sign in sign up
<<
Home , Agaricomycetes, Coprinopsis, Coprinopsis cinerea, Dacrymycetes, Hydrophobin, Schizophyllum

Downloaded by guest on October 2, 2021 www.pnas.org/cgi/doi/10.1073/pnas.1817822116 Krizs Krisztina fungi in multicellularity complex behind genes conserved reveals development of atlas Transcriptomic eie Johnson Jenifer Kiss Brigitta eoeIsiue SDprmn fEeg,Wlu re,C 94598; CA Creek, Walnut Energy, of Department US Institute, Genome a Grigoriev V. Igor n utial odsuc,wt aoal eiia properties medicinal favorable with important source, an food represent sustainable they and medicine; and in importance Fruiting ecology, immense agriculture, tetrapods. have fungi of mushroom-forming origin of bodies the with coinciding approximately com- of () fungi majority prise the mushroom-forming vast and The the () . contain () the of which lin- Agaricomycetes context independent of eight the least (2), at in in eages found mostly are which discussed kingdom, bodies, fruiting fungal is the multicellularity Within (1–4). florideophytes, complex algae brown embryophytes, laminarean animals, and multicellular complex F development body fruiting multicellularity complex organisms. eukaryotic complex to of point clades largest entry the in of an multicellularity one indepen- complex provides and study development among mushroom This studying multicellularity genetic lineages. complex to evolved plants solutions dently by multicellular convergent imposed reflecting in hurdles too, expanded animals convergently and/or which Agaricomycetes, of in many expansions showed kinases, factors, transcription , families, and expansin-like these proteins, of proteins F-box Several secreted including genes). small orphan and effector-like adhesion, (including degrada- protein transduction, targeted functions signal remodeling, covering tion, wall species, cell six fungal to to five related from genes regulated in tally development and body families gene conserved fruiting 300 Nearly (Agaricomycetes). and fungi mushroom-forming multicellularity complex of on comparisons based and of species formation six mushroom of data of transcriptome developmental atlas We complex reference conditions. to a laboratory simple under constructed from inducible transition is from that a bodies incom- multicellularity represents fruiting evo- thallus are fungal hyphal of the the multicellularity a development toward and The complex known. step origins of pletely evolutionary key the underpinnings a organisms, brown genetic handful being complex and a Despite of red in lution fungi. embryophytes, only and evolved animals, algae, has mul- including simple it lineages, the to cells, contrast of of In of one life. aggregates of been 2018) history 18, ticellular has October the review in multicellularity for transitions (received complex 2019 major 25, of February approved evolution and Canada, The NS, Halifax, University, Dalhousie Doolittle, Ford W. by Edited Netherlands; The and Utrecht, 3584 University, Utrecht Microbiology, France; Marseille, 13288 Biologiques, France; Hungary; Marseille, 6726, 13288 Szeged Aix-Marseille, Science, of Academy Hungarian Centre, B , Technical and Biotechnology ytei n ytm ilg nt nttt fBohmsr,Booia eerhCnr,HnainAaeyo cecs zgd62,Hungary; 6726, Szeged Sciences, of Academy Hungarian Centre, Research Biological Biochemistry, of Institute Unit, Biology Systems and Synthetic 0 hl eoe,t lcdt h oegntcprogram genetic core the elucidate to genomes, whole >200 nirpeetadvrelnaeo ope multicellular with complex compared of history evolutionary lineage unique a diverse with organisms a represent ungi k ilg eatet lr nvriy ocse,M 01610 MA Worcester, University, Clark Department, Biology 100seisadoiiae 5 ilo er g (5), ago years million 350 originated and species >21,000 a Bal , an ´ b b,j 0fntoa ruscnanddevelopmen- contained groups functional >70 naLipzen Anna , a z B azs ´ , ai .Hibbett S. David , v Alm Eva ´ | evolution alint ´ a,c asi ´ | ruaK Ursula , h fungi a eateto ilgclSine,Kn buai nvriy edh259 ad Arabia; Saudi 21589, Jeddah University, Abdulaziz King Sciences, Biological of Department g b sl Mer Zsolt , ntttNtoa el ehrh gooiu,UC10,Acietr tFnto e Macromol des Fonction et Architecture 1408, USC Agronomique, Recherche la de National Institut oi .Ohm A. Robin , se-nttt,Uiest fG of University usgen-Institute, ¨ | oprtv genomics comparative k n L and , ues ¨ enyi ´ d ereBarry Kerrie , aszl ´ a i eaSahu Neha , Istv , .Nagy G. o ´ f rhtcuee ocindsMacromol des Fonction et Architecture j eateto ln n irba ilg,Uiest fClfri,Bree,C 94720; CA Berkeley, California, of University Biology, Microbial and Plant of Department nNagy an ´ | c eoisLd,M Ltd., Seqomics tign G ottingen, b ¨ a,1 ui Cseklye Judit , a M , c amnPangilinan Jasmyn , ulse nieMrh2,2019. 22, March online Published 1073/pnas.1817822116/-/DCSupplemental. y at online information supporting contains article This at Archive GEO 1 transcrip- NCBI’s sequenced the the in no. of (accession deposited archive was (GEO) libraries Omnibus tome no. Expression (accession database Gene BioProject A Information PRJNA334780). Biotechnology for Center of National annotation the in and assembly Genome deposition: Data the under Published Submission.y Direct PNAS a is article This interest.y of conflict no declare authors The ...... n ...woetepaper. the wrote L.G.N. and D.S.H., I.V.G., K.B., Heged U.K., B. K.K., M.V., U.K., I.N., K.K., tools; B.B., R.A.O., reagents/analytic A.L., B.K., new J.J., S.M., T.K., Henrissat, contributed M.V., B. I.V.G. J.C., and K.B., Y.X., K.K., research; J.Y., research; performed J.P., I.N. designed and L.G.N. J.C., and B.B., M.V., D.S.H., K.K., contributions: Author im o yh-ohpaahso,cmuiain(.. via mecha- (e.g., group communication deploy this adhesion, fungi in -to-hypha development, multicellularity for body underpin- complex nisms fruiting genetic of the During 7–10), origins on refs. (2). the information e.g., of of (see, paucity nings studies a few in the surprisingly resulting of that to subject ancestor formation been common body recent fruiting most (2). of and the , origin Agaricomycetes, to single dates a probably Mushroom-forming share (6). fungi immunomodulatory) antitumor, (e.g., owo orsodnesol eadesd mi:[email protected] Email: addressed. be should correspondence whom To at ´ n,adrva infiatcnegnewt te complex other with convergence lineages. repro- significant multicellular reveal transcriptional and of splic- ing, alternative and role networks, coexpression the the gene gramming, including outline processes and , data developmental of bodies and These multicellularity-related origins. fun- fruiting major evolutionary conserved 200 multicellular a ancient to with in their families and dynamics gene genes species the expression find identified across fruit- genomes to of Comparisons gal species development bodies. the six during ing expression in of dynamic expression used origins a with We gene evolutionary multicellular known. of and hardly complex are readouts bases development diverse genetic multicellular most their the the yet of clades, one (Agaricomycetes) fungi represent Mushroom-forming life. in of innovation history evolutionary the major a is multicellularity Complex Significance riigbd eeomn nmsro-omn ug has fungi mushroom-forming in development body Fruiting Vir e tign Germany; ottingen, ´ ¨ rhlm68,Hungary; 6782, orahalom ´ agh ´ GSE125200 c oodHeged Botond , a Tam , NSlicense.y PNAS PNAS ). y sK as ´ clsBooius M 27 NS Universit CNRS, 7257, UMR Biologiques, ecules ´ e b nttt fBohsc,Booia Research Biological , of Institute uigYan Juying , | osz pi ,2019 9, April ´ o ´ us ¨ a d s .Hnist n ...aaye aa and data; analyzed L.G.N. and Henrissat, B. us, tpe Mondo Stephen , ¨ iiino oeua Wood Molecular of Division a,e enr Henrissat Bernard , b | iXiong Yi , o.116 vol. www.pnas.org/lookup/suppl/doi:10. iknlamellea i eateto Biology, of Department www.ncbi.nlm.nih.gov/geo | y b o 15 no. , b ecules ´ , .. .. N.S., Z.M., E.A., .. .. N.S., Z.M., E.A., ´ ´ a deposited was | f,g,h 7409–7418 b , Joint e ´

