An outbreak of Fusarium oxysporum f. sp. cubense Tropical Race 4 is currently threatening the global production of bananas. Due to the clonal nature of commercial banana plants, selecting resistant cultivars does not seem feasible; therefore, alternative approaches to crop protection must be developed. The 5´ → 3´ exoribonuclease XRN2/RAT1 is involved in 5´ → 3´ RNA decay. Fungal studies with XRN2 and conditional mutants have illustrated the crucial role of this enzyme, suggesting XRN2 should be considered as target to searching for novel inhibitors that might be used as fungicides to control Panama disease. Our in silico analysis of Tropical Race 4 XRN2 (FocTR4XRN2) revealed characteristic features of 5´ → 3´ exoribonuclease such as the catalytic domain that recognizes 5´-monophosphorylated RNA and catalyses the processed cleavage of mononucleotides. A delimited cavity showing the potential for substrate uptake appears prone to interacting with small molecules that might inhibit its activity. The catalytic domain in FocTR4XRN2 harbors a CCHC motif, which is conserved in orthologous proteins from filamentous fungi but lacking in yeasts. The residues involved in the interaction with the pyrophosphohydrolase RAI1 are also conserved. Molecular docking reveals the potential interaction of FocTR4XRN2 with the natural inhibitor adenosine 3´, 5´ bisphosphate, and suggests this approach is reliable to screen for novel enzyme inhibitors that could be help in suppressing the progression of causal agents of Panama disease.
Keywords: Fusarium oxysporum f. sp; Cubense Tropical race 4; 5Â´â3Â´ exoribonuclease; RNA processing; Enzyme inhibitor; Molecular docking
The Fusarium oxysporum species complex (FOSC) comprises soil-borne saprophytic fungi and plant pathogenic isolates that cause vascular wilt and root rot in plants, including some agronomically important species . The members of the FOSC complex are asexual, but like other pathogenic fungi, they are prone to acquiring DNA via horizontal gene transfer which acts as an alternative to provide the genetic information necessary for plant colonization . Although the FOSC complex causes disease in dozens of plant species, the host range of each virulent strain is limited to one or a few related species. The term forma specialis (f. sp.) is attributed to an individual strain to denote the host specificity . Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of the fusarium wilt in banana plants (Musa spp.), also known as Panama disease . Foc is classified into four races based on the banana cultivars affected. Foc Race 1 is a pathogen of Gros Michel bananas and related cultivars such as Pome, Silk, and Pisang Awak. It caused Gros Michel epidemics during the 19th Century that destroyed banana production, but it is avirulent in the resistant Cavendish cultivar on which current banana production is based. Foc Races 2 and 3 affect the banana cultivar Bluggoe and plants in the genus Heliconia, respectively. Foc Race 4 can cause disease in both Foc Race 1hosts and the Cavendish cultivar. Depending on the climate conditions under which the disease develops, Foc Race 4 is further classified into Subtropical Race 4 (FocSTR4) or Tropical Race 4 (FocTR4) when it occurs in the subtropics or tropics, respectively . Because they are parthenocarpic and propagated by vegetative methods, commercial banana cultivars have reduced genetic variability, which impedes selection for resistant cultivars and favors the establishment of novel enhanced virulent races such as FocTR4. Due to the wide host range of FocTR4 and the lack of resistant commercial cultivars, wilting caused by this race is considered the most deleterious factor affecting banana production worldwide, compromising the welfare of producers in developing countries . Thus far, there are no effective strategies to control or abolish FocTR4. Therefore, research focused on this pathogen is required to give rise to novel alternatives for protecting this important crop. Resistance genes have been identified in wild Musa plants, and although their expression in transgenic Cavendish banana plants conferred resistance to FocTR4, implementation is technically limited because there are roughly 5000 accessions of Musa spp. . The molecular docking approach is a tool used for investigating protein-ligand interactions that has been helpful for discovering and screening for new drugs and antibiotics [7,8]. Virtual screening of novel inhibitors is an early step in designing