The Genomics of 3 JoAnne Crouch, Richard O’Connell, Pamela Gan, Ester Buiate, Maria F. Torres, Lisa Beirn, Ken Shirasu, and Lisa Vaillancourt

top ten most important fungal phytopathogens 3.1 Introduction (Dean et al. 2012). Economically important diseases caused by The fungal genus Colletotrichum includes more Colletotrichum are widespread, occurring on than 100 species responsible for anthracnose , beans, strawberries, coffee, chili peppers, foliar blight and rot diseases of nearly every crop cucurbits, potatoes, and countless other culti- grown for food, fiber, and forage worldwide vated plants (e.g., Bergstrom and Nicholson (Cannon et al. 2012b; Hyde et al. 2009). 1999; Hyde et al. 2009; Lees and Hilton 2003; Because of their ubiquity, substantial capacity Legard 2000; Melotto et al. 2000; Prihastuti for destruction, and scientific importance as et al. 2009; Singh and Schwartz 2010; Than model pathosystems, fungi in the genus Collet- et al. 2008; Ureña-Padilla et al. 2002; Varzea otrichum are collectively ranked by the inter- et al. 2002; Waller 1992; Wasilwa et al. 1993; national plant pathology community among the Xie et al. 2010). Colletotrichum postharvest fruit rots are responsible for major economic losses, with severe infections resulting in up to 100 % J. Crouch Systematic Mycology and Microbiology Lab, loss during storage (Prusky 1996). Colletotri- USDA-ARS 10300 Baltimore Ave. Bldg. chum diseases also produce substantial damage 10A, Room 228, Beltsville, MD, on important subsistence crops including lentil, 20705, USA cowpea, yam, , , and cassava e-mail: [email protected] (Adegbite and Amusa 2008; Chona 1980; R. O’Connell Chongo et al. 2002; Finlay and Brown 1993; UMR1290 BIOGER-CPP, INRA-AgroParisTech, Avenue Lucien Brétignières, 78850, Thiverval-Grignon, France E. Buiate e-mail: [email protected] e-mail: [email protected] P. Gan K. Shirasu M. F. Torres Plant Science Center, RIKEN, Yokohama, e-mail: [email protected] Japan e-mail: [email protected] M. F. Torres Functional Genomics Laboratory, Weill Cornell K. Shirasu Medical College, Cornell University, Qatar e-mail: [email protected] Foundation-Education City, Doha, Qatar E. Buiate M. F. Torres L. Vaillancourt (&) L. Beirn Department of Plant Pathology, University of Department of Plant Biology and Pathology, Kentucky, 201F Plant Science Building, Lexington, Rutgers, The State University of New Jersey, 59 KY 40546, USA Dudley Road, New Brunswick, NJ 08901, USA e-mail: [email protected] e-mail: [email protected]

R. A. Dean et al. (eds.), Genomics of Plant-Associated Fungi: Monocot Pathogens, 69 DOI: 10.1007/978-3-662-44053-7_3, Ó Springer-Verlag Berlin Heidelberg 2014 70 J. Crouch et al.

Green and Simons 1994; Moses et al. 1996; substantially elucidated using Colletotrichum Moura-Costa et al. 1993). (Kubo and Takano 2013). Key components of Colletotrichum diseases can negatively the cyclic-AMP, MAP kinase, and calcium- impact many of the most important monocots mediated signaling pathways have been cloned targeted as candidate bioenergy crops, including and characterized from Colletotrichum species switchgrass, miscanthus, maize, sorghum, indi- (e.g., Chen and Dickman 2002, 2004; Dickman angrass, and sugarcane (Crouch 2013; Crouch and Yarden 1999; Ha et al. 2003; Kim et al. and Beirn 2009; Crouch et al. 2009a, b; Cortese 2000; Takano et al. 2000; Warwar and Dickman and Bonos 2012; Dahlberg et al. 2011; Hartman 1996; Yang and Dickman 1997, 1999a, b). et al. 2011; King et al. 2011; Waxman and Today, Colletotrichum species continue to serve Bergstrom 2011a, b; Zeiders 1987). Plants in a as important models for studies of the molecular wide variety of uncultivated terrestrial and and cellular basis of pathogenicity (Kubo and aquatic biomes may also be impacted by Col- Takano 2013; O’Connell et al. 2012; O’Connell letotrichum infections, including forests, grass- and Panstruga 2006; Perfect et al. 1999). lands, prairie, shrub land, savannahs, and deserts (Abang et al. 2006; Ammar and El-Naggar 2011; Crouch 2013; Crouch et al. 2009b; Damm et al. 3.2 Systematics of Colletotrichum 2012a; Dingley and Gilmour 1972; Lubbe et al. 2004; Soares et al. 2009). Colletotrichum is an asexual , with the Colletotrichum occupies a noteworthy place sexual state traditionally classified in the Asco- in the history of plant pathology and mycology. mycete genus Glomerella (; The first description of physiological races and ; ; Glome- cultivar specificity involved the causal agent of rellales) (Réblová et al. 2011). With the adoption bean anthracnose, C. lindemuthianum (Barrus of single name nomenclature for pleomorphic 1911), with that work leading to some of the first fungi established by the 2013 Melbourne Code of resistance breeding efforts using race differen- the International Code of Nomenclature for tials (reviewed in Geffroy et al. 1999). Sub- algae, fungi, and plants (www.iapt-taxon.org/ sequent work with the bean anthracnose nomen/main.php), it is unlikely that the Glome- pathosystem has greatly advanced our under- rella name will continue to be used in the future. standing of the gene-for-gene system (López Although several species in the genus are known et al. 2003; Melotto and Kelly 2001). Work with that produce the teleomorph readily (e.g., the teleomorph of C. gloeosporioides pioneered G. cingulata, G. acutata), Colletotrichum species early investigations of fungal sexual determina- are predominantly observed in the vegetative or tion and development (Lucas et al. 1944, Chilton asexual state, with the sexual morph rarely et al. 1945, Chilton and Wheeler 1949a, b; identified for most species (Vaillancourt et al. Driver and Wheeler 1955; Edgerton et al. 1945; 2000b). Since plant pathologists and mycologists Lucas 1946; Wheeler 1950, 1954; Wheeler et al. working with the fungus typically encounter the 1948; Wheeler and McGahan 1952). In the anamorph, the Colletotrichum name more accu- 1960s and 1970s, Colletotrichum studies were at rately communicates biological information the cutting edge of our understanding of the about the organism. Furthermore, Colletotrichum nature of systemic induced resistance, the is the older of the two genera and has priority chemistry of host defense, and the importance of (1831 vs. 1903; www.mycobank.org). Final res- phytoalexins in the defense response, and they olution of the sole adopted genus name will go enabled purification of elicitor molecules from through the formal channels established by the fungal cell walls for the first time (Kuc 1972; International Subcommission of Colletotrichum Sticher et al. 1997). The development and (www.fungaltaxonomy.org/ subcom- function of melanized appressoria has been missions) to ensure community consensus. In this 3 The Genomics of Colletotrichum 71 chapter we will use Colletotrichum to refer both characterized through multilocus phylogenies to the anamorphic and the teleomorphic phases. (Crouch et al. 2009a; Damm et al. 2009; 2012a, Colletotrichum is the sole member of the b; Weir et al. 2012), yielding a framework for Glomerellaceae, one of three families that col- understanding evolutionary relationships across lectively make up the order in the the genus as a whole (Cannon et al. 2012b). Sordariomycete subclass Hypocreomycetidae (Réblová et al. 2011). Earlier reports suggested Colletotrichum as a sister group to Verticillium 3.3 Colletotrichum Lifestyles (Zhang et al. 2006), but more comprehensive and Modes of Infection research has shown that this inferred relationship reflected insufficient sampling rather than an Fungi in the genus Colletotrichum display a actual close phylogenetic association, as Verti- range of nutritional strategies and lifestyles, cillium is a member of the Plectosphaerellaceae including plant associations that occupy a con- (Cannon et al. 2012a; Réblová et al. 2011; Zare tinuum from necrotrophy to hemibiotrophy and et al. 2000). endophytism. Some species employ a sapro- During the past several years, Colletotrichum trophic lifestyle to obtain nutrients from soil and taxonomy has been the subject of several sub- organic matter. Colletotrichum are also known to stantive revisions. Species concepts are still in a colonize organisms outside the plant kingdom, state of flux, but it is now well-established that including insects and humans. the genus consists of several major monophy- Plant-associated Colletotrichum species typi- letic clades that are referred to as species cally use a melanized appressorium to penetrate aggregates, described by the name and attributes host tissues (Kubo and Takano 2013) (Fig. 3.2). of the most prominent representative species in The melanin is required for appressorial func- the group (O’Connell et al. 2012; Cannon et al. tion, permitting the accumulation of significant 2012b; Fig. 3.1). To date, nine aggregates have turgor pressure that facilitates mechanical pene- been described based on multilocus molecular tration of the host cell wall (Bechinger et al. phylogenetics, namely acutatum, graminicola, 1999; Kubo and Furusawa 1991). The appres- spaethianum, destructivum, dematium, gloeo- sorium also secretes pectinases and cell wall- sporioides, boninense, truncatum, and orbiculare degrading enzymes that are likely to play diverse (Cannon et al. 2012b). Although the Colletotri- roles in preparing the infection court, adhesion, chum aggregates carry no formal taxonomic signaling, and softening the host cell wall rank, they provide a convenient way to connect (Kleemann et al. 2008; Mendgen et al. 1996). widely used, but outdated and overly broad The appressorium of Colletotrichum is morpho- species concepts with the revised taxonomy. For logically and functionally similar to that formed example, the gloeosporioides aggregate consists by Magnaporthe, in spite of the evolutionary of at least 22 species traditionally referred to as distance between these two fungal genera C. gloeosporioides, including C. gloeosporio- (Mendgen et al. 1996). Appressorial ultrastruc- ides sensu stricto (Weir et al. 2012). Under the ture is a taxonomically informative trait in Col- new, more accurate molecular-based taxonomy, letotrichum. Members of the destructivum and C. gloeosporioides sensu stricto is now known to graminicola aggregates have a ‘‘pore wall over- be much less common in the environment than lay’’ structure surrounding the penetration pore previously thought (e.g., Phoulivong et al. 2010; that is similar to that found in Magnaporthe Weir et al. 2012). The aggregate terminology (Howard and Valent 1996), whereas the orbicu- is especially useful for disease diagnostics that lare and gloeosporioides aggregates have a dis- still rely on ITS sequence similarity and/or tinctive cone-shaped structure associated with morphology to identify causal agents. Several the appressorial pore (Fig. 3.2). This cone is of the aggregate groups have been broadly surrounded by the appressorial plasma mem- 72 J. Crouch et al.