EVOLUTION cell−cell channels; ref. 11), cell differentiation, and defense, mental events of fruiting bodies except senescence. We defined and execute a developmental program that results in a geneti- two groups of developmentally regulated genes: those that show cally determined shape and size (2, 10). Fruiting bodies shelter greater than fourfold change and a fragment per kilobase per and protect reproductive cells and facilitate dispersal. million mapped reads of >4 between any two stages of fruit- Uniquely, complex multicellularity in fungi comprises short-lived ing body development (referred to as “FB development genes”) reproductive organs, whereas, in animals and plants, it com- and that show greater than fourfold increase in expression from prises the reproducing individual. Nevertheless, fruiting bodies vegetative mycelium to the first primordium stage (referred evolved complexity levels comparable to that of simple ani- to as “FB-init genes”). These definitions exclude genes that mals, with up to 30 morphologically distinguishable cell types show highest expression in vegetative mycelium and little or no described so far (10). Fruiting body development is triggered dynamics later on. Using this strategy, we could recover >80% by changing environmental variables (e.g., nutrient availabil- of previously reported developmental genes of ity), and involves a transition from vegetative mycelium to a (Dataset S2). To more broadly infer functionalities enriched in complex multicellular fruiting body initial. While the vegetative mushroom-forming fungi, we analyzed Interpro domain counts mycelium is composed of loosely arranged hyphae and shows lit- across 201 fungal genomes (including 104 Agaricomycetes), tle differentiation [hence, better regarded as a grade of simple which revealed 631 significantly overrepresented domains in multicellularity (1, 2)], the emergence of a fruiting body ini- mushroom-forming fungi (P < 0.01, Fisher exact test Benjamini tial involves a reprogramming of hyphal branching patterns to Hochberg adjusted P values, abbreviated as FET; Datasets S3 form a compact, three-dimensional structure in which hyphae and S4). adhere tightly to each other. The initial follows genetically encoded programs to develop species-specific morphologies (9, Dynamic Reprogramming of the Fungal Transcriptome. We detected 10), which, in the Agaricomycetes, ranges from simple crust-like 12,003 to 17,822 expressed genes, of which 938 to 7,605 were forms (e.g., ) to the most complex toadstools (e.g., developmentally regulated in the six species (Fig. 1A and Dataset bisporus). Previous studies identified several devel- S5). We found 192 to 7,584 genes that showed significant expres- opmental genes, including hydrophobins (12), defense-related sion dynamics during fruiting body development (FB devel- proteins (13), fungal cell wall (FCW) modifying enzymes (14– opment genes). Of developmentally regulated genes, 188 to 17), transcriptional regulators (8, 9, 18) (e.g., mating genes), 1,856 genes were upregulated at fruiting body initiation (FB- and light receptors (19) (e.g., white collar complex). Since all init genes), which represents a transition from simple to complex these studies focused on a single species, they provide little multicellular organization. Only P. chrysosporium had more FB- information on what genes comprise the conserved and species- init genes than FB development genes, which is consistent with specific toolkits of multicellularity and development in the its fruiting bodies being among the least complex types in the Agaricomycetes. Agaricomycetes. The number of genes significantly differentially Here we investigate the general evolutionary and func- expressed (DEGs) at fruiting body initiation further suggests that tional properties of fruiting body development using compara- the transition to complex multicellularity is associated with a tive transcriptomes of fruiting bodies of complex multicellular major reprogramming of gene expression (SI Appendix, Fig. S2). Agaricomycetes (mushroom-forming fungi). We sampled RNA The largest numbers of DEGs were observed in cap and gill from different developmental stages of six species that share tissues in all four species with complex fruiting bodies. On the a complex multicellular ancestor and represent the levels of other hand, the expression profiles of stipes changed little rela- fruiting body complexity found in the Agaricomycetes. We com- tive to primordium stages in , , and Rickenella, bine comparative analyses of developmental transcriptomes with which is explained by the completion of primordial early comparisons of 201 whole genomes and focus on conserved during development in these species [epinodular development developmental functions and complex multicellularity in fruiting (22)], as opposed to Coprinopsis, in which stipe and cap initials bodies. develop simultaneously inside the fruiting body initial [endon- odular development (22)]. Many Gene Ontology (GO) terms Results were partitioned between vegetative mycelium and fruiting body We obtained fruiting bodies in the laboratory for Coprinop- samples (P < 0.05, FET). Terms related to FCW, oxidoreduc- sis cinerea AmutBmut, commune H4-8, Phane- tase activity, and carbohydrate metabolism were enriched in rochaete chrysosporium RP78, and Lentinus tigrinus RLG9953-sp FB-init and FB development genes of all six species (SI Appendix, and, from the field, for Rickenella mellea SZMC22713 and pro- Fig. S3 and Dataset S6), suggesting that cell wall remodeling filed gene expression in three to nine developmental stages is a common function during fruiting body development. Other and tissue types (Fig. 1B). For C18/9, we commonly enriched terms cover functions such as DNA replica- used RNA-Seq data of five developmental stages and three tis- tion, transmembrane sugar transport, and ribosome, membrane, sue types from our previous work (20). Besides, we report the and lipid biosynthesis, while many others were specific to single de novo draft genome of R. mellea (). The species (SI Appendix, Fig. S3). phylogenetically most distant species in our dataset are Rick- To obtain a higher-resolution picture of developmental events, enella and Coprinopsis, spanning >200 million years of evolution we arranged developmentally regulated genes into coexpres- (5) and having a complex multicellular last common ancestor. sion modules using the Short Time-Series Expression Miner Species with the most complex fruiting bodies are A. ostoyae, (STEM) (23). Developmentally regulated genes grouped into C. cinerea, L. tigrinus (21), and R. mellea form fruiting bodies 28 to 40 modules, except Phanerochaete, which had 11. The with cap, stipe, and gills, whereas P. chrysosporium and S. com- largest modules in all species contained genes expressed at mune produce plesiomorphically and secondarily simple fruiting fruiting body initiation or in early primordia and genes with bodies, respectively. To construct a reference atlas of mushroom tissue-specific expression peaks, in young fruiting body caps, development, we performed poly(A)+ RNA-Seq on Illumina gills, stipes, mature fruiting bodies, and stipes or caps (Fig. 1C; platforms, in triplicates (totaling to >120 libraries; Dataset S1). only results for C. cinerea are shown; for the other species, see We obtained an average of 60.8 million reads per sample, of SI Appendix, Figs. S4–S9 and Supplementary Text). Many early- which, on average, 83.3% mapped to the genomes (SI Appendix, expressed modules show upregulation across multiple stages Fig. S1). For each species, the first and last developmental stages (hyphal knot, stage 1 and 2 primordia), suggestive of an early sampled were vegetative mycelium and mature fruiting body at expression program overarching multiple primordium stages. the time of spore release, respectively. This spans all develop- Coexpression modules display distinct functional enrichment

7410 | www.pnas.org/cgi/doi/10.1073/pnas.1817822116 Krizsan´ et al. Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 samcaimsae ihpat n osbyrflcscon- reflects possibly and Krizs expansion plants cell with by shared Growth mechanism (9). sig- numbers a without cell expansion is in cell by change and followed nificant nuclear events of early- wave division for early cellular an characteristic with were consistent 1C mitosis modules, expressed and Fig. replication in DNA five ple, shown least at as in regulated signatures, developmentally families gene of representation also UpSetR (see (D) module heatmap. the each fruiting of for side young given right YFB, GO) the also mycelium; enriched (see on vegetative no species given VM, means is stipe; genes term S, developmental (no primordium; key with terms 2 modules GO (Phane- 27 stage enriched right only P2, and the depict primordium; profiles on 1 expression shown stage simplified are P1, with bodies knot; Fruiting hyphal upregulated). HN, (C significantly gills; body. denotes producing (“up” spore species G, mushroom-forming body; six in stipe K.K.; and rochaete, cap of (DEGs) genes 1. Fig. A ne al. et an ´ B VM h itiuino eeomnal euae ee n infiatydfeetal expressed differentially significantly and genes regulated developmentally of distribution The (A) transcriptomes. developmental the of Overview nlsso oxrsinmdlsin modules coexpression of Analysis ) P2 B Rickenella, i.S10). Fig. Appendix, SI

chrysosporium Phanerochaete mellea Rickenella tigrinus Lentinus cinerea Coprinopsis commune Schizophyllum ostoyae Armillaria