novel compounds with antifungal activity that could be used to protect banana plants, regardless of the cultivar or environmental growth conditions. This approach was used to identify compounds that could function as G-protein coupled receptor (GPCR) inhibitors in Fusarium graminearum, the causal agent of head blight in wheat . Enzymes involved in RNA degradation, an essential molecular process in all organisms, including fungi, are possible targets to control Panama disease and other F. oxysporum-caused diseases . Endo- and exo-nucleases are required for eliminating unnecessary mRNAs as a part of general turnover, specific transcript regulation, and aberrant mRNA degradation to control RNA quality. Nucleolytic activity also participates in the processing, maturation, and decay of functional noncoding RNAs [10,11]. Using divalent cations as cofactors, 5´ → 3´ exoribonucleases catalyze the removal of nucleoside monophosphates from 5´-monophosporylated RNA . These enzymes were initially identified in the yeast Saccharomyces cerevisiae, which has two 5´ → 3´ exoribonucleases referred to as XRN1  and XRN2, originally known as Rat1 (ribonucleic acid trafficking protein 1) . XRN1 and the orthologous proteins in eukaryotes generally participate in the 5´ → 3´ decay of mRNAs in the cytoplasm . XRN2 localizes to the nucleus, and is involved in noncoding RNA processing such as the exoribonucleolytic trimming of rRNA precursors , snoRNAs biogenesis  and degradation of hypomodified tRNAs that lack complete functionality to protein synthesis . These activities suggest XRN2 influences protein biosynthesis by processing the noncoding RNAs involved in translation. In yeast, Rat1 is involved in the degradation of the 3´-end products of transcription termination, and it might collide with RNA polymerase II to induce its dissociation from DNA . The telomeric repeat-containing RNA (TERRA) is produced from telomere sequences located at the end of the chromosomes . The accumulation of TERRA inhibits telomere elongation in S. cerevisiae, possibly by repressing telomerase activity. Telomere elongation, and thus, chromosome stability is maintained by the Rat1- mediated degradation of TERRA . The lethality of the XRN2 (rat1, hke1, tap1) mutant in the fission yeast Schizosaccharomyces pombe suggests that the reactions catalysed by this enzyme are essentialfor fungal development. Expressing the XRN2 gene (driven by the inducible GAL7 promoter) in the human pathogen Cryptococcus neoformans suggests this exoribonuclease is essential for cell viability,as growth is established upon XRN2 induction and abolished under restrictive conditions . Thus far, there have been no reports concerning the role of this enzyme in phytopathogens. Although this is an unexplored topic, the evidence suggests that understanding XRN2’s structure and its mode of action in phytopathogens could be helpful in proposing approaches to suppress invasions by economically important phytopathogens such as the causal agents of Panama disease. The availability of fungal genome sequences, including FocTR4, and the crystal structures of 5´ → 3´ exoribonucleases facilitates this endeavor. In this study, we performed an in silico analysis of FocTR4XRN2 as an initial step to elucidate its structural features, which could lead to the screening and identification of novel inhibitors.
Protein sequences and phylogenetic analysis
The sequences of the 5´ → 3´ exoribonucleases XRN1 and XRN2 from representative fungal species were analyzed. The protein sequences were retrieved from the Universal Protein Resource (UniProt) database  by using the XRN1(P40383) and XRN2/ Rat1 (P40480) sequences from S. pombe as queries. Human XRN1 (Q8IZH2) and XRN2 (Q9H0D6) were used as controls. The protein annotation in UniProt automatically predicts the catalytic domain from the InterPro 5´ → 3´ exoribonuclease IPR027073. The Fusarium oxysporum f. sp. cubense TR4 strain 54006 sequence was subjected tofurther analysis as presented below. The software cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) was used to identify putative nuclear localization signals (NLS). The list of species and ID entry names for each protein are included in the Supplementary Table 1. Protein sequences were aligned using the MUSCLE algorithm . PhyML was used to estimate a maximum likelihood phylogenetic tree with 100 bootstrap replicates . The MUSCLE algorithm was also used to align S. pombe Sp Rai1 (O13836) and the homolog FOC4TR4RAI1 (X0K375).