Fig. 3.1 Bayesian C. acutatum (21 hosts) Colletotrichum species with C. chrysanthemi (2 hosts) phylogenetic tree of the genome sequence(s) 1 C. walleri (1 host) 0.89 C. cosmi (1 host) 0.25 Colletotrichum described in this chapter 1 C. paxtonii (1 host; monocot) genus, 1 C. simmondsii (6 hosts) showing the evolutionary 0.51 C. sloanei (1 host) 1 1 C. scovillei (1 host)

0.56 C. guajavae (1 host) relationship between the C. indonesiense (1 host) 1 0.25 C. laticiphilum (1 host) four Colletotrichum Key to taxa 0.63 0.83C. nymphaeae (14 hosts) C. brisbanense (1 host) acutatum species with genome Species associated with dicots C. cuscutae (1 host) 0.55 0.35C. lupini (4 hosts) 0.79 aggregate sequences, as described in 0.4 C. tamarilloi (1 host) Species associated with dicots & 1 1 0.42C. limticola (1 host) this chapter. The non-graminicolous monocots C. costaricense (1 host) C. melonis (1 host) phylogeny was constructed Species associated with 1 C. fioriniae (28 hosts; 1 monocot) C. fioriniae BB-NJ10 from DNA sequences of non-graminicolous monocots C. salis (10 hosts) 1 1 C. rhombiforme (2 hosts) Species associated with 1 C. acerbum (1 host) four markers (chitin 0.99 graminicolous monocots 0.47C.kinghornii (1 host; monocot) synthase, actin, b-tubulin 1 0.98 C. phormii (1 host; monocot) C. australe (2 hosts) 1 2, rDNA ITS; 1514-bp 1 C. pyricola (1 host) 1 C. johnstonii (2 hosts) C. godiae (21 hosts) 1 total). The analysis was C. orchidophilum (4 hosts; monocot) performed using BEAST C. pseudoacutatum (1 host) 1 C. trichellum (1 host) for 20 million generations, C. rusci (1 host) 1 C. graminicola M1.001 under the following model: 1 C. navitas (1 host; monocot) 1 C. paspali (1 host; monocot) GTR + i, empirical base 0.9 C. nicholsonii (1 host; monocot) 1 C. miscanthi (1 host; monocot) 0.95 graminicola C. eleusines (1 host; monocot) frequencies, and yule 0.891 C. sublineola (1 host; monocot) aggregate 0.98 C. eremochloae (1 host; monocot) process mode of 1 C. falcatum (1 host; monocot)

1 1 C. hanaui (1 host; monocot) speciation. The analysis is C. jacksonii (1 host; monocot)

1 C. cereale (16 hosts; monocots) summarized as a maximum 1 C. spaethianum (3 hosts; monocots) C. lilii (1 host; monocot) spaethianum 1 clade credibility tree from 0.86C. tofieldae (3 hosts) 1 C. liriopes (1 host; monocot) aggregate 0.52 20,001 trees, with the first C. verruculosum (1 host) 1 1 C. higginsianum IMI349063 2,000 trees ignored (10 %) 1 C. fuscum destructivum 1 0.39 C. tabacum 1 aggregate as burn-in. Posterior C. destructivum C. linicola probability support values 1 C. coccodes 1 C. nigrum 1 C. dematium (5 hosts) are listed on branches. 0.88C. lineola (12 hosts; 3 monocots) 1 dematium Branches with thick lines C. fructi (1 host) 1 C. anthrisci (1 host) aggregate 1 C. spinaciae (4 hosts) were highly supported by C. circinans (4 hosts; 3 monocots) posterior probability C. chlorophyti (2 hosts; 1 monocot) 1 C. fructicola (10 hosts) 0.48C. fructicola values, relative to branches 0.88 C.NARAgc5 nupharicola (2 hosts) 0.7 C. aeschynomenes (1 host) with thin lines. Datasets for C. salsolae (3 hosts) 0.41 0.87 0.94C. alienum (3 hosts) included species were C. aenigma (2 hosts) 0.41 C. queenslandicum (3 hosts) generated from alignments 0.961 C. siamense (12 hosts; 3 monocots)

0.95 C. asianum (2 hosts) C. tropicale (3 hosts) gloeosporioides of sequences from type 1 C. musae (1 host) aggregate specimens, following 0.65 C. gloeosporioides (6 hosts) 0.35 C. alatae (1 host; monocot) Cannon et al. (2012a, b). 0.88 C. xanthorrhoeae (1 host; monocot) 1 C. cordylinicola (1 host; monocot) 1 C. ti (1 host; monocot) Number of hosts is given 0.75 C. aotearoal (15 hosts; 1 monocot) 0.69 0.54 C. psidii (1 host) following the species name 1 1 C. clidemiae (2 hosts) 1 C. kahawae (10 hosts) where known. Hosts are C. theobromicola (8 hosts) dicots except where C. horii (1 host) 1 C. dacrycarpi (1 host) C. constrictum (2 hosts) otherwise noted C. hippeastri (1 host; monocot) 1 0.55 1 1 C. parsonsiae (1 host) C. brasiliense (1 host; monocot) C. novae-zealandiae (2 hosts) 1 C. brassicicola (1 host) 0.671 boninense aggregate 1 C. colombiense (1 host) 1 C. beeveri (1 host)

1 C. torulosum (2 hosts) 1 1 1 0.83C. oncidii (1 host; monocot) C. cymbidiicola (1 host; monocot)

1 C. annellatum (1 host) 1 C. phyllanthi (1 host) C. petchii (1 host; monocot) 0.98 1 C. truncatum (20 hosts; 1 monocot) truncatum aggregate C. cucurmae (1 host; monocot)

1 C. dracaenophilum (1 host; monocot) C. yunnananense (1 host) C. cliviae (1 host; monocot) 0.99 1 C. orbiculare T104 orbiculare aggregate C. lindemuthianum Monilochaes infuscans CBS86996

20.0 brane, and consists of modified cell wall material The cone and the pore wall overlay are both lacking chitin that is similar in structure to the continuous with the cell wall of the penetration pore wall overlay (O’Connell and Ride 1990). peg (Fig. 3.2). The functions of the cone and 3 The Genomics of Colletotrichum 73

Fig. 3.2 Appressoria. a Melanized C. orbiculare appressoria (A) formed on glass. Bar = 10 lm, C = conidia. b Penetration peg (PP) emerging from the base of a C. orbiculare appressorium (A) and entering cucumber epidermal cell wall (CW). Bar = 1 lm. c In C. orbiculare, a cone-shaped elaboration of the appressorial cell wall (arrows) surrounds the penetration pore (asterisk). Bar = 0.5 lm. d In C. higginsianum, a pore wall overlay (arrows) surrounds the penetration pore (asterisk). Bar = 0.5 lm. Photos by Richard O’Connell

pore wall overlay structures are unknown, but necrotrophic Colletotrichum diseases (Liao et al. they may serve to reinforce the penetration pore, 2012; Prusky 1996; Walker 1921). to focus turgor pressure, or to direct secretion of At the opposite end of the plant-associated enzymes and other proteins to the host-pathogen lifestyle continuum are Colletotrichum that col- interface. onize host tissue using intracellular hemibiotro- At one extreme of the spectrum of plant- phy (IH), a stealthy, highly orchestrated and associated lifestyles, some Colletotrichum fungi remarkably effective infection strategy. Intra- rely on a primarily necrotrophic lifestyle to obtain cellular hemibiotrophic Colletotrichum produce nutrients. These species include most of the causal specialized infection structures called primary agents of fruit rots. Necrotrophic Colletorichum hyphae that are used to invade living host cells, do not appear to colonize living host tissue; with or without the initial formation of an instead, they first become established either as infection vesicle (O’Connell et al. 2000; Perfect latent infections confined to unpenetrated et al. 1999; Perfect and Green 2001). Primary appressoria, or as a subcuticular mycelium, before hyphae are thickened or bulbous and are sur- eventually switching to a pathogenic lifestyle, rounded by a host-derived membrane separating killing host tissue in advance of colonization and the fungal cell wall from the living host cyto- inciting significant damage. Colletotrichum spe- plasm (Mims and Vaillancourt 2002; O’Connell cies from the acutatum and gloeosporioides et al. 2000; Perfect et al. 1999; Perfect and aggregates are the most common causes of Green 2001; Wharton and Julian 1996). The host 74 J. Crouch et al.

