by PenFKBP01 (13.4%-61.2%), PenFKBP03 (36.4%-51.1%), and PenFKBP04 (14.8%-16%) (Supplementary Table S5a-d). Of the different P. oxalicum FKBPs, the maximum similarity with hFKBP12 was observed for PoxFKBP12-1 (64.5%), followed by PoxFKBP12-2 (61.2%), PoxFKBP14 (51.1%) and PoxFKBP52 (15.1%) (Supplementary Table S5e). The similarity among different FKBPs in P. oxalicum ranges between 15.3% -73.6% (Supplementary Table S5e).
Interspecific variability observed in the MWs and pIs of FKBPs in each orthogroup in Penicillium suggests divergence (Table 5, Supplementary Table S6a-d). The MWs of FKBPs in P. oxalicum differ from 12.93 kDa to 52.72 kDa, with a pI range of 4.38–9.36 (Table S1). The FKBPs in Penicillium spp. were predicted to localize to different subcellular organelles. While members of the orthogroups PenFKBP01 and PenFKBP02 might localize to the cytosol, the PenFKBP03 and PenFKBP04 FKBPs are likely to be present in the ER and nucleus, respectively (Table 3). An ER retention sequence (KDEL) (Supplementary Fig. S5) might be responsible for the likely presence of PenFKBP03 proteins in the ER. Contrary to the PenFKBP01, PenFKBP02 and PenFKBP03 proteins, which consist of only FKBP domain, the PenFKBP04 members also exhibited a nucleoplasmin like (NPL) domain (Table 3). The FKBP domain, consisting of four to six antiparallel beta-sheets surrounding the alpha-helix and represented as βⅠβⅡβⅢαⅠβⅣαⅡβⅤαⅢβⅥ in hFKBP1224, is conserved in all Penicillium FKBPs except for few members of orthogroup PenFKBP01 that lack the β1-sheet (Supplementary Fig. S6). Of the 15 different motifs observed in Penicillium FKBPs, the motifs 1 and 3, part of the FKBP domain, were observed in all the proteins. (Supplementary Fig. S4). Comparison of the 13 key residues that are implicated in FK506-binding52 revealed that relative to hFKBP12, the members of orthogroups PenFKBP01, PenFKBP02, PenFKBP03 and PenFKBP04 showed conservation at six, eight, nine and ten positions respectively (Table 4, Supplementary Fig. S6).
On the basis of homology with human parvulins hPIN1 (Protein Interacting with NIMA) and hPAR14 (human parvulin 14), the Penicillium parvulins were grouped into two different orthogroups viz., PenPIN01 and PenPAR01, respectively (Table 2). Phylogenetic analysis also provided evidence for the evolutionary relationship of these proteins within each orthogroup (Fig. 3b). The genes encoding PenPIN01 (except PsuPIN21) and PenPAR01 showed one and two introns, respectively (Supplementary Fig. S7). Whereas, PenPAR01 proteins were observed in all Penicillium spp. analyzed, the PenPIN01 parvulins were not detected in P. antarcticum, P. arizonense, P. coprophilum and P. solitum (Table 2). The PenPAR01 and PenPIN01 parvulins showed 53.7%-68.5%, and 57.7%-66.7%, similarity with hPAR14 and hPIN1, respectively (Supplementary Table S7a, b). In P. oxalicum also, the PoxPAR14 and PoxPIN1 shared 66.9% and 65.7% similarity with their human orthologues hPAR14 and hPIN1, respectively. High similarity among members of PenPAR01 (65.4-100%) and PenPIN01 (76.5%-100%) groups implies conservation of parvulins among different species of Penicillium (Supplementary Table S7a, b). Except for PdePAR17 (17.67 kDa) in P. decumbens, the MWs of PenPAR01 proteins differed between 13.62 to 14.75 kDa, and that of PenPIN01 members between 19.30 to 21.88 kDa (Table 6). The pI values in orthogroups PenPAR01 and PenPIN01 varied between 9.41 to 9.68, and 5.72 to 6.46, respectively. The larger size of PdePAR17 is attributed to an extended N terminal 36 amino acid sequence containing mitochondrial localization signal53. Except for PdePAR17, which might be a mitochondrial protein, all members of the PenPAR01 orthogroup were predicted to localize to either cytosol or nucleus. Majority of the PenPIN01 proteins, on the contrary, appeared to be nuclear, with only P. steckii (PstPAR13) and P. subrubescens (PsuPAR14) depicting localization in the cytoplasm. Contrary to the PenPAR01 parvulins, that contain only the PPIase domain, the PenPIN01 members also exhibited an additional conserved N-terminal WW domain (Table 3). The PenPAR01 and PenPIN01 parvulins contain ten different motifs, with the motifs 1, 4 and 5 present in all members. (Supplementary Fig. S7). Whereas all the ten active site residues relative to hPIN1 are conserved in PenPIN01 parvulins, only five active site residues in PenPAR02 members showed conservation relative to hPAR14 (Table 4). As observed in hPAR14 and hPIN154, all Penicillium parvulins exhibited the presence of β1α1α2α3β2α4β3β4 elements in their PPIase domain (Supplementary Fig. S8a, b), suggesting that the secondary structure of these proteins is conserved across taxa. Though conservation of these proteins underlines their fundamental role in the cell, the absence of PenPIN01 members in P. antarcticum, P. arizonense, P. coprophilum and P. solitum also suggests redundancy in their functions.