Modelling of FocTR4XRN2
Once FocTR4XRN2 was identified (X0JIN1), the Robetta server (http://robetta.bakerlab.org/) was utilized to predict the tridimensional structure based on the full sequence surrounding the catalytic domain (IPR027073) with homology to 5´ → 3´ exoribonucleases . The previously reported structure SpRat1 (3FQD) was selected as the template to generate the FocTR4XRN2 models (pdb files), which were visualized in PyMOL (Schrödinger). The models were structurally similar, and one was selected for further analysis and detailed comparisons with SpRat1. The structure of the FocTR4XRN2 fragment with a potential for interacting with FocTR4RAI1 was generated in Swiss-Model by using the corresponding SpRat1 fragment as template . The sequence and structure of the activating partner SpRai1has also been previously reported .
The PatchDock algorithm was used to test adenosine 3´, 5´ bisphosphate (PAP) as a ligand of the FocTR4XRN2 protein predicted by the Robetta server . The PAP structure was retrieved from the ZINC database (http://zinc.docking.org/), and the 2D structure was drawn using ChemDrawR 17 (PerkinElmer). An energy minimization of the structures was performed with VEGA ZZ suite (http://nova. disfarm.unimi.it) prior to the molecular docking. The two solutions with the highest geometric score were selected for further analysis and visualization in UCSF Chimera .
Identification of XRN1 and XRN2
Differences in the XRN1 and XRN2 sequences and structures must be distinguishable, as each enzyme catalyzes the removal of mononucleotides from specific RNA substrates. The 5´ → 3´ exoribonuclease sequences from 26 representative fungal species were aligned, and a phylogenetic tree was constructed by maximum likelihood (Figure 1). XRN1 and XRN2 were clustered into two independent clades that were supported by the bootstrap value. While the analyzed protein sequences harbor the characteristic 5´ → 3´ exoribonuclease domain, the context surrounding the catalytic domain could influence substrate recognition and be the determining factor responsible for XRN2’s crucial role in fungal development.
Figure 1: The 5′→3′ exoribonucleases in fungi. A maximum likelihood phylogenetic tree shows the homologs of XRN1 and XRN2/RAT1. The UniProt entry ID and name of the selected species are shown at the right of the branches. Bootstrap support values > 50 are listed on the branches. A complete list of the species is included in the Supplemental Material.
Structural features of the predicted FocTR4XRN2
A more detailed analysis of the FocTR4XRN2 identified a CCHC motif and the potential NLS in the catalytic domain, which was absent in the FocTR4XRN1 (Figure 2). These additional motifs are lacking in the 5´ → 3´ exoribonucleases form yeasts [28,31]. A glycine-rich in the C-terminal portion of FocTR4XRN2 is evident, but thus far, the function of this region in 5´ → 3´ exoribonucleases is unknown. A MUSCLE alignment was performed to determine whether the catalytic domain is functional. Selected XRN2 sequences were aligned, including the already reported SpRat1 (Figure S1, supplementary material). The N-terminal of SpRat1 includes residues involved in the cleavage of mononucleotides and acidic residues involved in binding of cations such as Mn2+ or Mg2+, which is necessary for catalysis . All those residues are conserved. The alignment also reveals that the additional CCHC knuckle motif (C-X2-CX3GH-X4-C) that is unique to XRN2 from filamentous fungi, and present in plant 5´ → 3´ exoribonucleases . In S. pombe, the pyrophosphohydrolase Rai1 removes pyrophosphate form the first triphosphorylated nucleotide of a single stranded RNA . SpRai1interacts with SpRat1, and most of the residues of FocTR4XRN2 involved in the potential interaction are conserved (Figure S1, supplementary material), suggesting this interaction might also occur in FocTR4. Despite the slight differences and the presence of the CCHC motif, the highly conserved catalytic domain in 5´ → 3´ exoribonuclease homologs, including FocTR4XRN2, implies the formation of a tridimensional structure capable of removing mononucleotides from 5´-phosphorylated RNA, using divalent cations as cofactors. The structure of FocTR4XRN2 as predicted by the Robetta server is illustrated in Figure 3A. The overall structure highly resembles that of SpRat1 (Figure 3B). The 5´ → 3´ exoribonucleases possess 17 α-helices. The fourth α-helix, known as the tower domain, contains the basic residues critical for catalysis and are hypothetically oriented towards the pocket where the 5´-phosphate of the substrate is located, which agrees with the structure of the 5´ → 3´ exoribonuclease XRN1/ PCM in the fruit fly  (Figure 3C). As expected, the residues involved in metal binding are located near the tower domain (Figure 3C). The CCHC motif is a putative α-helix located at the surface of the predicted protein and does not seem to be directly involved in catalysis, but its conservation in XRN2 homologs suggests it might confer a specific biochemical function such as interacting with proteins involved in RNA processing (Figure S2, supplementary material).