Fig. 3.3 Three variants of intracellular hemibiotrophy. switch to necrotrophy, with secondary hyphae killing host Row 1 C. destructivum model (C. higginsianum). Bio- cells ahead of infection. Row 3 C. graminicola model. trophic primary hyphae (PH) colonize only one epidermal Biotrophic primary hyphae colonize multiple host cells, cell, followed by a complete switch to necrotrophy, with as with C. orbiculare, but biotrophy persists at the thinner secondary hyphae (SH) killing host cells ahead of advancing colony edge while necrotrophy is confined to infection. Row 2 C. orbiculare model. Biotrophic primary the colony center. AP = appressorium; IV = infection hyphae colonize multiple host cells (first infected cells vesicle. Diagrams by Guillaume Robin dead, later infected cells alive), followed by a complete cell remains alive for a variable period of time O’Connell et al. 1996; Pain et al. 1994; Perfect that may last for just a few hours up to several and Green 2001; Perfect et al. 2001; Wharton days, depending on the interaction (Mims and et al. 2001). These necrotrophic stage hyphae Vaillancourt 2002; O’Connell et al. 2000; Per- secrete substantial amounts of lytic enzymes, fect et al. 1999; Perfect and Green 2001). During resulting in host tissue collapse and symptom biotrophy, the fungus appears to evade plant development (O’Connell et al. 2000). defenses (Vargas et al. 2012). The transient There are at least three major variants of IH symptomless phase is followed by a shift to a utilized by pathogenic Colletotrichum (Fig. 3.3). destructive form of necrotrophic development, The first IH strategy is typified by C. higgin- accompanied by production of a distinct hyphal sianum and other members of the destructivum morphology that is thinner, not surrounded species aggregate. In this model, there is a lim- by a membrane, and exhibits a different wall ited biotrophic phase confined to the first composition (Mims and Vaillancourt 2002; infected epidermal cell, followed by a complete 3 The Genomics of Colletotrichum 75 switch to necrotrophy, marked by development of narrower secondary hyphae, killing of host tissues in advance of fungal colonization, and development of symptoms (Latunde-Dada and Lucas 2007; O’Connell et al. 2004, 2012). The second IH model is employed by members of the orbiculare species aggregate (O’Connell et al. 2000; Perfect et al. 1999; Perfect and Green 2001). Here, the biotrophic phase persists during sequential colonization of several cells. Cells behind the advancing colonization front die gradually, but there is no widespread destruction of cells, and the infection remains symptomless. At some point, the colonizing fungus switches to production of narrow necrotrophic hyphae that kill cells in advance of colonization, and symp- toms appear. The third IH strategy is typified by the graminicola species aggregate (Mims and Vaillancourt 2002; Wharton and Julian 1996; Wharton et al. 2001). The graminicola IH model is initially similar to that of orbiculare IH, but differs at the switch to necrotrophy. In the gra- minicola IH model, narrow secondary necro- trophic hyphae are produced as branches from the thicker primary intercalary hyphae in the Fig. 3.4 Trypan-blue stained hyphae of C. graminicola cells behind the advancing colony front, which colonizing maize leaf sheath epidermal cells during the necrotrophic phase of development. a In the center of the continues to invade new cells biotrophically. colony, the thicker primary hyphae (white arrow) give Thus, in the graminicola model, biotrophy and rise to thinner necrotrophic hyphae (black arrows) that necrotrophy exist simultaneously in different define this stage of development. b The edge of the same parts of the colony (Fig. 3.4). Disease symptoms colony is still being colonized biotrophically, evidenced by the ability of the newly invaded and surrounding cells appear soon after the emergence of necrotrophic to plasmolyze (black arrows). Scale bars = 50 microns. hyphae, as the centers of the colonies collapse Photos by Maria Torres and die. Relatively few Colletotrichum fungi have been subjected to detailed cytological species, especially members of the gloeosporio- analysis, and so it is not clear if these models are ides aggregate (Cannon and Simmons 2002; found in other species aggregates, or even if they Arnold et al. 2003). Similarly, a recent survey of are typical of all members of a single aggregate, wild Arabidopsis thaliana populations identified or of all tissues in a single host. Much more five different Colletotrichum species as foliar work is needed in this area. endophytes (García et al. 2013). Asymptomatic Beyond their notoriety as destructive agricul- colonization of host tissue by Colletotrichum tural pathogens, members of the genus Colleto- endophytes may lead to any of several outcomes trichum are among the most common endophytic for the plant, including enhanced growth, drought fungi associated with plants (e.g., Crouch et al. and heat tolerance, and/or disease resistance 2009c; Gazis et al. 2011; Hyde et al. 2009; (e.g., Arnold et al. 2003; Prusky et al. 1994; Rodriguez and Redman 2008; Rojas et al. 2010; Redman et al. 2001, 2002). Based on environ- Vega et al. 2010). For example, the most com- mental cues such as host senescence, wounding, mon endophytic fungi recovered from asymp- or other factors associated with changes in plant tomatic leaves of forest trees are Colletotrichum physiology (Rodriguez et al. 2009), endophytic 76 J. Crouch et al.

