Fungal secreted proteins (secretome) play an important role in fungal biology and adaption to various ecological niches, and environmental interactions ranging from mutualism to parasitism and interference competition (Druzhinina et al., 2012; Pellegrin et al., 2015; Lu and Edwards, 2016). Genes encoding secreted proteins typically encompass 4–15% of the total gene numbers in fungal genomes (Girard et al., 2013; Pellegrin et al., 2015). These proteins are classified into various functional groups such as carbohydrate-active enzymes (CAZymes), proteases, lipases and oxidoreductases needed for nutrient acquisition, self-protection and biotic interactions with microbes, plants and animals (Kim et al., 2016; Guzmán-Guzmán et al., 2017; Feldman et al., 2020). Moreover, 40–60% of predicted fungal secretome proteins are typically shorter than 300 amino acids (aa) and are referred to as small secreted proteins (SSPs; Pellegrin et al., 2015; Kim et al., 2016). SSPs are often cysteine-rich, lack known protein modules or catalytic domains and certain members act as effectors mediating communication between organisms ranging from beneficial to detrimental interactions (Kim et al., 2016; Selin et al., 2016).

SSPs are mainly studied in the context of pathogenic fungal-plant interactions, and their composition and role in parasitic fungal-fungal and beneficial fungal-plant interactions is still poorly investigated. In fungi used for biological control of plant diseases, for example members of the Trichoderma and Clonostachys genera, the function of certain SSPs including hydrophobins, cerato-platanins and LysM module-containing proteins in regulating interactions with fungal hosts (mycohosts) and plant hosts are shown (Dubey et al., 2012, 2014; Guzmán-Guzmán et al., 2017; Ramírez-Valdespino et al., 2019; Jensen et al., 2021). In addition, a SSP family with Common in Fungal Extracellular Membranes (CFEM) modules has been identified in T. atroviride (Kulkarni et al., 2003; Druzhinina et al., 2012; Zhang et al., 2015; Guzmán-Guzmán et al., 2017). Certain members of this family have been shown to be induced during interactions with host plants indicating their roles as putative effector proteins (Guzmán-Guzmán et al., 2017). A role of CFEM-containing proteins in fungal pathogenesis has been demonstrated in the plant pathogenic fungi Botrytis cinerea, Magnaporthe oryzae, and Colletotrichum graminicola (Kou et al., 2017; Zhu et al., 2017; Gong et al., 2020).

Clonostachys spp. including C. rosea, C. byssicola, C. chloroleuca, C. rhizophaga and C. solani, are filamentous ascomycetes fungi with a multi-trophic mode of lifestyle. These fungi can be found as saprotrophs in various ecological niches including soil and dead organic matter (Moreira et al., 2016; Jensen et al., 2021). Certain species can thrive in rhizospheres where they can colonize the plant root surfaces and establish a beneficial relationship with the plant host as endophytes (Sutton et al., 2002; Karlsson et al., 2015; Saraiva et al., 2015; Maillard et al., 2020; Jensen et al., 2021). In addition, these species can live as necrotrophic mycoparasites by killing and feeding on their mycohosts (Alvindia and Natsuaki, 2008; Dugan et al., 2012; Sun et al., 2018; Jensen et al., 2021). The antagonistic ability of certain strains of C. rosea against plant parasitic nematodes has also been reported (Iqbal et al., 2018a,b, 2020). To succeed in these distinct ecological niches, Clonostachys spp. have evolved capabilities for decomposition of organic materials, competition with other microorganisms for nutrients and space in soil and rhizosphere, and interference competition through antibiosis and mycoparasitism (Morandi et al., 2000; Li et al., 2002; Saraiva et al., 2015; Sun et al., 2017; Fatema et al., 2018). Due to these properties, certain Clonostachys strains are used as efficient biological control agents against fungal plant diseases in agricultural and horticultural production systems (Jensen et al., 2000; Sutton et al., 2002; Xue et al., 2008; Cota et al., 2009; Salamone et al., 2018).

