Vacuolar proteases and autophagy in phytopathogenic fungi: A review

Autophagy (macroautophagy) is a survival and virulence mechanism of different eukaryotic pathogens. Autophagosomes sequester cytosolic material and organelles, then fuse with or enter into the vacuole or lysosome (the lytic compartment of most fungal/plant cells and many animal cells, respectively). Subsequent degradation of cargoes delivered to the vacuole via autophagy and endocytosis maintains cellular homeostasis and survival in conditions of stress, cellular differentiation, and development. PrA and PrB are vacuolar aspartyl and serine endoproteases, respectively, that participate in the autophagy of fungi and contribute to the pathogenicity of phytopathogens. Whereas the levels of vacuolar proteases are regulated by the expression of the genes encoding them (e.g., PEP4 for PrA and PRB1 for PrB), their activity is governed by endogenous inhibitors. The aim of the current contribution is to review the main characteristics, regulation, and role of vacuolar soluble endoproteases and Atg proteins in the process of autophagy and the pathogenesis of three fungal phytopathogens: Ustilago maydis, Magnaporthe oryzae, and Alternaria alternata. Aspartyl and serine proteases are known to participate in autophagy in these fungi by degrading autophagic bodies. However, the gene responsible for encoding the vacuolar serine protease of U. maydis has yet to be identified. Based on in silico analysis, this U. maydis gene is proposed to be orthologous to the Saccharomyces cerevisiae genes PRB1 and PBI2, known to encode the principal protease involved in the degradation of autophagic bodies and its inhibitor, respectively. In fungi that interact with plants, whether phytopathogenic or mycorrhizal, autophagy is a conserved cellular degradation process regulated through the TOR, PKA, and SNF1 pathways by ATG proteins and vacuolar proteases. Autophagy plays a preponderant role in the recycling of cell components as well as in the fungus-plant interaction.


Identification of conserved sequences and phylogenetic analysis of Atg8 and PrB proteins from different organisms
The amino acid sequences for the Atg8 and PrB of the following organisms were downloaded from the NCBI database: S. cerevisiae (NM_001178318) (NM_001178318), C. albicans (XP_019330653) (XP_715244), Aspergillus fumigatus (KAH3637871) and A. niger (XP_001391470.1),Magnaporthe oryzae (ACJ06588) (XP_003716216.1),Alternaria alternata (XP_018382869) (XP_018382013), Cryptococcus amylolentus (XP_018996636) and C. neoformas (OXG28165.1)and U. maydis (XP_011391873) (XP_011391098).Once the sequence of each enzyme was downloaded, it was selected and subjected to multiple alignment in the Clustal Omega program (Aiyar, 2000).The motif domains were located and visualized with the web server WebLogo (http://weblogo.berkeley.edu/logo.cgi).Based on the sequences, a phylogenetic tree was constructed on the MEGA6 program (Tamura et al., 2013) by utilizing the maximum likelihood method and the WAG + G model.Finally, 100 bootstrap replicates were used to evaluate the reliability of the phylogenetic tree.

Generation of 3D PrA, PrB, and Atg8 from S. cerevisiae and fungal phytopathogens through homology modeling
The following sequences were downloaded from the NCBI database (http: //www.ncbi.nlm.nih.gov)(Sharma et al., 2018): the PrA enzyme of Homo sapiens (NP_001900); PrA, PrB, and Atg8 of S. cerevisiae (NP_010854, NM_001178318, and NM_001178318), M. oryzae (XP_003718037, XP_003716216.1,and ACJ06588), A. alternata (XP_018380546, XP_018382013, and XP_018382869) and U. maydis (XP_011391245, XP_011391098, and XP_011391873).The percentage of identity was determined for each of the sequences of PrB and Atg8 of fungal phytopathogens, as well as of the target PrB protein of Bacillus amyloliquefaciens and Atg8 of S. cerevisiae with the Emboss Water server.The sequences were used for generating the 3D models with the homology modeling technique on the Modeller 9.23 program (Webb and Sali, 2014), with the crystallized structures of S. cerevisiae (PDB: 1FMX), B. amyloliquefaciens (PDB: 1S01), and S. cerevisiae (PDB: 6WY6), deposited in the protein data bank (http://www.rcsb.org/pdb/)(Berman et al., 2000), serving as templates.The models were overlapped in the Discovery Studio Visualizer.Among the 3D models obtained for PrA, PrB, and Atg8, the one with the minimum score was chosen and evaluated with the discrete optimized protein energy method (DOPE) (Shen and Sali, 2006).

Protein-peptide interactions
The study of protein-peptide interactions was carried out with the best 3D models of PrB proteins from S. cerevisiae, U. maydis, M. oryzae, and A. alternata previously afforded by the homology modeling technique.Likewise, the amino acid sequences of the peptides (PBI2 and Um10059) and propeptides (PrB1 and Um4400) in FASTA format were transformed into PDB with the Open Babel GUI program (O'Boyle et al., 2011).Subsequently, the PrBs and peptide-propeptides mentioned were subjected to a protein-protein interaction study with the HDOCK server (Yan et al., 2020).The results of the interactions were described and organized in tables, and the graphs of such interactions were elaborated in the Discovery Studio Visualizer.

Conservation of the Atg8, PrA, and PrB proteins from different organisms
After retrieving the protein sequences of the organisms mentioned in the previous section, the percentage of identity and similarity was determined (Supplementary Table 1).These values suggests that the Atg8, PrA, and PrB proteins of the phytopathogens herein studied are likely to be conserved.1. Percentage of identity and similarity of the sequences of the PrA, PrB, and Atg8 proteins of S. cerevisiae with the fungal phytopathogens.

3D models of PrA, PrB, and Atg8 from S. cerevisiae and the fungal phytopathogens (U. maydis, M. oryzae, and A. alternata)
Fifteen models of each of the proteins (PrA, PrB, and Atg8) from S. cerevisiae and the fungal phytopathogens were generated by homology modeling, selecting in each case the best model based on its DOPE potential.The overlap of the PrAs of different organisms is shown in Supplementary Figure 1, while the overlap of the Atg8s and PrBs is included in the main text.1. Overlap of the best models of PrA of S. cerevisiae and the three fungal phytopathogens, represented as flat ribbons.PrAHs (orange), PrASc (green), PrBUm (blue), PrBMo (fuchsia), and PrBAa (yellow).

Protein-peptide interactions
Considering the similarity between the endogenous inhibitor of S. cerevisiae PrB (PBI2) and that of the U. maydis homolog (Um10059), as well as between the two respective propeptides (PrB1 and Um4400) (Supplementary Figure 2), a protein-protein interaction study was carried out between the PrBs analyzed presently and the PBI2 inhibitor of S. cerevisiae, the Um10059 of U. maydis, and the PrB1 and Um4400 propeptides.

3 .
Intermolecular interactions of the inhibitor PBI2 (purple) with PrBSc (A), PrBUm (B), PrBMo (C), and PrBAa (D).Dotted lines indicate the type of interactions.For the purpose of clarity, only the interactions between the amino acid residues of the PrB catalytic triad (Asp, His, and Ser) and PBI2 are depicted.