G C HN S YFB ceai ftedvlpetltm eisdt with data series time developmental the of Schematic (B) L.G.N.). others, Dima; alint c ´ c 0gns(ee to (refer genes >50 FB-genes Expressed genes and 3000 60009000 12000 15000 18000 P1 C o exam- For S7. Dataset eta f745dvlpetlyepesdgnsi ragdbsdo oueassignment, module on based arranged is genes expressed developmentally 7,475 of Heatmap cinerea . C. S is S4–S9 Figs. Appendix, SI FB-init genes FB # ofgenes Dev. reg.genes Cap DEG(up) Phanerochaete Schizophyllum Coprinopsis Rickenella Armillaria o h opeels fmdlsaddt o te pce) h itiuinof distribution The species). other for data and modules of list complete the for Lentinus D

ye ntesxseisuigrgo-etitdprobabilistic region-restricted using tissue species and six stages the developmental in across types isoforms Development. with transcript Associate in Patterns walls Splicing cell rigid evolved independently groups. these by imposed straints Gene families 100 50 Stipe DEG(up) GTP bind(RAS GTPase) score Row Z- Transmembrane trp. Energy d.metaltrp. Transcription factor Transcription factor Transcription factor Transcription factor Major Fac. Superfam. Major PNAS Carbohydr. metab. Carbohydr. metab. Prot.-prot. interact. C Prot.-prot. interact. His biosynth.proc. Signalling (GPCR) Cell wallremodell. Lipid metab.proc. Nucleosomal act. Prot. degradation Cell cycle+repl. Transmembr. tp. Oxid.-red. proc. Fungal cellwall Fungal cellwall Amino acidtrp. Microtub. proc. -2 Cell cyclereg. Energy prod. | .cinerea C. Translation Translation Translation F-box prot. Cytoplasm 0 pi ,2019 9, April CytP450 CytP450 CytP450 CytP450 CytP450 Mitosis Mitosis 2 sa xml.C a;F,fruiting FB, cap; C, example. an as 1090 141 193 128 232 400 61 717 51 288 67 624 503 65 88 210 81 92 412 412 461 240 468 56 | 55 55 78 .W graphically We S7). Dataset

o.116 vol.

VM

ereconstructed We HN P1

| P2 YFB-S

o 15 no. YFB-C

YFB-G FB-S

| FB-C 7411

EVOLUTION modeling, a strategy developed for gene-dense fungal genomes (32). Although generally linked to cellulose degradation (33, 34), (24). We found evidence of alternative splicing for 36 to 46% expansins, lytic polysaccharide monooxygenases, and cellobiose of the expressed genes (Dataset S9), which is significantly higher dehydrogenases have recently been shown to target chitin poly- than what was reported for fungi outside the Agaricomycetes (25, mers (35, 36) or to be expressed in fruiting bodies of 26) (1 to 8%). This transcript diversity was generated by 6,414 to (37) and (38), suggesting a role in fruiting body 13,780 splicing events in the six species. Of the four main types development. In addition, developmental expression of two algi- of events, intron retention (44.3 to 60.5%) was the most abun- nate lyase-like families (SI Appendix, Table S2) were shared by dant in all species, followed by alternative 30 splice site (22.9 six species, while that of a β-glucuronidase (GH79 1) was shared to 30.1%), alternative 50 SS (15.6 to 24.1%), and exon skipping by four species (Armillaria, Coprinopsis, Rickenella, and Lenti- (0.8 to 2.9%) (SI Appendix, Fig. S11A), consistent with obser- nus). The targets of these families in fruiting bodies are currently vations made on other fungi (25–27). No substantial difference unknown, yet their conserved expression pattern suggests roles in the proportion of spliced genes and of splicing events was in polysaccharide metabolism during development (39). Com- observed across developmental stages, tissue types, or species. parison across 201 genomes revealed that 24 of these families Nevertheless, we found that several genes with nearly constant have undergone expansions in the Agaricomycetes (SI Appendix, overall expression level had developmentally regulated tran- Table S2 and Dataset S3). In summary, CAZymes might be script isoforms (SI Appendix, Fig. S11 B and C). The six species responsible for producing fruiting body-specific FCW architec- had 159 to 1,278 such genes, the highest number in Rickenella tures, conferring adhesive properties to neighboring hyphae or (1,278) and the lowest in Phanerochaete (159) (SI Appendix, plasticity for growth by cell expansion. We, therefore, suggest Fig. S11D and Dataset S9). Based on their expression dynam- that FCW remodeling comprises one of the foundations of ics, these transcripts potentially also contribute to development, the transition to complex multicellularity during the life cycle expanding the space of developmentally regulated genes through of fungi. alternative splicing. A significant fraction of conserved developmentally regulated genes carry extracellular secretion signals and were predicted Conserved Transcriptomic Signatures of Mushroom Development. to be glycosylphosphatidylinositol (GPI) anchored (Fig. 2A Our transcriptome data are particularly suited to detecting and SI Appendix, Fig. S14). These include diverse FCW-active shared patterns of gene expression across species. We analyzed proteins, such as laccases (AA1), glucanases (GH5, GH16, common functional signals in the six species by estimating the Kre9/Knh1 ), and NodB homologs (chitooligosaccharide percent of developmentally regulated genes shared by all or sub- deacethylases), but also lectins, A1 aspartic peptidases, and sets of the species based on Markov clustering (28) of protein sedolisins, among others (Dataset S11). Homologs of rhizobial sequences. We found 100 clusters containing developmentally NodB genes were developmentally regulated in all six species, regulated genes from all six species, and 196 in five species whereas, in the context of ectomycorrhizal symbioses, they were (Fig. 1D and Dataset S10). These are enriched for GO terms discussed as potential elicitors of plant immune responses (40). related to oxidation−reduction processes, oxidoreductase activ- GPI-anchored proteins often mediate adhesion in filamentous ity, and carbohydrate metabolism, among others, corresponding and pathogenic fungi (41), but it is not known whether simi- to a suite of carbohydrate active enzymes. Of the 100 fami- lar mechanisms are at play in fruiting bodies (2). Laccases and lies shared by six species, 15 can be linked to the FCW, while glucanases could facilitate adhesion by oxidative cross-linking the remaining families cover diverse cellular functions such as or other covalent modifications of neighboring hyphal surfaces, transmembrane transport (6 families), cytochrome p450s (5 fam- although more data are needed on the biochemistry involved. ilies), targeted protein degradation (5 families), or peptidases Nevertheless, it seems safe to conclude that FCW-active proteins (3 families). One hundred and four gene families are shared may bind neighboring hyphae through covalent FCW modi- by five species excluding Phanerochaete (Fig. 1D), which comes fications in fruiting bodies, which would represent a unique as no surprise, as this species produces the simple crust-like, adhesion mechanism among complex multicellular organisms. fruiting bodies. Besides these highly conserved families, genes Homologs of the dystroglycan-type cadherin-like domain con- containing another 73 InterPro terms are developmentally reg- taining protein of Schizophyllum cerevisiae [Axl2p (42)] were ulated in six or five species but didn’t group into gene families enriched in Agaricomycetes compared with other fungi (P = due to their higher rate of evolution. These include most tran- 1.1 ×10−4, FET; Dataset S3) and were developmentally regu- scription factors (TFs), kinases, aquaporins, certain peptidase lated in all species (Dataset S11). These proteins share signif- families, and enzymes of primary carbohydrate metabolism (tre- icant sequence similarity with animal cadherins (Blast E value halose and mannitol; SI Appendix, Fig. S13), among others of <10−30) and, although fewer in numbers than in animals, (Dataset S10). their convergent expansion in complex multicellular fungi and Shared developmentally regulated gene families included a metazoans could indicate recurrent cooption for developmental conserved suite of CAZymes active on the main chitin and β- functions. 1,3- and β-1,6-glucan polymers as well as minor components of Fruiting body secretomes contained a rich suite of genes the FCW. These included various glycoside hydrolases (GH), encoding small secreted proteins (SSPs, <300 amino acids, hydrophobins, expansin-like proteins, and cerato-platanins, with extracellular secretion signal). Of the 190 to 477 SSPs among others. A large suite of β-glucanases, chitinases, lac- predicted in the genomes of the six species, 20 to 61% are cases, endo-β-1,4-mannanases, and α-1,3-mannosidases were developmentally regulated, with 20% being conserved across developmentally regulated, many of which are also expanded in the six species (Fig. 2B and SI Appendix, Fig. S15). Con- Agaricomycetes (SI Appendix, Table S2 and Fig. S12). The served and annotated genes comprise various FCW-related expression of glucan-, chitin-, and mannose-active enzymes is families, such as hydrophobins, cerato-platanins, cupredoxins, consistent with active FCW remodeling during fruiting body lectins, Kre9/Knh1, GH12 and LysM domain proteins, among formation and recent reports of similar genes upregulated in others (Fig. 2C and SI Appendix, Fig. S16). Hydrophobins and the fruiting bodies of (16, 17, 29), Flammulina (30), cerato-platanins are SSPs that self-assemble into a rodlet layer and Coprinopsis (31). Kre9/Knh1 homologs are developmentally on the cell surface, conferring hydrophobic surfaces to hyphae regulated in all species and are overrepresented in mushroom- that hinder soaking of fruiting bodies with water. They are forming fungi (P = 1.45 ×10−5, FET; Dataset S3). This family is hypothesized to mediate adhesion, the aeration of fruiting bodies involved in β-glucan assembly in Saccharomyces and has puta- (12, 43), or pathogenicity (44). As reported previously (12), most tive signaling roles through an interaction with MAP kinases hydrophobin genes are developmentally regulated (Fig. 2D and