The ability of Clonostachys spp. to inhabit broad ecological niches is reflected by their genomic characteristics (Karlsson et al., 2015; Broberg et al., 2021). For example, copy number of genes coding for enzymes associated with biosynthesis of specialized metabolites such as polyketide synthases, non-ribosomal peptide synthetase and cytochrome P450 monooxygenases are expanded in C. rosea compared to plant pathogenic Fusarium spp. and mycoparasitic Trichoderma spp. (Karlsson et al., 2015; Broberg et al., 2021). Similarly, the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) membrane transporter gene families, associated with efflux of endogenous and exogenous specialized metabolites, are also expanded (Karlsson et al., 2015; Nygren et al., 2018; Broberg et al., 2021). Among the CAZyme gene families, auxiliary activity (AA) family 9 lytic polysaccharide monooxygenases, AA7 gluco-and chitooligosaccharide oxidases, AA3 glucose-methanol-choline oxidoreductases, polysaccharide lyase family 1 (PL1) pectin/pectate lyases and certain proteases are also evolving under selection for increased gene copy numbers (Karlsson et al., 2015; Atanasova et al., 2018; Iqbal et al., 2018a,b; Broberg et al., 2021). However, analysis of gene family evolution of genes specifically coding for secreted proteins in Clonostachys is yet to be comprehensively investigated.

In this study, we performed prediction and in-depth analysis of the secretomes of six Clonostachys spp., including C. byssicola, C. chloroleuca, C. rhizophaga, C. rosea, C. solani and Clonostachys sp. CBS 192.96 with the hypothesis that the composition of Clonostachys spp. secreteome is shaped to accomplish their saprotrophic, mycotrophic and plant endophytic lifestyles. Our analysis was focused on predicted lipases, proteases, oxidoreductases, CAZymes, cysteine-rich SSPs, putative effectors and CFEM proteins and their possible roles in mycoparasitic and beneficial fungal-plant interactions. This revealed the presence of many proteins with a known role in antagonism against mycohosts and nematodes, including chitinases, endopolygalacturonases, subtilisin-like peptidases and phospholipases A. Moreover, several enzyme classes including hemicellulose and cellulose degradation enzymes, SSPs and effectors with putative role in fungus-plant interactions were identified.

The predicted secretomes of the considered Clonostachys spp. amount to 7.7% of their proteomes on average, more than what was predicted for Trichoderma spp. The secretomes comprise a similar number of predicted proteins in all the considered Clonostachys spp., with enriched GO terms relating to proteolysis, catabolism of carbohydrates and response to fungus. We therefore hypothesize a role of secreted proteins in nutrient acquisition and fungal antagonism, which correlates well with the fact that 18% of the C. rosea secreted proteins are encoded by genes that are differentially expressed during the response to plant pathogenic fungi (Lysøe et al., 2017; Demissie et al., 2018, 2020; Nygren et al., 2018). The proportionally higher number of proteases, lipases and oxidoreductases in Clonostachys spp., compared with Trichoderma spp., together with the proportionally lower number of CAZymes, suggests different evolutionary trajectories in the two genera, driven by differences in their ecological strategies. The Clonostachys secretome included many proteins involved in fungal cell wall catabolism, such as C. rosea chitinase ChiC2 (CRV2T00000260_1), whose gene deletion cause a reduction in the growth inhibitory activity of culture filtrates against B. cinerea and Rhizoctonia solani (Tzelepis et al., 2015). An ortholog of this gene is present in all Clonostachys spp. considered in this study. However, the total number of identified GH18 chitinases range between six and nine genes in Clonostachys spp., which is lower compared to the gene copy number (12–21 genes) predicted in Trichoderma spp. More specifically, the major difference between Clonostachys and Trichoderma relates to the number of subgroup C killer toxin-like chitinases, hypothesized to be involved in permeabilization of mycohost cell walls for toxin entry (Tzelepis and Karlsson, 2019), suggesting intrinsic differences in the mode of action of these mycoparasites. Another class of secreted CAZymes operating on fungal cell walls are GH16 endo-β-(1,3)-glucanases, with potential roles in cell wall morphogenesis and catabolism (Mouyna et al., 2016). This class is evolving for increased gene copy numbers in the analyzed Clonostachys spp. with on average nine genes, while only five are found in Trichoderma spp. It is possible that the abundance of GH16 enzymes in their secretomes allow Clonostachys spp. to better modify and adapt their cell wall for the interaction with their hosts, or simply indicate that GH16 endo-β-(1,3)-glucanases are involved in cell wall degradation of the fungal prey.

