G3: Genes|Genomes|Genetics Early Online, published on August 15, 2018 as doi:10.1534/g3.118.200656 1 2 3 4 5 Early diverging insect-pathogenic fungi of the order Entomophthorales possess diverse 6 and unique subtilisin-like serine proteases 7 8 9 Authors 10 Jonathan A. Arnesen1, Joanna Malagocka1, Andrii Gryganskyi2, Igor V. Grigoriev3, Kerstin 11 Voigt4,5, Jason E. Stajich6 (orcid 0000-0002-7591-0020) and Henrik H. De Fine Licht1* 12 (orcid 0000-0003-3326-5729) 13 14 15 1Section for Organismal Biology, Department of Plant and Environmental Sciences, 16 University of Copenhagen, Thorvaldsenvej 40, 1871 Frederiksberg, Denmark. 17 2Department of Biology, Duke University, Durham, North Carolina, USA. 18 3US Department of Energy Joint Genome Institute, Walnut Creek, California, USA. 19 4Jena Microbial Resource Collection (JMRC), Leibniz Institute for Natural Product Research 20 and Infection Biology - Hans Knoell Institute, Adolf-Reichwein-Str.23, 07745 Jena, 21 Germany. 22 5Institute of Microbiology, Friedrich Schiller University, Neugasse 25, 07743 Jena, Germany. 23 6Department of Plant Pathology and Microbiology, University of California, Riverside, 24 California, USA. 25 26 27 *Author for correspondence: 28 Section for Organismal Biology, Department of Plant and Environmental Sciences, 29 University of Copenhagen, Thorvaldsenvej 40, 1871 Frederiksberg, Denmark. Email: 30 [email protected], Tel: +45 35320097 31 32 1 © The Author(s) 2013. Published by the Genetics Society of America. 33 Abstract 34 Insect-pathogenic fungi use subtilisin-like serine proteases (SLSPs) to degrade chitin- 35 associated proteins in the insect procuticle. Most insect-pathogenic fungi in the order 36 Hypocreales (Ascomycota) are generalist species with a broad host-range, and most species 37 possess a high number of SLSPs. The other major clade of insect-pathogenic fungi is part of 38 the subphylum Entomophthoromycotina (Zoopagomycota, formerly Zygomycota) which 39 consists of high host-specificity insect-pathogenic fungi that naturally only infect a single or 40 very few host species. The extent to which insect-pathogenic fungi in the order 41 Entomophthorales rely on SLSPs is unknown. Here we take advantage of recently available 42 transcriptomic and genomic datasets from four genera within Entomophthoromycotina: the 43 saprobic or opportunistic pathogens Basidiobolus meristosporus, Conidiobolus coronatus, C. 44 thromboides, C. incongruus, and the host-specific insect pathogens Entomophthora muscae 45 and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants 46 (Formica polyctena), respectively. In total 154 SLSP from six fungi in the subphylum 47 Entomophthoromycotina were identified: E. muscae (n = 22), P. formicae (n = 6), B. 48 meristosporus (n = 60), C. thromboides (n = 18), C. coronatus (n = 36), and C. incongruus (n 49 = 12). A unique group of 11 SLSPs was discovered in the genomes of the obligate biotrophic 50 fungi E. muscae, P. formicae and the saprobic human pathogen C. incongruus that loosely 51 resembles bacillopeptidase F-like SLSPs. Phylogenetics and protein domain analysis show 52 this class represents a unique group of SLSPs so far only observed among Bacteria, 53 Oomycetes and early diverging fungi such as Cryptomycota, Microsporidia, and 54 Entomophthoromycotina. This group of SLSPs is missing in the sister fungal lineages of 55 Kickxellomycotina and the fungal phyla Mucoromyocta, Ascomycota and Basidiomycota 56 fungi suggesting interesting gene loss patterns. 57 58 Keywords 59 Subtilase; insect-pathogen; early-diverging fungi; proteases; phylogenomics 60 61 62 2 63 1. Introduction 64 Insect pathogenic fungi use a broad array of enzymes to penetrate the host cuticle and gain 65 entry to the soft tissues inside (Charnley, 2003; St. Leger et al., 1986b). In many cases, serine 66 proteases are among the first enzymes to be secreted in the early stages of infection in order 67 to cleave and open up chitin-associated proteins in the procuticle (St. Leger et al., 1986a; 68 Vega et al., 2012), which later is followed by extensive lipase and chitinase enzymatic 69 secretions (Charnley, 2003). In particular, subtilisin-like serine proteases (SLSPs) have been 70 considered important virulence factors in pathogenic fungi (Muszewska et al., 2011). The 71 first SLSPs from insect pathogenic fungi were identified in Metarhizium anisopliae 72 (ARSEF2575), which secretes SLSPs as some of the key proteases during fungal growth on 73 insect cuticle (Charnley, 2003; St. Leger et al., 1986a). Comparative genomic approaches 74 have identified significant expansions of the SLSP gene family in the genus Metarhizium 75 (Bagga et al., 2004; Hu et al., 2014), the insect pathogenic fungus Beauveria bassiana (Xiao 76 et al., 2012), two nematode-trapping fungi Monacrosporium haptotylum and Arthrobotrys 77 oligospora that are able to penetrate the chitinaceous cell wall of soil nematodes (Meerupvati 78 et al. 2013), and dermatophytic fungi such as Arthroderma benhamiae and Trichophyton 79 verrucosum that can cause nail and skin infections in humans and animals (Burmester et al., 80 2011; Desjardins et al., 2011; Martinez et al., 2012; Sharpton et al., 2009). Fungi that are able 81 to utilize chitin-rich substrates, including many insect pathogenic fungi, thus appear to often 82 be associated with a diversified and expanded set of SLSPs. 