7412 | www.pnas.org/cgi/doi/10.1073/pnas.1817822116 Krizsan´ et al. Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 Krizs duplica- tandem plants recent higher from in predominantly (50), seen resulting numbers proteins copies and the F-box 1,199 (51) to fil- to 90 comparable and 67 to 274), have (mean: 60 fungi while and mushroom-forming example, 20 respectively, For possess 3A). fungi (Fig. amentous yeasts filamentous related and genomes with the fungi compared in fungi overrepresented mushroom-forming strongly of also are families stage-specific gene and 3 developmental (Fig. or peaks Several tissue expression all displaying complex. in often and regulated ligase species, developmentally zinc-finger are RING-type ubiquitin proteins and domain E3 BTB/POZ proteins F-box the encoding of genes Mush- components in of Expansion Striking Shows saprotrophic rooms. Degradation of Protein This repertoires Targeted effectors. SSP body of rich some fruiting the that as (49). explain Agaricomycetes possibility act also the might raises could SSPs 48), (46), detected (47, interactions the part- species pathogenic across within and signaling (45) in in or role ectomycorrhizal function their in their known, ners Although not is role development. 45) bodies a body fruiting (5, have fruiting proteins SSPs in secreted -induced also species-specific in that sim- suggests observed is and that proportion This and/or to 2B). annotations ilar (Fig. functional orphans no species-specific had genes, were SSPs conserved regulated in to mentally (except addition regulated In developmentally rochaete). and S3) Dataset genomes (P the in fungi overrepresented mushroom-forming is family of the and S10), Dataset genes species. orphan six with the SSPs, in regulated SSPs developmentally of and annotation 4 all Functional Fig. (D) of in species. Numbers as (B) legend subsets. (color regulated differently shaded developmentally conserved and regulated developmentally 2. Fig. eaopaaisaeas xadd(P expanded also are Cerato-platanins

# of genes BD Coprinopsis 100 200 300 400 500 GH superfamily ne al. et an ´ Glyc. hydr./deacetyl, β/ɑ-barrel 0 A

h itiuino P-nhrdscee rti eetie n its and repertoire, protein secreted GPI-anchored of distribution The (A) bodies. fruiting in regulated developmentally are proteins secreted Diverse # of genes efudasrn inlfrdvlpetlexpression developmental for signal strong a found We 20 40 AsparticKre9/Knh1 peptidase family d. 60

Cupredoxin 0

Armillaria Peptidase family A1

AsparticNodB peptidase homology A1d.

Rickenella FB-initiation A

lgnsFB-dev. regulated ll genes Peptidase S8/S53 d.

Alpha/Beta hydrolase fold

Lentinus CFEM domain .These ). S17–S19 Figs. Appendix , SI

Schizophyllum FAD/NAD(P)-bindingPeptidase S53 d. <

Orphan SSPs GMC oxidoreductase; N-term.

10 GH13; catalytic domain C Sedolisin domain

.(C ). Phanerochaete

−300 Beta-glucuronidase; C-term. 1.56 = oynme itiuinaddvlpetlrglto fhdohbn n eaopaaisi h six the in cerato-platanins and hydrophobins of regulation developmental and distribution number Copy ) FET; ,

0 fdevelop- of >40% ɑ-amylase; DUF1966 Six-hairpin glycosidase-like Dev. reg.conservedin5/6species Developmentally regulated Genes encodingGPI-anchoredproteins ×10

aae S3). Dataset Serine carboxypeptidase Peptidase S10 Fungal lipase-liked. −50 NodBhomology d. Cerato-platanin FET; , Phane- Immunoglobulin-like fold AsparticPepsin-like peptidase act.domain site Pectin lyase fold/virulence f. Cupredoxin Not annotated

ct fteE bqii iaecmlx(-o,RN,and F-box RING, parallels (F-box, This complex Agaricomycetes. ligase in ubiquitin proteins) and E3 speci- BTB/POZ expansion target the define striking of that a genes ficity of observed patterns we expression body-forming distinctive together, fruiting in the Taken expan- PCD of in fungi. an evolution the genes observe proteins of F-box independent PCD-related not of is expansion of tissue the did that certain upregulation suggesting We Agaricomycetes, to systematic (55). limited or bodies be sion fruiting to seems in programmed which types with (PCD), connection (54). death neddylation a Ascomycota cell has of the also for patterns ubiquitylation reported expression Protein as specific genes deneddylation detect and not did fungi regulated we mushroom-forming developmentally 3D; in neither (Fig. overrepresented are HECT- significantly and ligases nor SKP1, ubiquitin cullins, type enzymes, (E1) ubiquitin-activating other the developmentally On in are fungi. only in regulated enzymes proven regula- (E2) be transcriptional to ubiquitin-conjugating as yet hand, is act this also although a (53), can tors enables proteins which F-box (51). 52), plants, development during In (50, proteolysis ligases selective of ubiquitin regulation tight E3 tar- of the of specificity define evolution genes get the These and morphologies. genes body these fruiting of complex expansion the suggesting cor- between 3C), (Fig. link poor genes a a expressed but of number complexity the body with relation fruiting with show correlation species six good the a in found genes containing and fruiting domain numbers zinc-finger, BTB/POZ The at RING-type 3B). F-box, (Fig. upregulated regulated stipes developmentally were and of gills, them caps, peak in of or single initiation many a body showed and mostly expression, They S20). in Fig. Appendix, (SI tions Galactose-b. d.-like Cellulose-binding d. Ricin Blectin ConcanavalinA-lectin/glucanase P A lpha/Betahydrolase fold Thaumatin > Glycosidehydr. superfam. Kre9/Knh1 family C .5 E) ihteecpinof exception the With FET). 0.05, 50 # of hydrophobin genes # of cerato-platanin genes ofcerato-platanin # # ofhydrophobin genes Hydrophobin, cons.site Coprinopsis, RlpA-like protein 40 PNAS Hydrophobin 02 10 20 30 | pi ,2019 9, April and Armillaria, 0 Rickenella Coprinopsis Phaneroc- Armillaria Schizop- hyllum Lentinus haete | o.116 vol.

