bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438069; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A Comprehensive Phylogenetic and Bioinformatics Survey of Lectins in the Fungal kingdom Annie Lebreton 1,†, François Bonnardel 2,3,4,†, Yu-Cheng Dai 1, Anne Imberty 2*, Francis M. Martin 1,5*, Frédérique Lisacek 3,4,6* 1 Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, Bei- jing, China; 2 University of Grenoble-Alpes, CNRS, CERMAV, 38000 Grenoble, France; 3 Swiss Institute of Bioinformatics, CH-1227 Geneva, Switzerland; 4 Computer Science Department, UniGe, CH-1227 Geneva, Switzerland; 5 Université de Lorraine, INRAE, UMR Interactions Arbres/Microorganismes (IAM), Laboratoire d'excellence ARBRE, Centre INRAE GrandEst-Nancy, Champenoux, France; 6 Section of Biology, UniGe, CH-1205 Geneva, Switzerland. † These authors contributed equally * Correspondence: [email protected], [email protected], [email protected], Abstract: Fungal lectins are a large family of glycan-binding proteins, with no enzymatic activity. They play fundamental biological roles in the interactions of fungi with their environment and are found in many different species throughout the fungal kingdom. In particular, their contribution to defence against feeders has been emphasized and extracellular lectins may be involved in the recogni- tion of bacteria, fungal competitors and specific host plants. Their carbohydrate specificities and qua- ternary structures vary widely, but evidence for an evolutionary relationship within the different clas- ses of lectins is provided by the high degree of amino acid sequence identity shared by the different fungal lectins. The UniLectin3D database contains 194 3D structures of fungal lectins, of which 129 are characterized with their carbohydrate ligand. UniLectin3D lectin classes from all origins were used to construct 107 lectin motifs in 26 folding configurations and to screen 1,223 species deposited in the genomic portal MycoCosm of the Joint Genome Institute. The resulting 33 485 protein sequences of putative lectins are organized in MycoLec, a publicly available and searchable database. The charac- terization of the lectin candidates in fungal genomes is based on systematic statistics regarding poten- tial carbohydrate ligands, protein lengths, signal peptides, relative motif positions and amino acid com- positions of fungal lectins. These results shed light on the evolution of the lectin gene families. Keywords: Lectins, MycoCosm, carbohydrate recognition bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438069; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Reynolds 2018). Pathogenic yeasts, such as Introduction Candida albicans, rely on adhesins to initiate Fungi are unicellular or multicellular organisms the colonization of epithelial hosts (Willaert found in terrestrial, marine and aquatic ecosys- 2018), while filamentous pathogenic fungi, such tems. They adopt a wide range of ecological as Aspergillus fumigatus, produce soluble lec- lifestyles (e.g., modes of nutrition) along the tins involved in host recognition and able to trig- saprotrophism/mutualism/parasitism continuum ger signaling pathways leading to lung inflam- (Naranjo-Ortiz and Gabaldón 2019; Richards et mation (Houser et al. 2013; Richard et al. 2018). al. 2015; Stajich 2017). Saprotrophic, symbiotic Less is known about the role of lectins in the es- and pathogenic fungal species compete or coop- tablishment of fungal-plant or fungal-animal erate with multiple bacteria, fungal, plant or an- mutualistic symbioses. A role of fungal lectins imal species. Specific signaling and develop- in the establishment of the mutualistic ectomy- mental pathways are underlying the establish- corrhizal symbiosis between the basidiomycete ment and maintenance of the relationships with Lactarius deterrimus and its host tree, Picea other organisms. Self/non-self discrimination is abies, has been suggested (Giollant et al. 1993) at the very core of the fungal hyphae develop- but not yet confirmed by genetic disruption of ment and involves surface-surface molecular in- the corresponding genes. A galactose-specific teractions (Fischer and Glass 2019). Similarly, galectin acts as a chemoattractant for the recruit- recognition of the animal or plant surfaces play ment and adhesion of the cyanobacterial Nostoc a key role in the pathogenic or mutualistic fun- symbiont to