Protein phosphatase 2A phosphatase activators (PTPAs)
The members of PenPTPA01 and PenPTPA02 orthogroups in Penicillium spp. were identified by BLAST analysis based on their similarity with their yeast orthologues YPA1 and YPA2, respectively. This analysis revealed that except for P. nalgiovense and P. steckii, which lack PTPA02 gene, all other Penicillium species contain both the PTPAs (Table 2, 7). Phylogenetic analysis also supported a close evolutionary relationship among proteins of each orthogroup (Fig. 3c). In silico studies further revealed that while all the genes encoding PenPTPA01 proteins contain two introns, the same is lacking in the PenPTPA02 genes (Supplementary Fig. S9). The YPA1 exhibited 44.3%-49.2% similarity with PenPTPA01 orthologues, compared to 53.1%-57.4% for YPA2 with PenPTPA02 members (Supplementary Table S8a, b). The molecular weights of PenPTPA01 and PenPTPA02 vary between 46.43 to 52.97 kDa, and 46.05 to 47.55 kDa, respectively, while the pI values for the two PPIases range between 5.81-7.20 and 5.84-6.44, respectively (Table 7). The PenPTPA01 and PenPTPA02 proteins in Penicillium spp. were predicted to localize to the cytosol, and consist of only PTPA domain of 283-331 and 293-295 amino acid residues, respectively. The two PTPA orthogroups revealed the presence of 15 different motifs, of which six (1-3, 6 and 9) are common to all members (Supplementary Fig. S9). High similarity among PenPTPA01 (69.3%-100%) and PenPTPA02 (77.1%-100%) members in Penicillium spp. suggests conservation, indicating an essential role for these proteins in the cell (Supplementary Table S8a, b).
Estimation of PPIase activity and expression analysis of PPIases genes in P. oxalicum
The total and specific PPIase activities under salt stress were significantly higher than control at all the stages of growth in P. oxalicum (Fig. 4a, b,c). Further, the PPIase activity under control conditions was not regulated temporally since no significant difference in the mycelial catalytic activity was observed at different growth stages. On the contrary, substantial enhancement in the specific PPIase activity was noticed between 4 and 7 days after inoculation (DAI) under salt stress that appeared to be due to induction of PPIases since decrease in total protein content during this duration was 40.3% (from 9.04 to 5.4 mg/g fresh weight) compared to 88.7% (from 8.3 to 15.68 nmol/sec/mg protein) increase in specific PPIase activity (Fig. 4c, d). FKBPs and cyclophilins' contribution to PPIase activity in P. oxalicum was evaluated by the extent of inhibition by their specific inhibitors FK506 and CsA, respectively. Whereas PPIase activity under control conditions was almost completely inhibited by CsA at all the growth stages, the CsA-induced inhibition in the presence of salt was about 85% and 87% at 4 and 7 DAI, respectively (Fig. 4e). These observations imply that PPIase activity in the mycelia of P. oxalicum was predominantly contributed by the cyclophilins. However, 15% and 13% abrogation of PPIase activity by FK506 at 4 and 7 DAI under salt stress also indicated the contribution of FKBPs to enzyme activity at these stages. We carried out real time-PCR analysis to further analyze the contribution of different PPIase genes to the mycelial PPIase activity. This analysis revealed that three cyclophilin (PoxCYP18, PoxCYP23 and PoxCYP41) and two FKBP genes (PoxFKBP12-2 and PoxFKBP52) were expressed at all stages of growth under both control and salt stress conditions (Fig. 5). Whereas the expression of PoxCYP18 at 4 and 10 DAI increased significantly under salt stress, the transcript levels of PoxCYP23, PoxCYP41, PoxFKBP12-2 and PoxFKBP52 at all stages of growth decreased substantially. However, the transcripts corresponding to parvulins and PTPAs were not observed at any of the stages analyzed.