0 FB-dev. regulated All genes FB-initiation whereas Rickenella, 15 2 | 3 o 15 no. Coprinopsis, 47 6 | 7413

EVOLUTION A B -140

400 200 400

# of genes 200 200 100

Mushroom-f. Basidiom. Fruiting body f. Ascom. ‘Early-div.’ Fungi Other Basidiomycota Other Ascomycota Nonfungal D C F-box BTB/POZ E2 Ubiquitin RING E1 Cullin Transcriptome E1 U 18000 E2 10000 300 E1 150 Rbx1 50 U U E2 U U 10 U Cullin 5 Target Skp1

# of dev. regulated genes # of dev. 1 F-box Coprinopsis Rickenella Schizophyllum Armillaria Lentinus Phanerochaete -2 02 Row Z-score Decreasing fruiting body complexity

Fig. 3. Expansion and developmental regulation of selective protein degradation pathways. (A) Copy number distribution of F-box, RING-type zinc finger, and BTB/POZ domain proteins showing their enrichment in mushroom-forming fungi compared with other analyzed organisms among 200 genomes. (B) Heatmap of developmentally regulated F-box proteins in C. cinerea. Genes were hierarchically clustered based on gene expression similarity using average linkage clustering. (C) The correlation between morphological complexity of fruiting bodies and the number of developmentally regulated elements of the E3 ligase complex. (D) Outline of the E3 ubiquitin ligase complex highlighting members expanded and developmentally regulated (solid) in mushroom- forming fungi. Transparent members are not expanded nor developmentally regulated.

gene expansion in plants which, combined with their widespread usually not conserved, we found five TF families that contained role in development (51, 56) similarly to the apoptotic-like cell developmentally regulated genes from five or six species (Dataset death and SUMOylation-related proteins, suggests that they S10). These included C2H2-type zinc fingers [including c2h2 of likely have key roles in complex multicellular development in Schizophyllum (18, 57)], and Zn(2)-C6 fungal-type and homeo- mushroom-forming fungi. box TFs [containing hom1 of Schizophyllum (18, 57)]. Two clus- ters of C2H2 and homeobox TFs showed expression peaks in Key Multicellularity-Related Genes Are Developmentally Regulated stipes of Coprinopsis, Lentinus, Armillaria, and Rickenella, con- in Fruiting Bodies. Complex multicellularity in fungi is imple- firming previous reports of Hom1 expression in Coprinopsis (58) mented by the reprogramming of hyphal branching patterns, and Schizophyllum (57, 59). Members of the light receptor white followed by their adhesion and differentiation (2). This assumes collar complex were developmentally regulated in all species mechanisms for cell-to-cell communication, adhesion, differen- except Phanerochaete, mostly showing a significant increase in tiation, and defense. We examined the expression dynamics expression at initiation. However, these genes did not group of gene families related to these traits, including TFs, protein into one family in the clustering, which was a common pattern kinases, adhesion and defense-related genes. Like other complex for TF families, perhaps caused by their high rate of sequence multicellular lineages, mushroom-forming fungi make extensive evolution. use of TFs in development. To identify development-related Communication among cells by various signaling pathways TFs, we manually curated TF candidate genes to exclude ones is paramount to the increase of distinct cell types in evolu- that nonspecifically bind DNA. The resulting TFomes contain tion. In accordance with the higher complexity of mushroom- 278 to 408 genes, of which 4.5 to 64% were developmentally forming fungi, their kinomes are significantly larger than those regulated (SI Appendix, Figs. S21 and S22). These were dom- of other fungi (P = 1.1 ×10−184, FET; Fig. 4B), due to expan- inated by C2H2 and Zn(2)C6 fungal type, fungal trans, and sions of the eukaryotic protein kinase superfamily. We clas- homeodomain-like TFs (Fig. 4A). Although TF families were sified protein kinases into nine groups (60) using published

7414 | www.pnas.org/cgi/doi/10.1073/pnas.1817822116 Krizsan´ et al. Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 Krizs any of enrichment general no the shows overlaying However, classification tissues. with gill network and cap in showed resem- members peaks family expression stages, CAMK Many of multiple S23–S25). Figs. modules Appendix, through expressed peaks expressed early expression bling highly early mostly with Kinases are 4 S23–S25 ). (SI (Fig. development Figs. in topology late Appendix, peak expression network an showing of kinases most driver main the groups. (P underrepresented are in expansion bicolor strong Tyro- a (7). show (P earlier also Agaricomycetes kinases reported (TKL) as kinase-like FunK1 significantly, sine Agaricomycetes-specific expanded and is The RGC and family missing. PKL, diverse with most kinases being composition, tyrosine families similar CAMK a and have CMGC, S12) (Dataset kinomes for of Agak1 classifications and kinome Funk1 families The classification. (E 0.825. kinase of by log cutoff split to coefficient species correlation transformed six a are with Axes the separately. visualized of shown were repertoires networks are (green) and group genes, kinome “Other” kinase the regulated of developmentally pair and each (gray) for calculated Kinome (Left were kinase similarity profile protein of expression eukaryotic kinases of Metazoa. of expressed and diagram 306 groups bar of fungal network Circular Coexpression other (B) in species. versus six fungi across forming genes (right) regulated not versus 4. Fig. B AD Mushroom-f. Basidiom. Other Basidiomycota iaecepeso ewr eeldtsu pcfiiyas specificity tissue revealed network coexpression kinase A CCAAT-binding fac. LAG1, DNA binding Zn-fin., NHR/GATA ne al. et an Homeodomain-like ´ HSF_DNA-binding DNA-binding RFX AT-rich interact.r. Lambda repr.-like Homeobox KNd. bZIP_1, bZIP_2 Ffml itiuinadtepootoso eeomnal euae (left) regulated developmentally of proportions the and distribution family TF (A) genes. kinase and TF of distribution number copy and Expression

300 Zn(2)C6 fungal Eukaryotic ProteinKinase Zinc fin.,C2H2 Yap1 redoxd. Homeobox d. HTH, APSES

200 Winged HTH Fungal trans p53-like TF Copper-fist 100 MADS-box Forkhead HMG box # ofdev. reg.genes Gt1/Pac2 P =1.2 JmjC d. JmjN d. Myb d. Others GATA TEA 6)adohrseis(2.Hsiiekinases Histidine (62). species other and (61) -185 < 75 10 02 550 25 0 25 50 Other Ascomycota 8.95 = −300 Fruiting bodyf. Ascom. Coprinopsis ,cnitn ihosrain in observations with consistent ), ×10 # ofnon-dev. reg.genes Coprinopsis −36 Phanerochaete Schizophyllum Rickenella A 90 Lentinu Coprinopsis

60 rmillaria eaiet te fungal other to relative ) 7.Tesxmushroom six The (7). 30 TK 75 .cinerea C. P <1 s ‘Early-div.’ Fungi L 0 125 100 Fg 1C (Fig. 0 C -300 and P Metazoa vradwt (C with overlaid auso vrersnaini uhomfrigfniaegvnfrbt lt.(C plots. both for given are fungi mushroom-forming in overrepresentation of values ,with D), C and E 128 128 2 64 16 32 YFB_C P2 P1 HN VM 64 16 32 4 0 8 2 scale. 4 0 8 2

SI Other Other TKL TKL iaecasfiain ariecreaincoefficients correlation Pairwise classification. kinase (D) and peak expression ) PKL PKL CAMK CAMK Coprinopsi etn hyocetaesga rndcinvaslbekinases soluble via transduction signal orchestrate sug- they fungi, mushroom-forming gesting TKL of genes in kinase signals Ser/Thr secretion other or and domains found extracellular par- we most of (65), to evidence while architectures no related receptor-like However, have be genes complexity. may TKL organismal plant and in (64)] from increases animals distinct allel of is [which expansion convergence 63) TK of (1, the plants case in multicellular remarkable family complex a with The TKL is the morphologies. fungi of simpler mushroom-forming expression with 4E) developmental species and (Fig. in expansion family not TKL but in expanded Rickenella, regulated highly developmentally regulated the of fam- developmentally are kinase of 33% other the of to Members of that 4 ilies. resembles 35% figure comprises to this FunK1 although 0.2 kinases, species, and fruit- our to kinome in In linked the upregulation (7). been an bodies has on ing family based FunK1 development, level The highest multicellular classification. the at kinase 4 development for of (Fig events tissues cooption or diverse ing stages developmental in family

etnsPhanerochaete CMGC Lentinus CMGC FB_S FB_C YFB_S YFB_G AGC AGC STE STE AtypicalCK1 AtypicalCK1 s FunK1 FunK1 AgaK1 AgaK1 128 128 64 16 64 16 32 32 4 0 8 2 4 0 8 Other 2 Other

PNAS TKL TKL Schizophyllum CAMKPKL CAMKPKL A

CMGC CMGC Rickenella rmillaria | n K ( TKL and ) AGC AGC CGMC CK1 CAMK A A pi ,2019 9, April