Colletotrichum may also adopt a saprotrophic details of timing dependent on the degree of host lifestyle (Promputtha et al. 2007, 2010), or resistance. Thus, high levels of resistance may induce disease symptoms or rots that are only result in latency/endophytism, while reduced manifested after an extended period of asymp- resistance could lead to IH, and a further reduction tomatic colonization (Freeman et al. 2001; in resistance could trigger a switch to necrotro- Photita et al. 2004; Rodriguez et al. 2009). phy. The duration of the initial biotrophic phase Latency/endophytism in association with plants could also partially explain the spectrum of life- may be an important component of the life cycle styles. The length of the biotrophic phase may be of many or most Colletotrichum fungi, but this dependent on the relative ability of the pathogen aspect of development is poorly understood. In to mask infection structures from detection. True particular, there are relatively few cytological endophytes that colonize extensively without studies and so the degree of host colonization by causing symptoms are likely to have highly Colletotrichum endophytes is usually unknown. developed stealth strategies, for example low It could range from unpenetrated appressoria expression of lytic enzymes, masking of chitin (latency) to extended systemic colonization. and other pathogen-associated molecular pattern Much more work is needed in this area. (PAMP) elicitors, and production of protein and Surveys of foliar epiphytes show that Collet- secondary metabolite (SM) effectors to suppress otrichum fungi are also widespread in the phyll- defense. osphere (Alvinidia and Natsuaki 2008; Freeman et al. 2001; Osono 2007, 2008; Santamaría and Bayman 2005). Several Colletotrichum species 3.3.1 Colletotrichum Genomics function as saprophytes, surviving in organic matter or soil; however, this free-living lifestyle Genome-scale analyses of Colletotrichum strains may be strictly facultative. In general, Colleto- with contrasting lifestyles could help us to trichum appears to be ill-equipped for long term identify commonalities and provide conserved survival in soil (e.g., Bergstrom and Nicholson targets for the management and control of these 1999; Ripoche et al. 2008), although there are fungi. Unfortunately, very few Colletotrichum notable exceptions (e.g., Dillard and Cobb 1998; species have been studied in depth at the Freeman et al. 2002), and melanized microscle- molecular level, and relatively little is known rotia have been observed in several species about those species of the greatest economic including C. truncatum, C. sublineola, and C. importance, or those species that cause the most coccodes (e.g., Boyette et al. 2007; Dillard and significant damage to subsistence crops. Indi- Cobb 1998; Sukno et al. 2008). In addition, vidual findings in one pathosystem have only members of the genus are occasionally reported rarely been validated or tested in other systems, as opportunistic pathogens of organisms outside so it is not clear to what extent mechanisms of the plant kingdom, including insects, turtles, cats, pathogenicity are similar across all lineages. As and humans (Cano et al. 2004; Manire et al. 2002; the genomes of other economically important Marcellino et al. 2008, 2009; O’Quinn et al. plant pathogenic fungi were sequenced, begin- 2001; Shivaprakash et al. 2011; Winter et al. ning with M. oryzae in 2005 (Dean et al. 2005), 2010). Colletotrichum remained in the background of The functional relationships between Colleto- the genomics revolution, primarily because it trichum lifestyles along the plant-associated was difficult to make a case for sequencing any continuum are unclear. The mechanisms regu- single species to represent all the others, without lating the developmental switches, and the role of understanding how the pathogenic models rela- host signals, are also mysterious. It is possible that ted to one another. Problematic taxonomy, and a all of the plant-associated lifestyles are manifes- woefully inadequate understanding of species tations of a similar underlying interaction, with boundaries and evolutionary relationships, also 3 The Genomics of Colletotrichum 77 initially limited our ability to identify the most is one of very few species in the genus in which suitable subjects for genome analysis. sexual crosses can be made (Vaillancourt and As genome sequencing technologies became Hanau 1991). C. graminicola causes one of the faster and cheaper, whole genome sequences most destructive diseases of maize, anthracnose were finally generated and published in quick stalk rot, resulting in annual losses of more than succession for four species of Colletotrichum: C. $1 billion in the United States (Bergstrom and higginsianum, C. graminicola, C. orbiculare, Nicholson 1999; Frey et al. 2011). Sequences for and C. fructicola (formerly C. gloeosporioides; C. graminicola M1.001 (Forgey et al. 1978) and Fig. 3.1; Gan et al. 2013; O’Connell et al. 2012). for M5.001 (aka CBS 130839), a strain that is According to our current understanding of Col- sexually compatible with M1.001 (Vaillancourt letotrichum taxonomy, these four species are and Hanau 1991), are available. each positioned within distinct monophyletic C. higginsianum strain IMI349063 causes lineages in the Colletotrichum phylogenetic tree anthracnose on Brassica and Raphanus crops, as (Fig. 3.1; Cannon et al. 2012a, b). C. gramini- well as wild cruciferous species. Although the cola is a member of the graminicola aggregate. fungus may occasionally cause significant crop C. higginsianum is part of the destructivum losses, C. higginsianum is generally of only aggregate, the sister clade to the graminicola minor importance in commercial agricultural aggregate. C. orbiculare is a close relative of the production (Horie et al. 1988; Lin and Huang bean anthracnose pathogen C. lindemuthianum, 2002). However, this species is of considerable and together they occupy the orbiculare species scientific interest because of its ability to cause aggregate. C. fructicola is a member of the disease on certain ecotypes of the model plant gloeosporioides aggregate, one of the most Arabidopsis (O’Connell et al. 2004; Narusaka diverse groups of Colletotrichum, encompassing et al. 2004). The sequenced strain of C. higgin- numerous species associated with a huge num- sianum was originally isolated from Brassica ber of hosts worldwide (Weir et al. 2012). chinensis, but it also readily infects and causes Although the Nara-gc5 strain was considered a disease in Arabidopsis (O’Connell et al. 2004, member of C. gloeosporioides sensu lato at the 2012). As such, C. higginsianum provides an time the genome sequence was published, recent experimental system in which both host and revisions to the taxonomy of the gloeosporioides fungal partners can be genetically manipulated. aggregate now enable a precise species identi- In particular, the availability of powerful genetic fication (Weir et al. 2012). As shown in the tools and resources on the plant side facilitate genus-wide multilocus phylogenetic tree in the analysis of host resistance and susceptibility Fig. 3.1, Nara-gc5 is a member of C. fructicola, (e.g., Birker et al. 2009; Narusaka et al. 2009). a globally distributed species within the gloeo- C. orbiculare 104-T (aka CBS 514.97, sporioides aggregate that has been isolated from LARS414; formerly known as C. lagenarium)is eight different plant families to date (Weir et al. a common and significant problem on cucurbits, 2012; Prihastuti et al. 2009). Accordingly, in this causing anthracnose lesions on vegetative tissue review we will refer to Nara-gc5 as C. fructicola and fruit (Westcott 2001; Kubo and Takano to reflect the revised, more accurate taxonomy. 2013). At one point, anthracnose was among the C. graminicola strain M1.001 (aka FGSC most common and destructive diseases of 10212, CBS 130836, M2; formerly known as cucumbers and melons in the United States ) was the first species of (Gardner 1918). Today, losses due to C. orbic- Colletotrichum to have a complete genome ulare are kept in check through improved crop sequence available, and is also one of the last of management strategies, although anthracnose the fungal genomes to be substantially remains a prevalent disease of commercial sequenced using Sanger dideoxy technology. C. watermelons grown in regions of high humidity graminicola is among the best characterized and (Maynard and Hopkins 1999). The sequenced most tractable of the Colletotrichum fungi, and strain of C. orbiculare also infects the model 78 J. Crouch et al. plants Nicotiana benthamiana and N. tabacum be made with caution, given that different (Shen et al. 2001), which are amenable to tran- sequencing strategies and methodologies were sient gene expression and silencing assays. used, and different computational tools were Techniques for genetic manipulation of C. employed to assemble and annotate the genomes. orbiculare have been established, including gene Three of the four sequenced Colletotrichum targeting or random gene insertion. In addition, strains, (C. graminicola M1.001, C. higginsia- C. orbiculare pathogenesis is stable and is well num IMI349063, and C. fructicola Nara-gc5), characterized cytologically, making this patho- yielded genome assemblies with estimated sizes system an attractive platform for experimental ranging from 53 to 58 Mb, somewhat larger than studies (Kubo 2012). the average 38 Mb sequenced Pezizomycota C. fructicola strain Nara-gc5 (formerly genome (Table 3.1; Cuomo and Birren 2010; Gan known as C. gloeosporioides) causes crown rot et al. 2013; Kelkar and Ochman 2012; O’Connell of strawberry (Okayama and Tsujimoto 1994, et al. 2012). At 88 Mb, the C. orbiculare 104-T 2007), a disease responsible for substantial los- assembly is considerably larger than the other ses for strawberry producers worldwide. Straw- three Colletotrichum species (Gan et al. 2013). It berry anthracnose can result in up to 80 % losses also dwarfs the genomes of most ascomycetes for nursery plants, and up to 50 % losses in the sequenced to date, surpassed in size only by the field (Howard and Albregts 1983; Xie et al. biotrophic powdery mildew fungi and Tuber 2010). Other members of C. fructicola are melanosporum (Cuomo and Birren 2010; Gan important pathogens of a broad range of com- et al. 2013; Spanu et al. 2010). The large genome mercially grown crops, including coffee, apples, of C. orbiculare strain 104-T is the result of yams, pears, and avocados (Prihastuti et al. blocks of low-complexity AT-rich sequences 2009; Weir et al. 2012). Comparisons between dispersed among the coding sequences, account- members of this species, and within the larger ing for nearly half of the genome assembly (Gan gloeosporioides aggregate, promise to be infor- et al. 2013). These AT-rich sequence blocks may mative in determining factors contributing to have arisen from modification of transposable adaptation to particular hosts and lifestyles. elements by repeat-induced point mutation (RIP; Galagan and Selker 2004). Despite the differences observed in overall 3.3.2 Colletotrichum Comparative genome size, predicted gene numbers were Genomics similar for the four Colletotrichum species, ranging from 12,006 (C. graminicola) to 16,172 Genome assembly statistics for the four (C. higginsianum); all were larger than the sequenced Colletotrichum strains are summa- average set of 11,281 genes observed in other rized in Table 3.1. Differences in the read lengths sequenced Pezizomycota (Table 3.1; Cuomo generated by the different sequencing methods are and Birren 2010; Gan et al. 2013; O’Connell reflected in the quality of the resulting genome et al. 2012). The reduced number of genes assemblies, with short read technologies produc- observed in C. graminicola, relative to the other ing more fragmented assemblies than those that three sequenced Colletotrichum genomes, was incorporated Sanger and Roche-generated data. largely due to the presence of fewer gene para- Despite the differences in sequencing approaches, logs, with the C. graminicola genome appearing overall gene coverage was high for all four to have undergone less gene duplication. In assemblies (Table 3.1; Gan et al. 2013; O’Con- particular, more than twice as many multicopy nell et al. 2012) when assessed using the CEGMA genes were identified in the C. fructicola gen- pipeline (Core Eukaryotic Genes Mapping ome as in that of C. graminicola (Table 3.1). Approach; Parra et al. 2007). Nonetheless, direct A remarkably low level of synteny was obser- comparisons between these four genomes should ved among the four sequenced Colletotrichum 3 The Genomics of Colletotrichum 79

