3.1 ITS sequence based phylogeny of FSSC members
The phylogenetic analysis based on ITS sequence separated three major clades of the FSSC members and the F. solani isolates SF104 and SF301 aggregated into the members belonging to Clade 3 (Fig. 1). F. solani SF104 was placed at the same branch with the Indian isolate F. ambrosium (FSSC19) and the Slovenian isolate Fusarium sp. (FSSC44), whereas F. solani SF301 was with the Italian isolate F. solani (FSSC5).
3.2 Molecular characterization of β-tubulin gene
Using the primer pair βOD F and βEx5 R, an approximately 1.6 kb DNA fragment was amplified from both the F. solani isolates. Nucleotide BLAST of the DNA sequences revealed that 98 % identical with partial β-tubulin genes of different F. solani strains/isolates: WNQ3 (GenBank accession no. MK441724), FJBX18-1 (MN295050), FJBX18-2 (MN295051), and FJBX18-3 (MN295052); 97 % identical with CMFS007 (KU983876), MICMW-30.1 (KX912242), PaR-1 (MN692927), PaR-2 (MN692928), PaR-3 (MN692929), and F. solani strain causing root rot and stem canker on storage roots of sweet pea in China (KF255996); 96 % identical with FS-01403 (KJ572782). Based on Fusarium MLST identification, the nucleotide sequences showed highest similarity (97.12 %) with partial sequence (1333 bp) of β-tubulin gene of F. solani species complex NRRL 46706. The β-tubulin gene sequences of F. solani SF0104 and SF0301 were deposited in GenBank under accession numbers MZ409524 and MK900720, respectively.
By comparison with other fungi it was inferred that the full length (1619 bp) β-tubulin gene of both the F. solani isolates was organized into five exons and four introns (Fig. 3A). The intron positions were conserved and occurred after the codons for amino acids 5, 12, 54 and 316, but they varied in their lengths. All introns contained 5' and 3' consensus splice junctions similar to those in β-tubulin genes of other fungi that conformed to the GT-AG rule. The G+C content of the coding region was calculated as 58.61 %. Both the β-tubulin genes encoded a 446 amino acid protein, with a calculated molecular mass 49.834 KD, extinction coefficient 41640 M-1cm-1 and an estimated isoelectric point of 4.64.
Sequence comparison between the carbendazim resistant and sensitive isolates revealed that they were all identical, indicating that the mechanism of carbendazim resistance in F. solani SF0301 was different from other filamentous fungi, where point mutation at different sites on the β-tubulin gene is responsible for the resistance.
Protein BLAST of the deduced amino acid sequences revealed that 100 % identical with that of F. euwallaceae (AMD38824), a member F. solani species complex; 99.78 % identical with that of F. heterosporum (KAF5674510); 99.55 % with F. oxysporum (XP_018241948), F. verticilloides (XP018748359), F. longipes (RGP77670), F. sacchari (AMD38831), F. denticulatum (KAF5673514) and F. pseudograminearum (XP009254731); 99.33 % with F. graminearum (PCD17759), F. fujikuroi (AAB18275), F. bulbicola (KAF5979982) and F. commune (AMD38822); 99.1 % with F. babinda (AMD388320) and F. poae (AMD38828); and 98.88 % with F. sporotrichioides (RGP75200).
3.3 Phylogenetic analysis of Fusarium species based on β-tubulin nucleotide sequences
The phylogenetic analysis of the Fusarium species including F. solani based on β-tubulin gene sequence revealed their position in nine clusters considered as species complexes (Fig. 2). The species complexes are F. sambucinum species complex (FSaSC, nine species), F. incarnatum-equiseti species complex (FIESC, two species), F. tricinctum species complex (FTSC, two species), F. heterosporum species complex (FHSC, one species), F. fujikuroi species complex (FFSC, 25 species), F. oxysporum species complex (FOSC, four sequences), F. redolens species complex (FRSC, one species), F. decemcellulare species complex (FDSC, one species) and F. solani species complex (FSSC, 10 sequences). The phylogenetic tree showed a well-supported relationship (99 % MP bootstrap) among the F. solani isolates of FSSC under the Clade 3 including our Indian isolates, SF0104 and SF0301. The formation of three clades by FSSC isolates was also confirmed by the analyses based on β-tubulin sequences. The sister groups FSaSC and FIESC, FTSC and FHSC also showed well-supported lineage with 100 % bootstrap support. F. decemcellulare formed lineage from F. solani was in accordance with Aoki et al. (2014). The 25 species under the FFSC were phylogenetically placed in three different clades as American, Asian and African - the Asian clade was derived from the African clade with 82 % bootstrap support and American clade from the Asian clade with 100 % bootstrap support.