Likewise, several CAZyme families with a putative role in deconstruction of plant cell walls evolve for increased paralog numbers in Clonostachys, possibly mediating nutrient uptake or plant host colonization. Family AA9 is involved in the degradation of cellulose and the high number of AA9 genes has already been observed in both C. rosea and C. byssicola (Karlsson et al., 2015; Gomes et al., 2020). In the present study, we identified a large difference, between 16 and 32 predicted AA9 enzymes, in the secretomes of different Clonostachys spp. indicating an involvement in ecological niche adaptation. Moreover, AA9 was the most frequent CAZyme class in C. rosea secreted proteins involved in the response to plant pathogens. AA9 enzymes need exogenous electron donors to function correctly, and it has been hypothesized that these could come from AA3 glucose-methanol-choline oxidoreductases (Vaaje-Kolstad et al., 2010), which is also the most numerous class of secreted oxidoreductases in the considered Clonostachys species. Additional gene families evolving for gene gene gains or losses include GH31 and GH3 where many members are putatively involved in hemicellulose degradation, including glucosidases, xylosidases and alpha-L-arabinofuranosidases. Enzymes of this class are also present in CAZyme family GH43, which is the most abundant in Clonostachys spp. with up to 39 members, while another abundant class, GH28 (13 to 16 members), include enzymes predicted to degrade pectin (Markovič and Janeček, 2001). Cell-wall degradation results in oligomers, such as xyloglucan, which can serve as damage-associated molecular patterns (DAMPs) and activate plant immunity reactions, including pattern-triggered immunity (PTI) and induced systemic resistance (ISR), resulting in the defense-inducing activity of Clonostachys spp. (Beliën et al., 2006; Souza et al., 2017; Claverie et al., 2018). Interestingly, Trichoderma spp. had on average a lower number of GH28, GH3, GH31, GH43, AA9 and AA3 enzymes, again emphasizing the different mechanistic strategies that underlie the ecological opportunism of these two genera. It is possible that Clonostachys spp. perform their biocontrol action through a greater induction of defense on host plants through partial plant cell wall degradation, while Trichoderma spp. have a greater capacity for the direct degradation of the fungal cell wall. Additional studies are needed to confirm this. Recent proteomic studies have shown an increased production of secreted plant cell wall degradation enzymes in Trichoderma spp. upon contact with the plant hosts Phaseolus vulgaris and Arabidopsis thaliana, and Clonostachys spp. might behave similarly (González-López et al., 2021; da Silva et al., 2022). Alternatively, these genomic adaptations may indicate a greater capacity for saprophytic growth of Clonostachys spp., which may also influence its usefulness in biocontrol applications.

Another process facilitated by secreted glycoside hydrolases is plant root colonization. In particular, all the considered Clonostachys spp. have a homolog of the PG1 protein, a class GH28 CAZyme involved in tomato root colonization in T. harzianum (Morán-Diez et al., 2009), and the C. rosea homolog of this gene (CRV2T00004567_1) is involved in the response to F. graminearum (Demissie et al., 2020). Another gene family with members involved in the interaction with plant hosts is the hydrophobins, necessary for plant colonization and pathogenicity in the pathogen M. oryzae (Talbot et al., 1996; Kim et al., 2005). Among the secreted C. rosea hydrophobins, we identified hyd3 (CRV2T00012494_1), a F. graminearum responsive gene (Demissie et al., 2020) whose deletion causes a reduction in root colonization (Dubey et al., 2014). This protein has a homolog in all considered Clonostachys spp., and it is similar (45% aa identity) to hydrophobin HFB2-6 of T. asperelleum, which has a function in root colonization and promotes jasmonic acid and salicylic acid signal transduction pathways in poplars (Huang et al., 2015). C. chloroleuca has almost twice as many hydrophobins, both secreted and otherwise, compared with other Clonostachys spp., suggesting that hydrophobins have evolved specific functions in the preferred ecological niche of this species.