STE STE typical GC AtypicalCK1 AtypicalCK1 FunK1 FunK1 Coprinopsis,

Right AgaK1 AgaK1 128 128 TK STE PK Othe 64 16 32 64 16 32 | 4 0 8 2 eetie fmushroom- of repertoires ) 4 0 8 2 L L o.116 vol. Other Other

TKL TKL r CAMKPKL CAMKPKL

and Armillaria, CMGC CMGC | ,indicat- C–E),

o 15 no. AGC AGC STE STE AtypicalCK1 AtypicalCK1 | and FunK1 FunK1

7415 AgaK1 AgaK1 D) )

EVOLUTION or other mechanisms different from those of multicellular plants Discussion and animals. We charted the transcriptomic landscape of multicellular Fungal immune systems comprise innate chemical defense development in six phylogenetically diverse mushroom-forming mechanisms against metazoan predators as well as bacterial species and performed comparative analyses of >200 genomes. and fungal infections (66). We cataloged 11 families of defense We pinpointed nearly 300 conserved gene families, and another effector proteins and their expression to assess the conserva- 73 gene groups with developmentally dynamic expression in five tion of the defensive arsenal of Agaricomycetes. Genomes of or more species, as well as 631 domains significantly overrep- mushroom-forming fungi harbor highly species-specific combi- resented in mushroom-forming fungi. These are enriched in nations of defense-related genes encoding pore-forming toxins, cell wall-modifying enzymes, various secreted proteins (includ- cerato-platanins, lectins, and copsins, among others, with most of ing GPI-anchored and SSPs), components of the ubiquitin ligase them being developmentally expressed and upregulated at fruit- complex, kinases, or TFs. Lectins and defense effectors, on ing body initiation (SI Appendix, Fig. S26). Of the 11 families, the other hand, showed species-specific repertoires, indicating a only 3 were conserved, and only 1 (thaumatins) was develop- higher rate of evolutionary turnover. These data provide a frame- mentally regulated in all six species, which can display either work for elucidating the core genetic program of fruiting body endoglucanase or antimicrobial activity, depending on the struc- formation and will serve as guideposts for a systems approach to ture of the mature protein. In silico structure prediction iden- understanding the genetic bases of mushroom development and tified an acidic cleft in fruiting body expressed thaumatins (SI multicellularity. Appendix, Fig. S26), consistent with an antimicrobial activity. Complex multicellularity evolved in five lineages, of which Several defense-related lectins have been reported from fruiting plants, animals, and fungi are the most diverse (1, 2). At the bodies (67), although lectins have been implicated in cell adhe- broadest level of comparison, all these lineages evolved solu- sion and signaling too. Agaricomycete genomes encode at least tions to cell adhesion, communication, long-range transport, 17 lectin and 2 lectin-like families, of which 7 are significantly and differentiation, although the exact mechanisms often dif- overrepresented (P < 0.05, FET; Dataset S3). Developmentally fer among lineages (1–3). As in animals and plants, protein regulated lectins belong to nine families, with four to seven kinases, putative adhesive proteins, defense effectors, and cer- families per species, but only ricin B lectins were developmen- tain TFs have expanded repertoires in mushroom-forming fungi tally regulated in all six species (SI Appendix, Fig. S27). Other and show developmentally dynamic expression patterns. Exam- lectin families show a patchy phylogenetic distribution, which ples for convergent expansions in mushroom-forming fungi, is also reflected in their expression patterns in fruiting bodies. plants, and/or animals include TKL family kinases, F-box pro- Several lectins are induced at fruiting body initiation, including teins, and cadherin-like proteins, indicating that ancient eukary- all previously reported nematotoxic Coprinopsis lectins (CCL1-2, otic gene families with apt biochemical properties have been CGL1-3, and CGL3). Taken together, the defense effector repeatedly coopted for complex multicellularity during evolu- and lectin-encoding arsenal of mushroom-forming fungi shows tion. F-box proteins showed the largest expansion in Agari- a patchy phylogenetic distribution, consistent with high gene comycetes across all gene families and were the largest devel- turnover rates or gains via horizontal gene transfer (66). Accord- opmentally regulated family, along with RING-type and BTB ingly, expressed defense gene sets are highly species-specific, domain proteins. Among fruiting body-forming fungi (2, 54), with most of the encoded genes upregulated in fruiting bod- Agaricomycetes share some similarity with the Pezizomycotina ies, suggesting that chemical defense is a key fruiting body (Ascomycota), many of which also produce macroscopic fruit- function. ing bodies. For example, laccases, lectins, several TFs, and signal transduction systems have also been implicated in fruit- Most Developmental Gene Families Are Older than Fruiting Body ing body formation in the Pezizomycotina, although, at the Formation. We investigated the evolutionary age distribution of moment, it is unclear whether the Pezizomycotina shares a developmentally regulated genes using phylostratigraphy (68), complex multicellular ancestor with the Agaricomycetes (2). and gene tree−species tree reconciliation analyses. We assigned Comparisons of developmental genes and transcriptomes across genes to phylogenetic ages, “phylostrata,” by identifying for the Agaricomycetes and the Pezizomycotina will be neces- each gene the most phylogenetically distant species in which sary to elucidate whether these two groups share a single a homolog could be detected. The phylostratigraphic profiles origin of or represent independent acquisitions of complex of all species show three peaks, corresponding to two major multicellularity. periods of fungal gene origin: the first containing genes shared Mushroom-forming fungi also show several unique solutions by all living species, the second containing genes shared by for multicellularity, as expected based on their independent evo- the (Ascomycota + Basidiomycota), and the third lutionary origin. These are, in part, explained by the very nature containing species-specific genes (SI Appendix, Fig. S28). In of fungi: Complex multicellularity comprises the reproductive terms of gene duplications and losses, we observed two major phase of the life cycle (except in sclerotia and rhizomorphs) expansion in 292 shared developmentally regulated gene fam- and so mechanisms have evolved for sensing when fruiting body ilies, one in the most recent common ancestor (mrca) of the formation is optimal (e.g., nutrient availability, light). Mush- Dikarya and the other from the mrca of Agaricomycetes and room development can be partitioned into an early phase of cell Dacrymycetes extending to basal nodes of the Agaricomycetes proliferation and differentiation and a growth phase of rapid (SI Appendix, Fig. S29). Notably, the expansion in the Agari- increase in cell size, a division evident on our gene coexpression comycetes roughly correlates with the origin of fruiting body profiles as well. Broadly speaking, this is similar to the develop- development in this . These data suggest that many devel- ment of fleshy plant fruits, although mechanisms are likely to be opmentally regulated genes have homologs in simple multicel- different. lular or unicellular organisms: The origin of 83.3% predate This work has provided a glimpse into the core genetic toolkit the origin of mushroom-forming fungi (SI Appendix, Fig. S28), of complex multicellularity in mushroom-forming fungi. Our indicating that several conserved gene families were recruited comparative transcriptomic and genomic analyses revealed sev- for fruiting body development during evolution. Neverthe- eral gene families with conserved developmental expression less, Agaricomycetes-specific phylostrata showed a characteristic in fruiting bodies, with scope to increase the resolution both enrichment for F-box genes, TFs, and protein kinases, indicating phylogenetically and among cell types (e.g., by single-cell RNA- an increased rate of origin for these in mushroom-forming fungi Seq). Such data should help define the conserved genetic pro- (Dataset S8). grams underlying multicellularity in mushroom-forming fungi,