3.4 Comparison of β-tubulin gene of F. solani isolates with other Fusarium species
The β-tubulin gene of carbendazim resistant F. solani SF0301 was compared with complete β-tubulin gene of other Fusarium species available in GenBank (Fig. 3B). All Fusarium species including F. solani had β-tubulin gene with five exons except F. graminearum (AY303689), F. praegraminearum (KX260131) and F. sambucinum (AF484166) as they lack 4th intron; hence, their total β-tubulin length became shorter with 1631 bp, 1629 bp and 1643 bp, respectively. Other species also showed variation in total length due to variation of intron length and among these, 1st intron showed maximum variation. The β-tubulin gene of the F. solani isolate was found to be shortest with 1619 bp in length and their introns were shortest among the Fusarium species. Out of the total length of 1619 bp, coding region consisted of 1341 bp and non-coding region with only 278 bp was made up of 1st, 2nd, 3rd and 4th introns with 139 bp, 49 bp, 44 bp and 46 bp, respectively. All the introns in other Fusarium species were larger, thus their β-tubulin lengths were larger and in the range from 1668 bp in F. redolens (MT011043) to 1684 bp in F. andiyazi (MT011059). The nucleotide length of 2nd, 3rd and 4th introns were almost similar among the Fusarium species with an average of 58 bp, 48 bp and 50 bp, respectively. However, shorter 2nd intron was found in F. sambucinum (57 bp) and F. solani (49 bp), and larger in F. verticillioides (59 bp). The length of 3rd intron also varied in few species such as, 49 bp in F. ramigenum and F. napiforme, 47 bp in F. redolens and 44 bp in F. solani. The length of 4th intron was 49 bp in F. brevicatenulatum, F. pseudoanthophilum, F. pseudonygamai, F. verticillioides and F. redolens, whereas in F. solani it was 46 bp. The variable 1st intron was largest in F. sambucinum (197 bp) and shortest in F. solani (139 bp). Among the total 1341 bp coding region, the length of exons of all Fusarium species was highly conserved and exon 1, 2, 3, 4 and 5 contained 12 bp, 24 bp, 123 bp, 791 bp and 391 bp, respectively. Thus, exon 4 was the largest. However, exon 4 of F. graminearum (AY303689), F. praegraminearum (KX260131) and F. sambucinum (AF484166) were larger than the average length (791 bp) due to lack of 4th intron and contained 1185 bp, 1182 bp and 1182 bp, respectively. The β-tubulin of all the Fusarium species encoded 446 amino acid protein except F. graminearum which encoded 447 amino acid protein. All these data were calculated only from β1-tubulin nucleotide sequence from those which had two β-tubulin genes.
3.5 Comparison of β-tubulin amino acid of F. solani isolates with other Fusarium species
The most frequent difference of β-tubulin amino acid sequence of F. solani isolates with other Fusarium species was found to be at 381st position with isoleucine (I) in F. solani isolates and valine (V) in other (Fig. 4). However, F. avenaceum and F. heterosporum also contained isoleucine at 381st position similar to the F. solani isolates. But they differed from F. solani isolates at 189th position where valine in F. solani was changed as isoleucine in these two species. Another difference was identified at 335th position where asparagine (N) in F. solani isolates was changed to serine (S) in other Fusarium species. However, this change was not detected in species such as F. oxysporum, F. fujikuroi, F. verticilloides, F. decemcellulare, F. avenaceum and F. heterosporum. The amino acid proline (P) was only found in F. oxysporum f.sp. cubense at 279th position, where as in others it was histidine (H). In addition, a few minor changes were found in different species (Fig. 4).