Protease subfamily S8A is highly represented in the Clonostachys secretomes and it has previously been shown to evolve for gene gains in C. rosea (Iqbal et al., 2018a,b) and the same is true for the whole S8 family in Trichoderma spp. (Druzhinina et al., 2012). This family contains the serine endopeptidase subtilisin and its homologs, which have proven roles in biocontrol of fungi (Fan et al., 2014; Zhang et al., 2017) and nematodes (Ahman et al., 2002; Fekete et al., 2008). Numerous proteases of this class are proven to be involved in the response to mycohosts and nematodes, and this class was the most abundant one among the secreted C. rosea proteases found to be responsive to plant pathogens, together with class S1A. For example, the protease genes prs6 and prs16 are induced in C. rosea during the response to F. graminearum (Iqbal et al., 2018a,b; Demissie et al., 2020), while prs11, prs14 and prs16 are induced during parasitism of the potato pathogen H. solani (Lysøe et al., 2017). The serine protease prC gene is expressed in C. rosea when the fungus is degrading nematode cuticle material and is also involved in resistance to oxidative stress (Zou C. et al., 2010; Zou C.-G. et al., 2010). Many more members of these classes were detected in the secretomes of Clonostachys spp. than Trichoderma spp., indicating that Clonostachys spp. rely more on this type of proteases for their proteolytic action against mycohosts. However, serine proteases were also observed to be secreted in greater quantities upon T. harzianum interaction with P. vulgaris, suggesting a role in the interaction with the plant (da Silva et al., 2022).

The most numerous lipases in Clonostachys spp. proved to be GDSL-like lipases, which can potentially contribute to ethylene-based resistance in plants (Kwon et al., 2009; Gottwald et al., 2012). C. rosea and other species are known for inducing defense responses in plant hosts (Kamou et al., 2020; Sun et al., 2020), and this class of lipases could bolster that action. A subfamily of this class, GDSL esteraselipases exl3, are among the lipase families predicted to evolve for gene gains in Clonostachys species. Notably, we detected more than six Clonostachys GDSL-like lipases for each Trichoderma sp. in the secretomes, giving another indication that Clonostachys spp. have a greater part of their secretome dedicated to influencing plant hosts defense reactions. Among the most represented lipase families are also phospholipases A2, normally involved in nutrient acquisition but also in the modulation of host’s immune response (Köhler et al., 2006). One such gene (JK757061.1) is induced during T. harzianum colonization of tomatoes (Mehrabi-Koushki et al., 2012), and phospholipase A activity is a key mechanism by which Trichoderma spp. rupture the biological membranes of other fungi (Minchiotti et al., 2021).

C. rosea LysM protein LYSM2 (CRV2T00011102_1) is also predicted to be secreted. Deletion of the lysm2 gene resulted in C. rosea mutants with impaired biocontrol capabilities towards B. cinerea and F. graminearum, and also altered the suppression of wheat defense genes PR1 and PR4 (Dubey et al., 2020). An ortholog of this gene is present in all considered Clonostachys spp. except for C. rhizophaga. Other secreted proteins of interest include homologs of the effector cerato-platanin protein EPL1, involved in induction of defense reaction in maize, cotton, beans and Nicothiana bentamiana (Djonović et al., 2006; Djonovic et al., 2007; Crutcher et al., 2015; Gomes et al., 2015; Cheng et al., 2018). The thioredoxin-like effector class is also present in the secretomes of all considered species, with three proteins present in all Clonostachys spp. and one (CRV2T00013356_1) involved in the response to H. solani in C. rosea (Lysøe et al., 2017). This class is normally involved in apoplastic reactive oxygen species scavenging to protect plant pathogens from oxidative stress during the interaction with the plant, and it could play a similar role in Clonostachys spp., which are known to withstand high amounts of oxidative stress (Viefhues et al., 2014; Li et al., 2016).