Table 3.1 Genome assembly statistics for the four sequenced Colletotrichum species (O’Connell et al. 2012; Gan et al. 2013) C. higginsianum C. graminicola C. fructicola C. orbiculare Assembly size (Mb) 53.4 57.4 55.6 88.3 Coverage 101x 9.1x 37x 55x Sequencing Roche 454 (25x) Sanger (7.9x) Illumina (37x) Roche 454 (22x) technology Illumina (76x) Roche 454 (1.2x) Illumina (34x) Sanger (0.2x) Number of scaffolds 653 367 1241 525 N50 contig length 265.5 579.2 112.8 428.9 Number of contigs 10269 1151 5335 10545 Gene space coverage 95.1 % 99.2 % 96.4 % 98.0 % Number of predicted 16172 12006 15469 13479 genes Overall GC content 55.1 % 49.1 % 53.6 % 37.5 % GC content of genes 58.4 % 59.3 % 56.0 % 57.1 % Number of 10 major, 2 ‘‘B’’ 10 major, 3 ‘‘B’’ Unknown 10 major, no ‘‘B’’ chromosomes chromosomes chromosomes chromosomes Multicopy genes 9713 6468 14933 7475 Conserved single copy 4725 4767 372a 4553 genes Repeat elements 1.2 % 12.2 % 0.75 % 8.3 % Public access to NCBI (Accession NCBI (Accession NCBI (Bioproject NCBI (Bioproject genome CACQ02000000); ACOD01000001); PRJNA171218) or PRJNA171217) or Broad Institute Broad Institute the Dryad Digital the Dryad Digital website website Repository Repository (doi:10.5061/ (doi:10.5061/ dryad.r4026) dryad.r4026) The number of multicopy genes was determined by clustering the predicted proteins within each genome using MCL and an inflation value of 2.0. To estimate the numbers of fungal conserved genes, BLASTp was performed against the 11 other fungal genomes in addition to the other three Colletotrichum genomes, with a cutoff of 1E-5 a The low number of conserved genes in C. fructicola is likely to be an artifact resulting from the much shorter average read length for that genome genomes, much less than that displayed between M1.001 genome as a reference for assembling members of two different genera (Botrytis and other Colletotrichum species may be limited. Sclerotinia; Amselem et al. 2011). Synteny In marked contrast with the low degree of between C. graminicola and C. higginsianum was synteny documented between Colletotrichum only 35 %, while the more distantly related C. species, intraspecific chromosomal rearrange- orbiculare and C. fructicola shared only 40 % ments may be rare. Thus, two strains of C. synteny (Gan et al. 2013; O’Connell et al. 2012). graminicola, one isolated in North America in These low levels of shared gene order, which 1972 (M1.001), and a second strain isolated in appear to be independent of the degree of taxo- South America in 1989 (M5.001), appeared to nomic relatedness, suggest that major genome be highly syntenic with relatively few sequence rearrangements have been a common feature dur- polymorphisms (O’Connell et al. 2012). This ing the history of the Colletotrichum genus. indicates that major chromosomal rearrange- Unfortunately, this also means that the value of the ments within species may be uncommon. This high quality assembly of the C. graminicola may also suggest that genome rearrangements 80 J. Crouch et al. play a role in speciation, perhaps by promoting 50 % of the unanchored scaffolds were com- reproductive isolation (Aguileta et al. 2009). posed of predicted TE sequences, while the primary chromosome assemblies contained only 5.5 % repetitive DNA. In the C. graminicola 3.4 Repetitive DNA genome, there was a statistically significant correlation between the location of TEs and Prior to the availability of genomic resources, paralogous gene families, genes encoding relatively little was known about Colletotrichum secreted proteins, and genes without orthologues transposable elements (TEs) and their impact on in C. higginsianum (O’Connell et al. 2012). the host genome. Even with the availability of Similarly, in the C. orbiculare genome, AT-rich genome assemblies, Colletotrichum TEs have blocks, which may represent relics of TEs been subject to little detailed analysis, but some mutated through repeat-induced point (RIP) general characterizations are possible. All four mutation, were associated with small unique sequenced Colletotrichum genomes contained secreted protein genes (Gan et al. 2013). signatures of common long terminal repeat Unfortunately, the C. fructicola and C. higgin- (LTR) and DNA transposon classes. Summary sianum assemblies were too fragmented to per- data showed that the percentages of these form a similar analysis. The observed transposon repetitive sequences were higher for C. gra- clustering in Colletotrichum may reflect selec- minicola and C. orbiculare (12.2 and 8.3 %, tion against harmful integration into gene-rich respectively) than for C. higginsianum (1.2 %) regions, as described for Saccharomyces cere- or C. fructicola (0.75 %), but this may be an visiae Ty3 LTR elements (Voytas and Boeke artifact derived from the more complete genome 1993). There is no obvious evidence that TE assemblies and sequencing strategies used for C. integration has played a role in the duplication graminicola and C. orbiculare (Gan et al. 2013; of adjacent genes. Regardless of the mecha- O’Connell et al. 2012). Several TE sequences in nism(s) responsible for TE clustering, the the Colletotrichum genomes were similar to the observed patterns suggest that TEs may play a Ccret1, Ccret2, Ccret3, and Cgret Metaviridae role in the generation of effector diversity and family LTR retrotransposons, the non-LTR novel genes in Colletotrichum. Further research LINE-like retroelement CgT1, and the Collect1 is needed to investigate these possibilities. DNA TE sequences described previously from TEs populating the genomes of C. gramini- C. cereale and C. gloeosporioides (Crouch et al. cola, C. fructicola, and C. orbiculare showed the 2008; He et al. 1996; Zhu and Oudemans 2000). signature of widespread RIP mutation, Clustering of TEs in the context of rapidly consistent with the mutation patterns docu- evolving genome regions undergoing high rates mented from elements described previously of duplication is a trait that Colletotrichum holds from C. cereale (Crouch et al. 2008). TpA and in common with other phytopathogenic asco- ApT dinucleotides were both amplified in the mycetes, including M. oryzae, Verticillium oxy- TEs of all three genomes, with the correspond- sporum, and V. dahlia (Amyotte et al. 2012; Gan ing depletion of CpA, CpG, and CpC dinucleo- et al. 201; Hua-Van et al. 2000; O’Connell et al. tides resulting in the canonical footprint of the 2012; Thon et al. 2006). In C. graminicola, TEs RIP process as first described in Neurospora were organized in clusters distributed through- crassa (Cambareri et al. 1989). Comparative out the genome. C. graminicola supernumerary genome profiles between C. graminicola and 48 minichromosomes (see below) and unanchored additional filamentous ascomycetes showed that scaffolds were particularly enriched in TEs, RIP distortion of dinucleotides exhibited by C. relative to the ten primary chromosomes. graminicola TEs was exceptionally pronounced Almost 23 % of the three minichromosomes and (Clutterbuck 2011). 3 The Genomics of Colletotrichum 81

3.5 Supernumerary Chromosomes graminicola (Rollins 1996). In both Colletotri- chum genomes, the minichromosomes possessed Supernumerary minichromosomes (aka B-chro- such poor quality sequence assemblies that it mosomes) are a common feature in the genus was not possible to determine if they were Colletotrichum. These small chromosomes are enriched for pathogenicity genes. typically conditionally dispensable for growth in fungi, and highly variable from one strain to the next (Covert 1998; Stukenbrock et al. 2010). In 3.6 Mating Type Genes some fungi, including Alternaria alternata, Cochliobolus heterostrophus, Fusarium oxyspo- Sexual reproduction is rarely documented from rum, M. oryzae, Mycosphaerella graminicola, the genus Colletotrichum. Some species for and Nectria haematococca, minichromosomes which the Glomerella morph has never been are enriched in secreted genes that encode pro- observed in nature, including C. graminicola, teins involved in niche or host adaptation have been induced to mate in the laboratory (Chuma et al. 2003; Coleman et al. 2009; Hatta (Politis 1975; Vaillancourt and Hanau 1991). et al. 2002; Ma et al. 2010; Stukenbrock et al. The genetics underlying Colletotrichum mating 2010). For several fungi, horizontal transfer of are perplexing, in that fungi in this genus do not minichromosomes has been demonstrated, often employ the canonical bipolar mating system resulting in expanded pathogenicity on new characteristic of other ascomycete fungi hosts (Mehrabi et al. 2011). In Colletotrichum (Vaillancourt et al. 2000a, b). In the standard for example, minichromosomes of C. gloeospo- bipolar model used by most ascomycetes to rioides sensu lato infecting Stylosanthes guian- regulate sexual compatibility, mating can occur ensis were transferred between different strains when both idiomorphs of the mating type gene, of the fungus, conferring novel pathogenicity Mat1, are present (Mat1-1 and Mat1-2). This (Masel et al. 1996). The sequenced strains requirement may be met in a single homothallic of C. higginsianum and C. graminicola are individual carrying both idiomorphs, or in a known to possess minichromosomes (Table 3.1; combination of two heterothallic individuals, O’Connell et al. 2012). However, the mini- each carrying one of the two different idiom- chromosomes in each case were very poorly orphs (Ni et al. 2011). Colletotrichum does not assembled due to high levels of repetitive conform to this system. To date, only the Mat1-2 sequences, extensive tracts of AT-rich sequen- idiomorph, with the characteristic conserved ces, and reduced gene density relative to the rest high mobility group (HMG) binding domain, is of genome. Although it is not known whether C. known from any Colletotrichum species sur- fructicola has minichromomes, C. fructicola veyed, regardless of whether the strains are sequences shared some similarity with those heterothallic or homothallic (Crouch et al. 2006; documented from the minichromosomes of Du et al. 2005, Rodríguez-Guerra et al. (2005); another member of the gloeosporioides aggre- García-Serrano et al. (2008); Vaillancourt et al. gate pathogenic to S. guianensis (Gan et al. 2000b). However, genetic evidence does point to 2013; Masel et al. 1996). In contrast, C. gra- at least two unlinked loci acting as mating minicola M1.001 minichromosome sequences determinants in crosses involving C. gramini- did not match the genome sequence from a cola strains M1.001 and M5.001 (Vaillancourt second strain of the same species, M5.001 et al. 2000a). (O’Connell et al. 2012), confirming earlier Early attempts to identify the Mat1-1 gene hybridization analyses that showed that the were made by using Southern hybridizations, minichromosomes were not conserved between degenerate primer pairs, and primer walking in the M1.001 and M5.001 strains of C. cosmid libraries. These experiments, performed 82 J. Crouch et al. by multiple laboratories, focused on detection of genes encoded by C. fructicola, C. graminicola, the highly conserved alpha DNA binding and C. orbiculare (726-, 840-, and 750-bp, domain that characterizes the Mat1-1 idiomorph respectively). C. fructicola and C. orbiculare in other ascomycete fungi. None of these share 66 % nucleotide identity at the Mat1-2 approaches provided any evidence for the pres- locus, consistent with their closer evolutionary ence of a Mat1-1 gene, even from homothallic relationship (Fig. 3.1). The more distantly rela- strains of Colletotrichum (Crouch et al. 2006; ted C. graminicola shared 51 % identity with C. Du et al. 2005, Rodríguez-Guerra et al. 2005; fructicola and C. orbiculare at this locus. How- García-Serrano et al. 2008). BLAST searches of ever, the C. higginsianum Mat1-2 coding the four Colletotrichum genome sequences sequence displays extensive sequence diver- confirmed the absence of any sequence with gence relative to the other three species, sharing significant identity to the Mat1-1 gene. The only 30–35.5 % identity. Differences between Mat1-1 gene was not found in the genomes of Mat1-2 encoded by C. higginsianum and the C. graminicola strains M1.001 and M5.001, other three Colletotrichum species is attributable even though these two strains can be mated to the presence of numerous insertions through- in vitro to produce fertile progeny (Vaillancourt out the predicted coding sequence—between 193 and Hanau 1991; Vaillancourt et al. 2000a). and 319 nucleotide gaps. Evaluation of 90 genes proximal to the Mat1- Despite the high level of interspecific differ- 2 gene (*134 Kb) showed that C. graminicola ences in the coding sequences of the Mat1-2 strains M1.001 and M5.001 share 99.8 % genes, the region surrounding the Mat1 locus nucleotide sequence similarity in genic regions shows a high level of conserved synteny (99.6 % overall for the region), and the ordering between C. fructicola, C. graminicola, and C. and orientation of genes in this region were orbiculare. Comparison of the genes proximal to identical between the two strains. The Mat1-2 the Mat1-2 locus shows that this region is 100 % gene is highly conserved between M1.001 and conserved in gene content and gene order in M5.001, sharing 99.5 % nucleotide identity. these three Colletotrichum species (Msa1/Cia30/ Only four of the 840 nucleotides vary between Apc5/Cox13/Apn2/Mat1/Sla2/L21e 60 s ribo- these two strains of C. graminicola, and none of somal protein/S4-9 40 s ribosomal protein /Slu7/ the Mat1-2 base changes are located within the Rev3/Tex2/Ami1). A similar comparison with the HMG box DNA binding domain. Three of the C. higginsianum genome could not be com- four variable Mat1-2 nucleotides are located in pleted, as the genome assembly is fragmented, the first intron, while the fourth base change with no more than three genes on a single contig, introduces an amino acid change from aspara- several genes incomplete/truncated, and some gine in M1.001 to aspartic acid in M5.001 in genes predicted that seem unlikely to actually exon 2. The M5.001 aspartic acid residue at this exist. site is also found in C. higginsianum IMI 349063, while the asparagine residue of M1.001 is also found at this site in the Mat1-2 genes of 3.7 Expanded Gene Families C. lindemuthianum and C.gloeosporioides (Du et al. 2005; García-Serrano et al. 2008). Several gene families are expanded in the gen- Pairwise comparisons of the Mat1-2 coding omes of the four sequenced Colletotrichum sequence from the four sequenced Colletotri- strains, relative to other sequenced ascomycetes. chum genomes show that outside of the con- Expansions included genes predicted to encode served HMG-box, the coding sequences are quite carbohydrate-active enzymes (CAZymes), sec- different among the four species. The Mat1-2 ondary metabolism (SM) enzymes, secreted exons are considerably longer in the C. higgin- proteases, and putative secreted effectors (Gan sianum genome: 987-bp, relative to the smaller et al. 2013; O’Connell et al. 2012). 3 The Genomics of Colletotrichum 83