3.6 Homology modeling and structural analysis
To build a tertiary structural model of β-tubulin, an appropriate template has been searched using Swiss-Model (https://swissmodel.expasy.org). Of the yielded templates, the crystal structure of Bos taurus (PDB ID: 4O4I) was found to be the best template with the sequence identity of 83.15 % with the query coverage of 96.64 % and the QMEAN score of the built model was -1.85. A tertiary model structure of β-tubulin was again built up by BIOVIA Discovery Studio package using the crystal structure. The three-dimensional model of the protein was found to be composed of different secondary structural elements (Fig. 5).
3.6.1 Proposed secondary structural conformation
The secondary structure of β-tubulin of F. solani had proposed to contain 21 α-helices, 16 β-strands and several coils (Fig. 5A). The α-helices were formed with the amino acid position from 10-27, 41-44, 47-49, 71-78, 87-89, 101-105, 108-126, 143-158, 181-195, 204-213, 222-241, 250-257, 276-279, 286-293, 296-298, 305-307, 323-336, 338-340, 372-389, 396-399 and 405-427. The β-strands were found with the amino acid position from 4-9, 30, 36, 51-54, 58-61, 63-68, 90-92, 131-138, 163-170, 198-203, 265-271, 299, 310-319, 341, 249-354 and 363-371. When the constructed secondary structure of β-tubulin of F. solani was compared with that of other species using POLYVIEW-2D, it was observed that all the Fusarium species had identical secondary structural elements. However, F. oxysporum f.sp. cubense contains 20 α-helices due to presence of proline (instead of histidine in other) at 279th position and as proline is a helix breaker, the α-helix formed by ARG276, GLY277, ALA278 and HIS279 in other Fusarium species is absent in F. oxysporum f.sp. cubense (Fig. 5B).
3.6.2 Model reputation
The stereo-chemical behavior of the predicted model of β-tubulin protein of F. solani was analyzed through ProSA and QMEAN server’s confirmation and evaluated by the Phi/Psi Ramachandran plot inspection. ProSA was used to confirm the three-dimensional model of β-tubulin protein for potential errors. The program displayed two attributes of the input structure: its Z-score and a plot of its residue energies. The ProSA Z-score of -10.11 indicates the overall model quality of β-tubulin protein (Fig. 6A). The deviation of total energy of the structure, measured by the Z-score, with respect to an energy distribution obtained from random conformations. The scores were well within the range of scores and indicated a highly reliable structure typically found for proteins of comparable size. The energy plot demonstrated the local model superiority by plotting the knowledge-based energies as function of the amino acid sequence position (Fig. 6B). QMEAN analysis was also employed to evaluate and validate the model. The QMEAN4 score of the model was 0.82 (Fig. 6C) and the Z-score was -1.85 which was close to zero and this confirmed the superior quality of the model. This is because the estimated consistency of the model was projected to be in between 0 and 1 (Table 1). Assessment between regularized QMEAN score (0.82) and protein size in non-redundant set of PDB structure in the plot revealed different set of Z-values for different parameters such as C-beta interactions (0.28), interactions between all atoms (0.24), solvation (0.85) and torsion (-2.31) (Table 1).
The constructed homology model was also assessed for structural and stereo-chemical competency. A Ramachandran Phi/Psi plot for β-tubulin (Fig. 6D) showed 97.436 % highly preferred observations, 1.865 % preferred observations and 0.699 % questionable observations. The analysis of the predicted structure provided strong evidence that the predicted three-dimensional structure of β-tubulin was of superior quality.