Figure 2: Structural comparisons of the 5′→3′ exoribonucleases in + and Schizosaccharomyces pombe showing the catalytic domain (green), CCHC motif (yellow), glycine-rich region (purple), and putative nuclear localization signal (NLS; blue). The putative NLS in S. pombe was reported in .
Figure 3: Predicted 3D model and ribbon diagram of FocTR4XRN2. (A) The surface is presented in transparent green, the tower domain involved in catalysis is indicated in red, the CCHC motif is in orange, and the putative nuclear localization signal is in blue. (B) Merging of the predicted FocTR4XRN2 and SpRat1 structures in green and cyan, respectively. (C) Lateral residue chains involved in catalysis in the predicted FocTR4XRN2 structure are highlighted. The tower domain is in orange, and the acidic residues for metal binding are in black. Hydrogen atoms were omitted to simplify the diagram; the oxygen and nitrogen atoms of the residues are shown in red and blue, respectively.
RAI1 as a potential interactor of FocTR4XRN2
The sequence alignment revealed conserved residues involved in the interaction with RAI1. To obtain some insight into the relevance of these residues, the SpRai1 and FocTR4RAI1 sequences were aligned. As fission yeast, the conservation of residues involved in interacting with Rat1/XRN2 suggests that both enzymes co-evolved to associate in FocTR4 (Figure 4A). The Figure 4B shows the structure of the tract of residues H819 to G826 located at the FocTR4XRN2 C-terminal that hypothetically interact with RAI1 is represented in the Figure 4B.
Figure 4: Residues on the surface of FocTR4XRN2 involved in the interaction with RAI1 are conserved. MUSCLE alignment of SpRai1 and the homolog FocTR4RAI1, black spots mark residues involved interacting with SpRat1, which are conserved in FocTR4RAI1 (A). Predicted model of the surface of FocTR4XRN2 interacting with SpRAI1 (B).
Virtual interaction between FocTR4XRN2 and adenosine 3´, 5´ bisphosphate
Visualizing the cavities that converge in the FocTR4XRN2 catalytic site reveals potential sites for inhibitors (Figure 5A). The in vitro relative activities of the S. cerevisiae 5´ → 3´ exoribonucleases XRN1p and Rat1p decreases by addition of adenosine 3’, 5’ bisphosphate (PAP), a bypass product of sulfate assimilation (Figure 5A) . As a proof of concept, molecular docking was performed with PatchDock to assess PAP as potential inhibitor of the 5´ → 3´ exoribonucleases. The two solutions produced by this virtual procedure with the highest geometric scores (4766 and 4702) demonstrated that PAP can be placed at the catalytic site as a competitive inhibitor, close to the tower helix and neighboring residues (Figure 5B).
Figure 5: Docking of adenosine 3′, 5′ bisphosphate (PAP) into the catalytic site of FocTR4XRN2. (A) The cavity in the FocTR4XRN2 structure in the proximity of the catalytic site is represented by dotted black mesh; the tower domain is in red, and the CCHC motif is in orange; PAP is illustrated at the right. (B) The two orientations of PAP that displayed the highest scores are represented in orange and yellow. The residues in FocTR4XRN2 involved in docking are represented by colored lines (grey, carbon; white, hydrogen; red, oxygen; blue, nitrogen); the tower domain is indicated by an arrow.