70 60 50 40 30 20 10 0

Fig. 3.5 Relative numbers of genes encoding CAZymes Fig. 3.6 Relative numbers of genes encoding putative targeting different plant cell wall components in Collet- SM-related enzymes in Colletotrichum, and in other otrichum, and in other related fungi with various fungi with various lifestyles. Black bars PKS and PKS- lifestyles. Black bars pectin. Gray bars pectin and like, Gray bars NRPS and NRPS-like, Speckled bars hemicellulose. Speckled bars hemicellulose. White bars PKS-NRPS hybrids, White bars DMATs. Aspergillus cellulose. Blumeria graminis (biotroph); Ustilago maydis nidulans, saprophyte; Stagonospora nodorum, hemibio- (biotroph); Botrytis cinerea (necrotroph); Sclerotinia troph; Sclerotinia sclerotiorum, necrotroph; Magnapor- sclerotiorium (necrotroph); Fusarium oxysporum (hemi- the oryzae, hemibiotroph; Neurospora crassa, biotroph); Fusarium graminearum (hemibiotroph); Col- saprophyte; Fusarium graminearum, hemibiotroph; Usti- letotrichum fructicola (hemibiotroph); Colletotrichum lago maydis, biotroph; Colletotrichum graminicola, orbiculare (hemibiotroph); Colletotrichum higginsianum hemibiotroph; Colletotrichum higginsianum, hemibio- (hemibiotroph); Colletotrichum graminicola (hemibio- troph; Colletotrichum fructicola, hemibiotroph; Colleto- troph); Neurospora crassa (saprophyte); Magnaporthe trichum orbiculare, hemibiotroph. Data from Gan et al. oryzae (hemibiotroph). Data from Gan et al. (2013), and (2013); O’Connell et al. (2012) Pamela Gan

reflecting an important adaptive trait for these CAZYmes Expanded enzyme arsenals capable pathogens. In particular, C. orbiculare and of degrading cellulose and other polysaccharides C. fructicola each encode more than 100 different contained within plant cell walls are a common pectinases, greatly surpassing other sequenced theme for hemibiotrophic and necrotrophic plant fungal genomes (Fig. 3.5). The genome of the pathogens, including Colletotrichum species, M. maize pathogen, C. graminicola, possesses a oryzae and Fusarium species (Cuomo et al. reduced cohort of pectinase genes compared with 2007; Dean et al. 2005; Ma et al. 2010). The the other Colletotrichum species: on average, expansion of cell wall degrading enzymes is a 46 % fewer than the dicot-infecting species. Also defining feature of the four Colletotrichum consistent with overall pectinase abundance, gene genomes (Fig. 3.5). The overall abundance of expression profiles during the necrotrophic phase these proteins in Colletotrichum is unmatched in revealed that 51 pectinases were deployed by any ascomycete sequenced to date, even the C. higginsianum during necrotrophy, versus only destructive necrotrophic gray and white rot fungi sixteen utilized by C. graminicola (O’Connell Botrytis cinerea and Sclerotinia sclerotiorum et al. 2012). (Fig. 3.5) (Amselem et al. 2011; Gan et al. 2013; Secondary metabolism genes. Another highly O’Connell et al. 2012). expanded class of genes in the Colletotrichum In dicots, pectin comprises approximately genome encodes putative SM enzymes 35 % of the cell walls, while the cell walls of (Fig. 3.6). SM enzymes are low molecular monocots such as maize generally contain only weight molecules that are not essential for 10 % pectin (Vogel 2008). There was an extre- growth and survival of the organism, but may mely large number of unique pectinases encoded become important for niche adaptation. Pro- by the genomes of the dicot-infecting C. higgin- duction of these metabolites is often associated sianum, C. fructicola, and C. orbiculare, likely with successful competition for host resources 84 J. Crouch et al. through toxic and/or inhibitory effects on other (PKS) and non-ribosomal peptide synthetases organisms (Bölker et al. 2008; Shwab and Keller (NRPS), resulted in decreased pathogenicity in 2008). There are numerous examples of SM C. graminicola, providing support for the idea genes that function in this manner, including the that SM genes play an important role in the well-known trichothecene gene cluster of regulation of pathogenicity to maize (Horbach Fusarium graminearum and the AAL toxin et al. 2009). Studies have also shown that some cluster of tomato-infecting Alternaria alternata Colletotrichum fungi, including C. gloeosporio- (Akagi et al. 2009; Procter et al. 2009). Large ides, are able to synthesize the plant growth numbers of SM-associated genes are usually hormone auxin, which is likely to be important found in the genomes of necrotrophic plant in host manipulation (Chung et al. 2003; Rob- pathogens (Amselem et al. 2011), and SM inson et al. 1998). All four sequenced Colleto- enzymes are often implicated as phytotoxins trichum species contain genes with the potential with direct roles in pathogenicity (e.g., Daub to encode for production and efflux of auxin 1982; Gengenbach et al. 1973; Matthews et al. (Gan et al. 2013; O’Connell et al. 2012). Genes 1979; Scott-Craig et al. 1992). In contrast, a for synthesis of auxin via an IAM intermediate reduced cohort of SM genes is commonly are present in C. fructicola and C. graminicola. observed in biotrophic fungi such as Blumeria Auxin synthesis by C. higginsianum and C. o- graminis (Spanu et al. 2010) and Ustilago biculare may occur via a different intermediate. maydis (Kämper et al. 2006; Bölker et al. 2008). Genes encoding putative polyketide syn- Relatively little is known about the role of thases (PKS) and PKS-like genes and dimeth- SM in hemibiotrophic plant pathogens (Colle- ylallyl transferases (DMATs) are especially mare et al. 2008; Böhnert et al. 2004). Colleto- abundant in the four sequenced Colletotrichum trichum species have been reported to produce a genomes (Gan et al. 2013; O’Connell et al. variety of SM genes, including flavones, pep- 2012). C. higginsianum and C. fructicola pos- tides, and terpenes, as well as the polyketide- sess many more PKS genes, in particular, than derived DHN (1,8-dihydroxynaphthalene) mel- any other sequenced fungi (although it is anin, an essential requirement for appressorium- important to point out that these two assemblies mediated host penetration (Kubo et al. 1991; had the lowest qualities, and therefore PKS Singh et al. 2010). Additional examples include numbers may be somewhat inflated by gene the siderophore ferricrocin, isolated from C. fragmentation). Apart from genes involved in gloeosporioides, which has phytotoxic activity the production of melanin, few of the predicted in grass cotyledons (Ohra et al. 1995), colleto- SM genes have orthologs outside of the Collet- trichins A, B, and C from C. nicotianae, which otrichum genus, and many appear to be specific produce symptoms resembling tobacco anthrac- to individual Colletotrichum strains. nose when infiltrated into tobacco leaves (God- In all fungal species, SM genes tend to be dard et al. 1976; Kimura et al. 1977, 1978; organized into clusters; often these clusters García-Pajón and Collado 2003), and a tetra- include additional enzymes, cytochrome P450 hydroxylated compound with antioxidant prop- genes, transcription factor genes, and transporter erties from C. gloeosporioides (Femenía-Ríos genes (Shwab and Keller 2008). The number of et al. 2006). Several secondary metabolites have SM clusters predicted from the four sequenced been characterized from C. graminicola, Colletotrichum strains is exceptionally high, including the antifungal compounds monorden ranging from 42 clusters in C. orbiculare to 56 and monicillins I, II, and III (Wicklow et al. clusters in C. fructicola. This abundance of SM 2009), and mycosporine-alanine, a spore ger- clusters is substantially greater than other plant mination inhibitor (Leite and Nicholson 1992). pathogenic fungi sequenced to date (Gan et al. It was recently reported that deletion of PPT1,a 2013; O’Connell et al. 2012). Many of the gene encoding a cofactor essential for the Colletotrichum SM clusters are not well con- enzymatic function of all polyketide synthases served among the four species (Table 3.2). 3 The Genomics of Colletotrichum 85