3.6.3 Determination of putative nucleotide binding sites
In order to explore the spatial relationships between nucleotide and β-tubulin we used a topological approach after homology modeling. The nucleotide-binding domain in β-tubulin was depicted in Fig. 7A and B. We assumed that the GDP-binding site of β-tubulin was composed of two components: a guanine-binding component and a phosphoryl-binding component. The guanine-binding component was composed of CYS12, GLN15, SER176, GLU181, ASN204, TYR222 and ASN226 and the phosphoryl-binding component was composed of GLN11, SER138, GLY141, GLY142 and THR143. The binding pattern and the H-bond formation are according to the Fig. 7A and B and Table 2.
3.6.4 Molecular docking to identify carbendazim binding site(s)
Molecular docking was performed to map the interactions between carbendazim and β-tubulin and to find out putative binding site(s). The molecular docking of carbendazim and β-tubulin detected 48 clusters in eight different sites of the protein. The top-score cluster exhibited best “full fitness” and those parameters were calculated by averaging the 30 % most favourable effective energies of a cluster’s element and lower free energy than those obtained for other potential binding sites (Grosdidier et al., 2007; Vela-Corcía et al., 2018). The pocket atoms of β-tubulin for carbendazim binding site were VAL193, SER196, ARG251, VAL255, VAL258, PHE260, ARG262 and HSE(Half Sphere Exposure)264. Carbendazim bound with β-tubulin with different kind of interactive forces such as classical hydrogen bond, non-classical hydrogen bond, electrostatic interaction (charged) and alkyl hydrophobic interaction. There were a conventional hydrogen bond between MBC:H18 and β:PRO261:O; two non-conventional hydrogen bonds between MBC:H16 and β:GLU194:O, MBC:H17 and β:GLU194:O; seven charged-electrostatic interactions between β:ARG156:HZ2 and MBC:C1, β:ARG156:HZ2 and MBC:C3, β:GLU194:CA and MBC:C1, β:ASN195:CA and MBC:C1, β:ASP197:CA and MBC:C3, β:PRO261:CA and MBC:C1, β:PRO261:CA and MBC:C3; alkyl hydrophobic interaction between β:ALA254 and MBC (Fig. 7C and D). All the putative bonds with their bond distance were in accordance with Table 3.
3.6.5 Dimerization of α- and β-tubulins
Hetero-dimerization of α- and β-tubulin subunits was also checked by protein-protein docking. The two monomers were interacted to each other with H-bonds and electrostatic interactions. The H-bonds were formed through salt bridge formation, conventional H-bonds and non-conventional with carbon-hydrogen bond formation. Out of total eleven bonds/interactions, the monomer β-tubulin contributed six amino acids as H-donor and five amino acids as H-acceptor. The amino acid representatives of α- and β-tubulin monomers with their actual positions in the peptide chain, the bond distance and bond angles are represented in Table 4. The β-tubulin of F. solani isolates had sites for the putative beta/alpha domain interface at ASP128, CYS129, ARG162, LYS252, ASN256, ARG331, ASN347, GLN350 and GLY429 (Fig. 7E).
3.6 Comparison of β-tubulin tertiary structure
Homology models of β-tubulin of all the Fusarium species were constructed and to identify any discrimination with the F. solani protein, superimposition was performed. Though few regions in amino acid residues were dissimilar among the species, the tertiary structural superimpositions were nearly identical. This could be due to substitution of the amino acids with the same class of other amino acids. The three-dimensional model of β-tubulin of F. oxysporum f.sp. cubense, however, differed from that of F. solani SF0301 and other species (Fig. 8A and B). In β-tubulin of F. oxysporum f.sp. cubense, PRO279 (yellow) strongly refused to form an α-helix and acted as a helix-breaker (Fig. 8D); where as in all other Fusarium species including F. solani, HIS279 (yellow) formed an extra α-helix (Fig. 8C). In Fig. 8C, the schematic diagram of β-tubulin of F. solani SF0301 showed ARG213 (blue) as a component of an α-helix while in F. oxysporum f.sp. cubense (Fig. 8D) ARG213 was not integrated into the α-helix rather it formed loop with its C terminal amino acid residue.