the hyphae of Peltigera canina gal symbioses. These interactions rely on a wide leading to a lichen symbiosis (Díaz et al. 2011). range of secreted proteins, including lectins. The involvement of fungal lectin has since been Lectins are ubiquitous carbohydrate-binding established for a large number of lichen associ- proteins playing a crucial role in self/non self- ations (Singh and Walia 2014). recognition through specific interactions with The paucity of knowledge on the biochemistry complex glycans present on the surface of pro- and role of fungal lectins is reflected by the low- teins, microorganisms or tissues (Lis and Sharon quality annotations of lectins in international 1998). These small proteins are therefore in- protein and genome databases. Moreover, a lim- volved in a wide range of biological processes ited number of 3D structures of fungal lectins is such as reproduction and development, the es- currently available. A recent review reported 26 tablishment of biotrophic associations, but also different fungal lectins from ten different folds in many pathogenesis-related mechanisms, such for 100 crystal structures (Varrot, Basheer, and as host-pathogen interactions and tumor metas- Imberty 2013). Using a classification based on tasis. A large number of lectins have been iden- 3D X-ray structures and protein sequences, the tified in filamentous fungi and yeasts (Goldstein curated UniLectin3D database (www.unilec- and Winter 2007; Guillot and Konska 1997; tin.eu/unilectin3D) (Bonnardel et al. 2019) can Nordbring-Herz and Chet 1986; Pemberton now be used to generate conserved sequence 1994; Singh, Bhari, and Kaur 2011). They are motifs based on known lectin classes. Those generally considered as defence-related pro- conserved motifs can further be used to charac- teins. This role is well documented in mush- terize the lectin repertoire, so-called lectomes, room-forming fungi where lectins are known to of a given species. This lectin repertoire is in- protect these reproductive structures from hy- dexed in LectomeXplore (Bonnardel et al. phal grazers, such as nematodes, slugs, snails 2021). The objective of this study was to de- and insects, through their nematotoxic and ento- velop a novel approach for efficiently and accu- motoxic activities (Sabotič, Ohm, and Künzler rately predicting and classifying lectin proteins 2016). in Fungi. We have used UniLectin3D-based mo- Except for their role in defence, the current body tifs to predict lectins in published fungal ge- of information on the biological and ecological nomes available at the Joint Genome Institute roles of lectins in saprotrophic, mutualistic and (JGI) MycoCosm genomics portal pathogenic fungi is scarce. Some yeasts produce (http://jgi.doe.gov/fungi) (Grigoriev et al. flocculins that play a role in the formation of 2014). This database aims to provide access of multicellular structures of interest for brewers genomes representative of most fungal lifestyles and winemakers (Goossens and Willaert 2010; and supports integration, analysis and bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438069; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. dissemination of fungal genome sequences and FUNGuild (Nguyen et al. 2016) and expert eco- other ‘omics’ data by providing interactive web- logical annotation was performed for the 125 based tools (Grigoriev et al. 2014). At the time Agaricomycetes strains available at the time of of this study in 2020 this database contained ap- this study. prox. 1500 genomes (from 1223 species). From these, the resulting 35 460 identified lectin do- Identification and scoring of candidate fungal mains on 33 485 sequences of putative lectins lectins can be searched and analyzed on the MycoLec For each class, the lectin 3D domain sequences web site (https://www.unilectin.eu/mycolec/). from which histidine tags were removed, were In order to identify possible relationships be- aligned with MUSCLE (Edgar 2004). The ob- tween the lectin repertoire and the diverse eco- tained alignments were used to generate a Hid- logical niches of fungal species, we focused our den Markov Model (HMM) for each domain. survey on the Agaricomycetes (Basidiomycota HMMSEARCH (Potter et al. 2018) was then run phylum). The rationale is the availability of a to identify lectins in the proteomic dataset. Re- large set of genomes and transcriptomes for this sults were uploaded