83 84 Although SLSPs are expanded among insect pathogenic fungi, this group of proteases is 85 ubiquitous among eukaryotic organisms. Most SLSPs are secreted externally or localized to 86 vacuoles, and especially in saprobic and symbiotic fungi SLSPs constitute an important 87 component of the secretome (Li et al., 2017). According to the MEROPS peptidase 88 classification, the S8 family of SLSPs together with the S53 family of serine-carboxyl 89 proteases make up the SB clan of subtilases (Rawlings et al., 2016). The S8 family of SLSPs 90 is characterized by an Asp-His-Ser catalytic triad (DHS triad), which forms the active site 91 and is further divided into two subfamilies S8A and S8B. Subfamily S8A contains most S8 92 representatives, including the well-known Proteinase K enzyme that is widely used in 93 laboratories as a broad-spectrum protease. The S8B SLSPs are kexins and furins which 94 cleave peptides and protohormones (Jalving et al., 2000; Muszewska et al., 2017, 2011). 95 Based on characteristic protein domain architectures and protein motifs surrounding the 96 active site residues, the large S8A subfamily of SLSPs is further divided into several groups 3 97 such as proteinase-K and pyrolysin. Besides these two major groups of proteinase K-like and 98 pyrolisin subfamilies, six new groups of subtilase genes designated new 1 to new 6 have 99 recently been found (Li et al., 2017; Muszewska et al., 2011). The analysis of fungal genome 100 data from a wide taxonomic range has shown that the size of the proteinase K gene family 101 has expanded independently in fungi pathogenic to invertebrates (Hypocreales) and 102 vertebrates (Onygenales) (Muszewska et al., 2017; Sharpton et al., 2009). Closely related 103 invasive human-pathogenic fungi, however, do not show the same expansions and related 104 pathogens and non-pathogens can show the same expansions (Muszewska et al., 2011; 105 Whiston and Taylor, 2016). This suggests that the number of SLSPs that a fungus possess is 106 not directly related to pathogenicity, but instead is associated with the use of dead or alive 107 animal tissue as growth substrate (Li et al., 2017; Muszewska et al., 2011). 108 109 Most anamorphic insect-pathogenic fungi in the order Hypocreales (Ascomycota) are 110 generalist species with a broad host-range capable of infecting most major orders of insects 111 (e.g. M. robertsii and B. bassiana) or specific to larger phylogenetic groups (e.g. the locust- 112 specific M. acridum or the coleopteran pathogen B. brongniartii) (Boomsma et al., 2014; Hu 113 et al., 2014). The above-mentioned inferences of fungal SLSP evolution rely almost 114 exclusively on insights from Ascomycota, and consequently have strong sampling bias 115 towards generalist insect-pathogenic fungi. In contrast, the other major clade of insect- 116 pathogenic fungi in the subphylum Entomophthoromycotina (Zoopagomycota, formerly 117 Zygomycota) consists almost exclusively of insect-pathogens and many are extremely host- 118 specific, naturally only infecting a single or very few host species (Spatafora et al., 2016). 119 The dearth of genomic data for Entomophthoromycotina has previously precluded their 120 inclusion in comparative genomic analyses (De Fine Licht et al., 2016; Gryganskyi and 121 Muszewska, 2014). Here we take advantage of recently available transcriptomic and genomic 122 datasets from four genera within Entomophthoromycotina: the saprobic Basidiobolus 123 meristosporus, the saprobic and opportunistic pathogens, Conidiobolus coronatus, C. 124 thromboides, C. incongruus, and the host-specific insect pathogens Entomophthora muscae 125 and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants 126 (Formica polyctena), respectively. We use phylogenetics and protein domain analysis to 127 show that the obligate biotrophic fungi E. muscae, P. formicae and the saprobic human 128 pathogen C. incongruous, in addition to more “classical” fungal SLSPs, harbor a unique 129 group of SLSPs that loosely resembles bacillopeptidase F-like SLSPs. 130 4 131 2. Materials and Methods 132 Sequence database searches