Table 3.2 Secondary metabolite gene clusters in Colletotrichum (O’Connell et al. 2012; Gan et al. 2013) C. orbiculare C. fructicola C. higginsianum C. graminicola Number of secondary metabolite clusters 42 56 43 44 With homologs in –352626 C. orbiculare With homologs in 29 – 26 24 C. fructicola With homologs in 22 21 – 20 C. higginsianum With homologs in 23 26 20 – C. graminicola Clusters were identified with a combination of SMURF prediction (www.jcvi.org/smurf/index.php) and manual annotation. A cluster was defined as being conserved if the best BLAST hit of at least two members within the cluster belong to a single cluster of another species (except where two genes match a single gene as happens to be the case for some of the hybrid enzymes). It should not be assumed that there is a one-to-one relationship in the number of conserved clusters because of the duplication or split of some of the clusters. For example, there are two noncon- tiguous C. higginsianum ACE1/SYN2 homologous clusters but only one of these clusters was detected in C. orbic- ulare and C. fructicola

3.7.1 The Colletotrichum Secretome S8A) that are known in some other pathosystems to function as effectors (Prusky et al. 2001; Fungal secreted proteins are known to have many Olivieri et al. 2002). The relative expansion of important roles in plant-fungal interactions, these in C. fructicola, C. higginsianum, and C. including making plant nutrients accessible to the orbiculare, but not in C. graminicola, may fungus, or inducing host cell susceptibility or host reflect the narrower host range of the latter cell death (Choi et al. 2010; Lowe and Howlett species. Some of the subtilisins appear to cluster 2012). The predicted secretomes of the four more closely with those of plant origin, sug- sequenced Colletotrichum fungi are large and gesting that they could have been acquired by diverse, ranging in size from 1650 proteins for C. horizontal transfer from their plant hosts (Gan graminicola (14 % of the proteome) to 2356 et al. 2013; Jaramillo et al. 2013). proteins for C. fructicola (15 % of the proteome) The Colletotrichum secretomes contain (Table 3.3). This is comparable to the predicted homologs of genes that encode known fungal average of 1798 secreted proteins for Pezizomy- pathogenicity effectors, including several cotina (Choi et al. 2010). Hemibiotrophic patho- necrosis-inducing proteins (Fellbrich et al. 2002; gens typically have a larger percentage of their Gijzen and Nuernberger 2006; Kanneganti et al. genomes ([10 %) devoted to secreted proteins 2006), and the biotrophy-associated BAS2 and than other fungi, and Colletotrichum fits this BAS3 proteins from M. oryzae (Mosquera et al. pattern (Lowe and Howlett 2012). Approximately 2009) (Table 3.4). Genes encoding members of 1,500 secreted protein genes were shared by all the necrosis- and ethylene- inducing peptide four Colletotrichum species. Many other secreted (NEP) 1-like protein family (Gijzen and Nu- proteins appeared to be species-specific, includ- ernberger 2006) were identified in C. higgin- ing 248 found only in C. orbiculare, 225 specific sianum (Kleemann et al. 2012). Only three of to C. fructicola, 227 found only in C. higginsia- these were able to cause cell death in N. benth- num, and 123 specific to C. graminicola. amiana: the others lacked crucial amino acids Compared with other sequenced ascomyce- and did not function to induce necrosis (Klee- tes, the three dicot-infecting species of Colleto- mann et al. 2012). Two NEP protein genes in C. trichum were highly enriched in genes encoding orbiculare also did not contain necrosis-induc- secreted proteases, particularly the serine prote- ing motifs (Gan et al. 2013). These two had peak ases known as subtilisins (MEROPS family expression levels in early biotrophy. 86 J. Crouch et al.

Table 3.3 Summary of Colletotrichum secretomes (compiled by Ester Buiate) C. graminicola C. higginsianum C. fructicola C. obiculare Total proteins 12006 16159 15463 13479 Secreted proteinsa 1650 2142 2356 2149 Percent secreted 14 13 15 16 SSPb 687 1173 933 925 Cysteine-rich SSPc 204 366 333 373 Species-specific cysteine-rich SSPd 32 13 42 88 a Secreted proteins, predicted by WoLF PSORT (www.wolfpsort.org) b SSP = small secreted proteins, 300 bp or less c Cysteine-rich SSP, [3 % cysteine d Species-specific: no BLAST hits to the other Colletotrichum species, or to the NCBI nr database with a cutoff of 1e-5

Table 3.4 Homologs of conserved some effectors in Colletotrichum Gene Accession C. graminicola C. higginsianum C. orbiculare C. fructicola CgDN3 AAB92221.1 0 2 1 1 NPP1 EGZ24512.1 3 4 7 7 NEP1 AF036580.1 4 5 7 9 NIS1 BAL70334.1 1 1 2 2 ToxB AAO49374.1 0 0 1 0 MSP1 AAX07670.1 24 22 23 26 CIH1 AJ271296.1 2 2 3 2 SIX1 ACY39281.1 0 0 5 0 SIX5 ACN87967.1 0 0 1 1 SIX6 ACN69116.1 0 1 2 0 BAS2 ACQ73207.1 2 3 2 1 BAS3 ACQ73208.1 1 1 0 0 Ctnudix HO663661.1 1 4 2 2 Conservation was determined by BLASTp searches with a cutoff of 1e-5. (Ester Buiate, and Gan et al. 2013; Kleemann et al. 2012; O’Connell et al. 2012)