Among the orthogroups detected with Orthofinder, 38 are evolving for gene gains and 16 of them contain genes involved in the C. rosea response to either F. graminearum or H. solani. Among these, nine contain CAZymes putatively involved in hemicellulose degradation and seven include putative effectors. Effector proteins in biocontrol fungi are typically necessary to resist and suppress the defense responses of plant hosts in order to allow plant colonization (Mendoza-Mendoza et al., 2018; Nogueira-Lopez et al., 2018; Romero-Contreras et al., 2019). Additionally, these orthogroups include proteases involved in the degradation of the plant cuticle, which is fundamental to initiate defense responses (Xia et al., 2009; Aragón et al., 2017). Orthogroup OG0000113 in particular included homologs of TvPG2, an endopolygalacturonase regulating the induction of plant defense in T. virens (Sarrocco et al., 2017). Yet other orthogroups consisted of trypsin proteases, which are a part of the biological control action of T. atroviride (Grinyer et al., 2005). C. rosea genes from these two orthogroups (CRV2T00016251_1 and CRV2T00014266_1) are both induced during the response to H. solani (Lysøe et al., 2017).

Several proteins with CFEM domains are predicted in the considered Clonostachys species. Such proteins are particularly numerous in fungal pathogens and they often act as cell-surface receptors, signal transducers, adhesion molecules or proteins involved in appressorium formation (Choi and Dean, 1997; Kulkarni et al., 2003; Zhang et al., 2015; Sabnam and Barman 2017). Interestingly, the considered Clonostachys spp. have higher numbers of CFEM proteins compared with Trichoderma species. In non-pathogenic fungi, CFEM can have a role in interactions with plants, and one member is upregulated in T. atroviride during plant host interaction (Guzmán-Guzmán et al., 2017), suggesting that their high number in Clonostachys spp. may be tied to a role in plant host perception and colonization. The high number of CFEM proteins could therefore help Clonostachys spp. to interact with a high variety of plant hosts, possibly by facilitating adhesion. Some of them, however, could also play a part in the interaction with mycohosts. In particular, the transmembrane CFEM protein CRV2T00016013_1 is part of a phylogenetic group evolving for gene gains in Clonostachys and is induced in C. rosea in response to F. graminearum (Demissie et al., 2020). Furthermore, the GPI-anchored protein CRV2T00009137_1 is the only CFEM protein encoded by a gene induced in response to both H. solani and F. graminearum (Lysøe et al., 2017; Demissie et al., 2020), indicating a general function in interspecific fungal interactions. This could be related to the known role of CFEM proteins as signal transducers (Kulkarni et al., 2003; Sabnam and Barman 2017).

This work investigates the composition of the predicted secretome of Clonostachys spp. and highlights its potential role in the mycoparasitic lifestyle and ecological opportunism of these commercially important fungi. Presence of proteins with a known role in fungal antagonism, including the chitinase CHIC2, the LysM protein LYSM2 and the endopolygalacturonase PG2 homolog, as well as several subtilisin-like peptidases and phospholipases A, emphasize the potential contribution of antibiosis to the biocontrol property of Clonostachys. Clonostachys spp. secretomes contained more CAZymes with a predicted function to degrade hemicellulose compared with Trichoderma spp., which however contained more chitinases. This may suggest that Trichoderma spp. are more adapted to degrade the cell wall of their mycohosts but less suited to induce defense reactions on their plant hosts. Moreover, we detected an unexpectedly high number of CFEM proteins in Clonostachys spp., typically more frequently found in plant pathogens, which also highlight the intimate interaction between Clonostachys spp. and plants, with potential consequences for their biocontrol activity. In summary, Clonostachys and Trichoderma species superficially share the same ecological lifestyle as rhizosphere-competent mycoparasites and opportunistic plant mutualists. Together with previous studies (Karlsson et al., 2015; Nygren et al., 2018; Broberg et al., 2021), the current work emphasizes several differences in the genomic characteristics of these two genera that show that convergent evolution resulted in adaptation of different underlying mechanisms for these apparent ecological similarities. This may have important consequences for the commercial exploitation of these fungi for biocontrol applications.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

MD, MK, and DJ conceived and designed the analysis. EP and MG performed the analyses with inputs from MK and MD. EP, MG, DJ, MK, and MD discussed and interpreted the results. EP wrote the first draft of the manuscript. MD and MK revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was financially supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS; grant numbers 2018-01420, 2021-01461), and Carl Tryggers Stiftelse för Vetenskaplig Forskning (CTS 19: 82).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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