Currently, the most outstanding factor that hampers the world production of bananas is the emergence of FocTR4. Both natural and synthetic products have been tested as FocTR4 inhibitors. Treatment with demethylation-inhibiting fungicides decreases symptom development in banana plants inoculated with FocSTR4, however, these compounds can be toxic to mammals . Bacterial volatiles have been demonstrated to act as suppressors of spore germination and mycelial growth in FocTR4 [35,36]. Crude extracts obtained from Streptomyces violaceusniger are also inhibitors of FocTR4 spore germination of . Synthetic Cu(II) complexes with benzophenone derivatives have also shown antifungal activity against FocTR4 . Although these results are promising, more compounds need to be screened to increase the repertoire of innocuous-to-humans and environmentally friendly fungicides. Molecular docking is an emergent approach to screening for novel antibiotics with high affinity, including compounds to control the agents of Panama disease. Research aimed at determining which enzymes are indispensable for fungal growth and viability is necessary to accomplish this task. In this study, the 5´ → 3´ exoribonuclease XRN2 was selected to perform an in silico structural analysis. In fungi, nuclear-localized XRN2 is the predominant 5´ →3´ exoribonuclease, performing the RNA trimming required for the maturation of rRNAs and snoRNAs . Thus, its activity is an early event that secures protein synthesis. The degradation of TERRA by Rat1p in S. cerevisiae is indicative of a requirement for 5´ → 3´ exoribonuclease activity for maintaining chromosome integrity . Furthermore, the failed attempts to generate XRN2 knockout mutants in filamentous fungi suggest that the disruption of this gene is lethal [22,40]. These reports strengthen the proposition that XRN2 might have unique features that confer its prominent role in RNA processing. The differences in the fungal XRN1 and XRN2 primary structures were notable and allowed the phylogenetic reconstruction of 5´ → 3´ exoribonucleases into two clades. Thus, detailed about the structure of XRN2 can be utilized to design novel fungicides. Furthermore, the catalytic residues of XRN2 are under selective pressure that would limit the probability to select a functional enzyme resistant to competitive inhibitors. As a pre-requisite to accomplishing this task, we predicted the in vitro structure of FocTR4XRN2. The protein has a quite similar structure to that previously reported for SpRat1p, including the tower domain that harbors the residues required for catalysis and the acidic residues involved in binding divalent cations. When XRN1p was targeted to the nucleus in S. cerevisiae, it fulfilled the functions of Rat1p . This indicates that the nuclear localization of XRN2 is critical for it to play its role. The identification of a putative NLS in FocTR4XRN2 is in silico evidence that it localizes to the nucleus to rapidly and efficientlyrecognize substrates such pre-rRNAs. The CCHC motif is specific to XRN2 in filamentous fungi, including FocTR4, and according to the predicted tridimensional model, it is an α-helix exposed at the surface of the protein. Thus far, the function of the CCHC motif is unknown, but in S. cerevisiae, the CCHC-containing protein Mpe1is a subunit of the cleavage and polyadenylation factor (CPF) involved in terminating transcription . Specific subunits of the holo-CPF immunoprecipitates with snoRNA genes , which leads us to suggest that the CCHC motif might be involved in binding of the RNA substrate or interaction with other proteins that mediate its activity or recruitment into nuclear complexes. It is feasible to search for small molecules that could interact with this exposed motif, and thus interrupt protein-protein interactions and cause deleterious effects in the fungus. Since the catalytic site of FocTR4XRN2 was resolved in silico, it might be possible to test the docking of small molecules.
Knocking out the hal2 gene in yeast caused the accumulation of its substrate PAP, consequently inhibiting XRN1p and Rat1p activity and provoking the accumulation of pre-rRNAs and pre-snoRNAs. There is no evidence that 5´ → 3´ exoribonucleases recognize PAP, but as it is a nucleotide, it is plausible that PAP’s inhibitory effect might be due to its insertion into the XRN1p and Rat1p catalytic sites, a process that might be conserved in fungi. The virtual result of the molecular docking suggests that the catalytic sites of 5´ → 3´ exoribonucleases, especially in XRN2, are prone to being docked by small molecules that disrupt their function, which could provide a way to screen for novel natural products that could be utilized as fungicides.
LDMB is supported by the Cátedras CONACYT Program, Grant No. 538. MACO is supported by a PhD Scholarship grant from PRODEP-DSA-DGESU (Beca para estudios de posgrado de alta calidad 2017).