7416 | www.pnas.org/cgi/doi/10.1073/pnas.1817822116 Krizsan´ et al. Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 0 K 10. 1 a erA,e l 20)Tespa oecpi nognleta ucin nveg- in functions that organelle an is cap pore septal The (2009) al. et AF, Peer Van 11. 2 ar ,Amnad ,Giar I ud ,Latg M, Sunde JI, Guijarro V, Aimanianda J, Bayry 12. 3 lz F i W a e ednN,Ab ,K M, Aebi NS, Velden der van CW, Lin DF, Plaza 13. 4 ue ,LzrZ K Z, Lazar R, Buser 14. 5 i -,e l 21)De N eunigrvasahg rqec falterna- of frequency high a reveals sequencing RNA Deep (2015) al. et B-B, gene Xie series time 25. short of analysis the for tool A STEM: (2006) Z Bar-Joseph J, Ernst 23. of development Cl and 22. Genomics (2018) al. et fungal in innovations B, genetic for lineage-specific Wu and molecules 21. expansion Genome (2017) Sensory al. et photoreceptors: G, Sipos Fungal 20. (2007) LM Corrochano 19. W C, Bekker de JF, Jong protein de RA, thaumatin-like Ohm a 18. encodes tlg1 edodes Lentinula (2006) al. et Y, Sakamoto 17. endo-β An (2011) Y Sakamoto N, Konno laccase 16. of regulation Transcriptional (2000) DA Wood CF, Thurston N-S, Cho S, Ohga 15. 4 eranT ta.(2016) al. et T, Gehrmann 24. eoclainaayi a efre oivsiaeteeouinr age evolutionary genes. the regulated investigate developmentally (49). to of tree distribution al. performed tree−species gene was et mod- and analysis Pellegrin Phylostratigraphy coexpression reconciliation (23). of into v1.3.11 pipeline STEM clustered using the were ules of genes version regulated modified Developmentally a per- was using SSPs of Prediction formed We metabolism. composition. in involved domain kinases InterPro classical on DNA-binding excluded based sequence-specific predicted with were Kinases domains activity. Interpro on were TFs based Pred-GPI. using identified pipeline. performed was CAZy proteins the GPI-anchored using of Prediction annotated were puta- enzymes encoding carbohydrate-active Genes tive (24). modeling isoforms, probabilistic transcript region-restricted reconstruct used To we enrichment genomes. fungal their 201 and across domains, regulated analyzed Interpro was developmentally by identify characterized were to these used genes; was profiling expression Gene RP78, of complex scriptomes see major methods, additional For a Methods of and Materials origins eukaryotes. evolutionary the in lineage the multicellular uncover and Krizs .OmR,e l 21)Gnm euneo h oe mushroom model the of sequence K Genome 9. (2010) al. et RA, assem- Ohm the 8. from fungi multicellular of evolution into Insights source (2010) rich al. A et Mushrooms: JE, (2017) Stajich RB 7. Beelman A, Calcagnotto JP, Richie MD, Kalaras 6. of evolution independent the and genome Seb Ectocarpus The 4. (2010) al. et JM, Cock 3. multicellularity. complex Kov LG, of Nagy origins 2. multiple The (2011) AH Knoll 1. .Khe ,e l 21)Cnegn osso ea ehnssadrpdtroe of turnover rapid and mechanisms decay of losses Convergent (2015) al. et A, Kohler 5. cinereus. commune. USA Sci mushroom Acad Natl the of chromosomes bled glutathione. and ergothioneine antioxidants the of algae. brown in multicellularity both? or origin, single convergence, 39:217–239. ois .Mrhlgclapcso development. of aspects Morphological 1. bodies? mutualists. mycorrhizal in genes symbiosys perspective. ttv rwhadmsro omto ftewo-o wood-rot the of formation mushroom commune. and growth etative uglproteins. fungal roisadacnevdcrutyfrsxa development. sexual for circuitry conserved a mushroom and armories model the of transcriptomics in n isnhsso oe -lcnmdfiaini h riigbd fthe of body fruiting the in modification N-glycan novel a basidiomycete of biosynthesis and tion, ieslcn vnsi h fungus the in 16:54. events splicing tive repertoire. splicing alternative data. expression Tables 12 with bodies. fruiting dimorphic with 10:3250–3261. mushroom wood-decaying rot Armillaria. fungi pathogenic forest the behaviour. and development Microbiol Mol of genes tor senescence. body fruiting and degradation 141:793–801. lentinan in involved is that autolysis. body fruiting edodes Lentinula of mycelium the substrate. in sawdust-based formation a body on fruit to relation in cellulase and e 20)Lf itr n eeomna rcse ntebasidiomycete the in processes developmental and history Life (2000) U ues e ,Navarro-Gonz U, ues ne al. et an ¨ ¨ mno ,Emt ,Emt E(2012) EE Emmett V, Emmett H, emencon ´ ´ e-Pedr ´ .mellea R. irbo o ilRev Biol Mol Microbiol sA ennB,Ri-rloI(07 h rgno eao:Aunicellular A Metazoa: of origin The (2017) I Ruiz-Trillo BM, Degnan A, os ´ a Biotechnol Nat nio Microbiol Environ ciohlu commune Schizophyllum a e Genet Rev Nat c M Krizs GM, acs ´ .cinerea C. 81:1433–1445. ornpi cinerea. Coprinopsis SZMC22713, LSPathog PLoS M Bioinformatics BMC 107:11889–11894. srS K S, aser ¨ lzM(05 o oAaioyee hp hi fruiting their shape Agaricomycetes do How (2015) M alez ´ plMcoilBiotechnol Microbiol Appl nK(08 ope utcluaiyi ug:Evolutionary fungi: in multicellularity Complex (2018) K an ´ 28:957–963. ciohlu commune Schizophyllum AmutBmut, 18:498–512. nlrM eiM(00 dnicto,characteriza- Identification, (2010) M Aebi M, unzler 12:833–844. 8:e1002700. htce htbo Sci Photobiol Photochem IAppendix SI ¨ Mycoscience .tigrinus L. c Rep Sci Nature Cae erBrtagrVr uh,Stuttgart). Buchh, Verl Borntraeger Gebr (Cramer 64:316–353. ilChem Biol J se A,LgnsL 21)Tasrpinfac- Transcription (2011) LG Lugones HAB, osten ilRev Biol ¨ rcoem longibrachiatum . Trichoderma 7:191. novdi euaino uhomformation. mushroom of regulation in involved 6:33640. ornpi cinerea Coprinopsis a clEvol Ecol Nat 465:617–621. 16guaaeivle nLniuaedodes Lentinula in involved -1,6-glucanase .commune S. ornpi cinerea Coprinopsis a Genet Nat L95-p and RLG9953-sp, eaaye eeomna tran- developmental analyzed We . 41:149–153. 93:1778–1794. 285:10715–10723. yooyadPetlg fthe of Plectology and Cytology 91:1365–1373. uglBo Rev Biol Fungal P(02 Hydrophobins-unique (2012) JP e ´ odChem Food a netnieadfunctional and extensive an has 1:1931–1941. nlrM(04 Comparative (2014) M unzler 47:410−415. , ¨ 6:725−736. white- A tigrinus: Lentinus H4-8, ornscinereus). ( M Genomics BMC eel tissue-specific reveals 233:429–433. .ostoyae A. .chrysosporium P. eoeBo Evol Biol Genome 29:63–97. at lntSci Planet Earth M Genomics BMC Schizophyllum Schizophyllum ln Physiol Plant 15:492. Coprinus C18/9. Proc 9 elgi ,MrnE atnF,Vnal-ore 21)Cmaaieaayi of analysis Comparative (2015) C Veneault-Fourrey FM, commu- Martin guides E, protein Morin adhesion C, Pellegrin Fungal (2013) 49. X Lin R, Gyawali X, Tian secreted L, small Wang for role 48. A (2017) Y Hadar O, Yarden NL, Glass DJ, Kowbel D, Feldman proteins. 47. effector Fungal (2009) the PJGM Unearthing Wit (2016) de DS I, Stergiopoulos Hibbett C, 46. Veneault-Fourrey C, Murat A, Kohler of F, Martin bodies 45. fruiting fam- in protein fungal A channels Cerato-platanins: (2014) air V Seidl-Seiboth line K, Bonazza Hydrophobins R, Gaderer (1999) 44. al. et in LG, domains Cadherin-like Lugones (2002) 43. CP Ponting S, Beatson NJ, Dickens 42. to guide biochemical A (2007) PN Lipke JE, Coronado JM, Rauceo AM, Dranginis 41. An in KR, maturation Cope body P-M, fruit to Delaux relation K, in Garcia metabolism Glycogen 40. (1993) D Moore J, Ji 39. Osi dehydro- 38. cellobiose and laccase between interaction Novel (1999) C Eggert oxidases U, polysaccharide Temp lytic 3400 37. of analysis bioinformatics fungus A white-rot (2017) al. the et from N, protein Lenfant expansin 36. novel extracellular A (2015) related al. et and OE, Tovar-Herrera oxidases basidiomycetes. 35. from enzymes radical Plant-polysaccharide-degrading (2014) Copper al. et J, (2014) Rytioja D 34. Cullen Le.DRMIP, P, partner, interacting its Kersten and Le.MAPK 33. (2007) HS Kwan GS, Leung CY, Szeto syn- 32. and characterization Purification, (2015) S Yuan J, Wang Z, exo-beta-1,3- Liu W, novel Zhang Y, a Zhou of 31. characterization and Purification (2008) al. et K, Fukuda 30. Endo-β (2011) N Konno K, large-scale Nakade for Y, algorithm Sakamoto efficient An by 29. (2002) revealed CA Ouzounis fungi S, in Dongen Van transcripts AJ, Enright polycistronic Widespread 28. (2015) al. et SP, Gordon of transcriptome 27. the of Survey (2010) al. et B, Wang 26. h ainlCne o itcnlg nomto BioProject no. Information (accession Biotechnology database for Center National of the annotation and assembly Genome Availability Data neUe aiiy sspotdb h fc fSineo h SDEunder DOE Sci- US the of of Office Science of DOE Office a the DE-AC02-05CH11231. by Contract Institute, supported Genome is Facility, US Joint User innova- the ence (DOE) by and work Energy The research of 716132. 2020 and Department 758161 Horizon Agreements the Grant the by programme under Develop- tion and Council Research, GINOP-2.3.2-15-2016-00001, Research National Grant Hungarian European Office by the Innovation LP2014/12, of and Contract Program ment Momentum Sciences by of supported Academy was the work for This Agriculture) study. of Department (US ACKNOWLEDGMENTS. Phanerochaete). at transcriptome GSE125195, Archive GSE125184, sequenced GEO NCBI’s accessions: the the in of ncbi.nlm.nih.gov/geo deposited was archive libraries (GEO) Omnibus aallmN sequencing. mRNA parallel ertmsfo coyoria ug iha mhsso ml-ertdproteins. small-secreted on emphasis Microbiol an Front with fungi ectomycorrhizal from secretomes manner. paracrine a in autoinduction 110:11571–11576. and behaviors in nity regulation enzyme Ligninolytic lifestyle: fungal saprophytic ostreatus . a in (SSPs) proteins 47:233–263. symbioses. ectomycorrhizal of roots potential. application and 98:4795–4803. properties intriguing with ily commune Schizophyllum α occasions. antisocial and 71:282–294. social for Glycoproteins adhesins: symbioses. ectomycorrhizal of 79–87. maintenance and establishment the cinereus. Coprinus Polish isolated 65:295–305. newly from ies fungus rot white Microbiol the Environ in Appl synthesis pigment during genase AA9. family from commune. Schizophyllum Rev Biol Mol Microbiol Agaricomycetes. wood-decay of oxidoreductases edodes. Lentinula in development body fruiting in 1,3-β of cinerea group a of ( autolysis in Enoki ergism mushroom edible the of body fruiting velutipes). the family. from hydrolase 1,6-glucanase glycoside new a to belongs edodes, Microbiol Environ Lentinula of body ing families. protein of detection sequencing. mRNA single-molecule /ξ saJrsu ,e l 21)Cmlxbohmclaayi ffutn bod- fruiting of analysis biochemical Complex (2016) al. et M, nska-Jaroszuk ´ srolcnadyatadbceilproteins. bacterial and yeast and -sarcoglycan riigbodies. fruiting isiBoeho Biochem Biotechnol Biosci GSE125198, Rickenella; 6:1278. abhd Res Carbohydr 77:8350–8354. yo Res Mycol c Rep Sci 78:614–649. 65:389–395. Microbiol h uhr hn ailCle n ilGaskell Jill and Cullen Daniel thank authors The PNAS and LSOne PLoS ne ceso S150 (individual GSE125200 accession under uli cd Res Acids Nucleic 7:14553. 97:283–289. uli cd Res Acids Nucleic grcsbisporus. Agaricus PRJNA334780 448:166–174. | GSE125190, Coprinopsis; lmuiavelutipes Flammulina 161:1978–1989. LSOne PLoS pi ,2019 9, April 10:e0122296. a e Microbiol Rev Nat gua yrlssfo h ie of pilei the from hydrolases -glucan 72:3107–3113. - 21)Mlclrsgasrqie for required signals Molecular (2015) J-M e ´ Phanerochaete 38:5075–5087. 10:e0132628. GSE125199, Schizophyllum; 30:1575–1584. 13GuaaeGU,fo h fruit- the from GLU1, -1,3-Glucanase .mellea R. yo Res Mycol uglGntBiol Genet Fungal urBiol Curr Gene | .AGn Expression Gene A ). seglu oryzae Aspergillus o.116 vol. 14:760–773. 393:87–93. strains. plMcoilBiotechnol Microbiol Appl ynprscinnabarinus. Pycnoporus 12:R197–R199. 103:635–640. rcNt cdSiUSA Sci Acad Natl Proc irbo o ilRev Biol Mol Microbiol a eoie in deposited was nuRvPhytopathol Rev Annu tanue nthis in used strain | oihJMicrobiol J Polish 72:124–130. o 15 no. e Phytol New α -dystroglycan, i massively via Lentinus; Flammulina Coprinopsis | www. 7417 Appl 208:

EVOLUTION 50. Liu TB, Xue C (2011) The ubiquitin-proteasome system and F-box proteins in 59. Pelkmans JF, et al. (2017) Transcription factors of involved pathogenic fungi. Mycobiology 39:243–248. in mushroom formation and modulation of vegetative growth. Sci Rep 7:310. 51. Xu G, Ma H, Nei M, Kong H (2009) Evolution of F-box genes in plants: Different modes 60. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase of sequence divergence and their relationships with functional diversification. Proc complement of the human genome. Science 298:1912–1934. Natl Acad Sci USA 106:835–840. 61. Kosti I, Mandel-Gutfreund Y, Glaser F, Horwitz BA (2010) Comparative analysis of 52. Metzger MB, Pruneda JN, Klevit RE, Weissman AM (2014) RING-type E3 ligases: Mas- fungal protein kinases and associated domains. BMC Genomics 11:133. ter manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim 62. Zhao Z, Jin Q, Xu JR, Liu H (2014) Identification of a fungi-specific lineage of protein Biophys Acta-Mol Cel Res 1843:47–60. kinases closely related to tyrosine kinases. PLoS One 9:e89813. 53. Chae E, Tan QK-G, Hill TA, Irish VF (2008) An Arabidopsis F-box protein acts as 63. Lehti-Shiu MD, Shiu S-H (2012) Diversity, classification and function of the a transcriptional co-factor to regulate floral development. Development 135:1235– plant protein kinase superfamily. Philos Trans R Soc B Biol Sci 367:2619– 1245. 2639. 54. Poggeler¨ S, Nowrousian M, Teichert I, Beier A, Kuck¨ U (2018) Fruiting-body develop- 64. Suga H, Torruella G, Burger G, Brown MW, Ruiz-Trillo I (2014) Earliest Holozoan ment in ascomycetes. Physiology and Genetics (Springer Int, Cham, Switzerland), pp expansion of phosphotyrosine signaling. Mol Biol Evol 31:517–528. 1–56. 65. Shiu SH, Bleecker AB (2001) Plant receptor-like kinase gene family: Diversity, function, 55. Lu BC (1991) Cell degeneration and gill remodelling during development and signaling. Sci Signaling 2001:re22. in the fungus Coprinus cinereus. Can J Bot 69:1161–1169. 66. Kunzler¨ M (2015) Hitting the sweet spot-glycans as targets of fungal defense effector 56. Dharmasiri N, et al. (2005) Plant development is regulated by a family of auxin proteins. Molecules 20:8144–8167. receptor F box proteins. Dev Cel 9:109–119. 67. Schubert M, et al. (2012) Plasticity of the β-trefoil protein fold in the recognition 57. Pelkmans JF, et al. (2016) The transcriptional regulator c2h2 accelerates mush- and control of invertebrate predators and parasites by a fungal defence system. PLoS room formation in . Appl Microbiol Biotechnol 100:7151– Pathog 8:e1002706. 7159. 68. Domazet-Losoˇ T, Brajkovic´ J, Tautz D (2007) A phylostratigraphy approach to uncover 58. Muraguchi H, et al. (2015) Strand-specific RNA-seq analyses of fruiting body the genomic history of major adaptations in metazoan lineages. Trends Genet development in . PLoS One 10:e0141586. 23:533–539.

7418 | www.pnas.org/cgi/doi/10.1073/pnas.1817822116 Krizsan´ et al. Downloaded by guest on October 2, 2021