A screen for C. orbiculare proteins that (Stephenson et al. 2000). During infection, the induced cell death in N. benthamiana led to the CgDN3 transcript accumulated in biotrophic identification of NIS1, a protein secreted by infection vesicles. CgDN3 knockout mutants primary hyphae (Yoshino et al. 2012). Cell death failed to penetrate or form primary infection induced by NIS1 is mediated by interaction with hyphae, and they rapidly induced localized cell the plant heat shock protein 90 (Hsp90), known death. Homologs of CgDN3 were found in the to be important in R-gene mediated HR- genomes of C. fructicola, and also in C. orbicu- response (Zhang et al. 2010). Homologs of the lare, and C. higginsianum, but not in C. gra- NIS1 effector gene are found in all four minicola. The C. orbiculare and C. higginsianum sequenced Colletotrichum species. homologs suppressed cell death induced by the Screening of an EST library derived from necrosis-inducing effector NIS1 when they were nitrogen-starved mycelium of C. gloeosporioides transiently expressed in N. benthamiana leaves resulted in the identification of CgDN3, a gene (Kleemann et al. 2012; Yoshino et al. 2012). predicted to encode a small secreted protein that Another conserved secreted effector is the is required for the successful establishment of Nudix hydrolase previously identified as an this pathogen on Stylosanthes guianensis leaves induced gene during the transition to 3 The Genomics of Colletotrichum 87 necrotrophy in the cowpea anthracnose pathogen suggesting that the annotated effectors in the four C. truncatum (Bhadauria et al. 2013). Overex- Colletotrichum genomes may represent only the pression of CtNudix in C. truncatum induced tip of the iceberg. localized host cell death and loss of pathoge- Overall, the genome sequences of the hemi- nicity. Localization studies in N. benthamiana biotrophic Colletotrichum fungi are more similar indicated that the protein is located in the plant to the genomes of necrotrophic fungi rather than plasma membrane, suggesting that it might alter biotrophs, having expanded families of second- integrity of host cells by affecting stability of the ary metabolites and CAZymes. Indeed, Colleto- host plasma membrane. Homologs of the Nudix trichum fungi may have some of the largest and effector are present in other hemibiotrophic most diverse repertoires of lytic enzymes and pathogens including M. oryzae, and P. infestans, secondary metabolites yet found among the but absent in biotrophic and necrotrophic pathogenic fungi. In common with necrotrophs, pathogens, suggesting that it might be important Colletotrichum also encode secreted toxin specifically for this lifestyle. effectors associated with the induction of cell Compared with other sequenced ascomyce- death. At the same time, Colletotrichum also tes, all four Colletotrichum genomes contain an encode large and diverse repertoires of putative expanded family of genes encoding proteins small, lineage-specific secreted effectors, a hall- containing CBM50 carbohydrate binding mod- mark of biotrophic fungal genomes, that may ules, also known as LysM motifs. These genes have a similar function, to manipulate host appear to be highly divergent among the species defenses and induce compatibility. We can and thus to be evolving rapidly. They may act as speculate that this combination of gene arsenals chitin-binding lectins and serve to ‘‘mask’’ the reflects the ‘‘schizophrenic’’ hemibiotrophic biotrophic hyphae from host recognition by existence of Colletotrichum, in which they must binding to the fungal wall chitin (de Jonge and function almost as two distinct organisms at Thomma 2009). In C. lindemuthianum, a LysM different stages of their lifecycles. protein called CIH1 was localized to the surface of biotrophic hyphae using a monoclonal anti- body (Pain et al. 1994; Perfect et al. 1998). All 3.7.2 Colletotrichum Transcriptomics four sequenced species have homologs of CIH1. During Biotrophy Biotrophic plant pathogens are known to and Necrotrophy produce large numbers of effector candidates, in the form of small secreted proteins (SSP) that act The availability of whole genome sequences to establish a compatible interaction with the host enabled genome-wide analysis of the Colletotri- by suppressing host defenses and reprogramming chum transcriptome at different stages of hemi- host cells to accommodate the pathogen (Göhre biotrophic infection. Deep Illumina RNA and Robatzek 2008). These SSP effectors are sequencing was performed for C. higginsianum typically less than 300 amino acids, cysteine-rich, and C. graminicola at three stages of development and lineage-specific (Stergiopoulos and de Wit in planta: prepenetration (appressoria); bio- 2009). All four Colletotrichum genome annota- trophic hyphae; and necrotrophic hyphae tions include many SSP effector candidates, (O’Connell et al. 2012). For C. orbiculare, whole including a large number that are cysteine-rich genome microarrays were produced based on the and/or unique to each species (Table 3.3). Inter- annotated genome assembly, and were used to estingly, numerous additional candidate effectors investigate gene expression during prepenetra- were identified after deep 454 pyrosequencing of tion, biotrophic, and necrotrophic growth phases the in planta transcriptome of C. higginsianum (Gan et al. 2013). In earlier studies, C. gramini- (Kleeman et al. 2012). It was observed that only cola biotrophic hyphae were isolated by laser- about a quarter of these transcripts had been capture microscopy (LCM) and analyzed using annotated in the initial genome-based analysis, microarrays designed from a limited gene set 88 J. Crouch et al. based on the preliminary 2X shotgun sequence of et al. 2013). In C. orbiculare, most of the genes strain M5.001 produced by DuPont (Tang et al. that were upregulated in planta were located in 2006). C. higginsianum appressoria formed on GC-rich, rather than AT-rich, regions (Gan et al. artificial surfaces, and primary hyphae isolated 2013). It is clear that Colletotrichum is able to from the host tissues by fluorescence-activated detect and respond to plant signals, although the cell sorting (FACS), were also analyzed by nature of these signals remains mysterious. sequencing expressed sequence tags (ESTs), and The dome-shaped melanized appressoria of by Roche 454 sequencing (Kleemann et al. 2008; Colletotrichum function in penetration of the host Kleemann et al. 2012; O’Connell et al. 2012; cuticle and epidermal cell wall (Fig. 3.2). Cutin- Takahara et al. 2009). ases were over-represented in prepenetration In interpreting and comparing these various appressoria in comparison with later phases of Colletotrichum transcriptome datasets, it is development, as might be expected (O’Connell important to recall that in C. higginsianum, only et al. 2012). Genes involved in cAMP signaling the first invaded cell contains biotrophic hyphae, were upregulated in the preinvasion stage of C. followed by a complete switch to necrotrophy. orbiculare, in agreement with previous reports In C. orbiculare and C. graminicola, the nec- showing that this is an important signaling path- rotrophic switch is delayed until several cells way in the regulation of germination and appres- have been colonized. Thus, the biotrophic phase sorium formation (Yang and Dickman 1997, in these two species consists of a heterogeneous 1999a, b; Yamauchi et al. 2004; Gan et al. 2013). cell population that includes hyphal tip cells Melanin deposited in the appressorial wall allows advancing into living host cells and intercalary accumulation of glycerol and high turgor pres- fungal cells occupying dead or dying host cells. sures that facilitate mechanical rupture of the host Moreover, in C. graminicola, the necrotrophic cell wall (Bechinger et al. 1999; Bastmeyer et al. phase is also heterogeneous, composed of nec- 2002). Transcriptome analysis of appressorial rotrophic colony centers and biotrophic colony stages of C. higginsianum, C. graminicola, and C. margins. This variation in the timing and extent orbiculare confirmed increased expression of of host cell death caused by Colletotrichum genes of the PKS SM cluster involved in melanin species is likely to be reflected in the represen- production in all three species (Gan et al. 2013; tation of biotrophy- and necrotrophy-related O’Connell et al. 2012). genes in their transcriptomes. A large number of genes encoding putative A common theme that has emerged from secreted effector proteins are also expressed in studies comparing transcription in vitro to tran- unpenetrated Colletotrichum appressoria (Klee- scription in vivo is that a large number of genes in mann et al. 2008; 2012; O’Connell et al. 2012; Colletotrichum are plant-induced (Gan et al. Gan et al. 2013). In C. graminicola, homologs of 2013; Kleemann et al. 2012; O’Connell et al. the M. oryzae BAS2 and BAS3 effectors were 2012; Tang et al. 2006). For example, comparison among the most highly expressed genes in of gene expression in morphologically identical appressoria (O’Connell et al. 2012). Lineage- C. higginsianum appressoria produced in vitro specific SSP effectors were particularly enriched versus in planta revealed that more than 1,500 during the early stages of development (appres- genes were significantly induced in planta com- sorial and biotrophic) versus necrotrophy, sug- pared with their expression in vitro (O’Connell gesting they might play a role in establishment of et al. 2012). Many of these induced genes encoded a compatible interaction (Gan et al. 2013; Kle- secreted proteins, including SSP and putative eman et al. 2012; O’Connell et al. 2012). In C. effectors, and many others encoded SM enzymes. orbiculare, 28 of the 100 most highly upregu- Gene Ontology (GO) categories that were over- lated genes at the appressorial stage were SSPs represented in planta included those involved in (Gan et al. 2013). C. higginsianum appressoria carbohydrate binding and degradation, protein were shown to deliver candidate effectors by degradation, and transmembrane transport (Gan targeted secretion through pores at the host- 3 The Genomics of Colletotrichum 89 appressorial interface (Kleemann et al. 2012). higginsianum function primarily as organs for Numerous SM gene clusters were also expressed nutrient uptake (O’Connell et al. 2012). Consis- during prepenetration and early penetration tent with this view, recent evidence indicates that stages in the appressoria of C. graminicola, C. carbohydrate supply by the host is dispensable higginsianum, and C. orbiculare (Gan et al. for the biotrophic growth of C. higginsianum 2013; O’Connell et al. 2012). These findings (Engelsdorf et al. 2013). Thus, the primary role suggest that in addition to mechanical breaching of biotrophic hyphae seems to be as organs for of the host cell wall, appressoria play an impor- the secretion of effectors and SM that presum- tant role in the secretion of protein and small ably modulate host responses and suppress host molecule effectors, which may prepare the cell death. The SM enzymes and effectors of infection court for subsequent invasion. biotrophic hyphae differ from those expressed in Analysis of genes expressed during biotrophy appressoria, with distinct ‘‘waves’’ of these fun- in the three Colletotrichum species identified gal modifiers produced over the course of path- hundreds of differentially regulated genes, ogenic development (Gan et al. 2013; Kleemann including more than 300 upregulated genes in C. et al. 2012; O’Connell et al. 2012). graminicola, and more than 700 upregulated The switch from biotrophy to necrotrophy is genes in C. higginsianum. Although the data were marked by the production of narrow secondary generally consistent, shifts in gene expression infection hyphae that are not separated from the were not as pronounced in either C. graminicola host cell by a membrane (Fig. 3.4). Analysis of or C. orbiculare as they were in C. higginsianum, gene expression in all three species during necro- and this is likely because of the more synchronous trophy revealed the induction of a large array of development of the latter species. A previous genes encoding secreted proteases and CAZymes, LCM-enabled study of C. graminicola focused on producing a cocktail of enzymes that is probably analysis of biotrophic hyphae, but unfortunately highly efficient for degrading plant cell walls (Gan did not provide a full account of the genes that et al. 2013; O’Connell et al. 2012). Between 23 and were upregulated in those cells (Tang et al. 2006). 25 % of the 100 most highly expressed genes Thus analyses of the C. higginsianum biotrophic upregulated by C. graminicola and C. higginsia- phase, including cells colonizing living host cells, num during the necrotrophic phase are CAZymes and also biotrophic hyphae isolated by the FACS (O’Connell et al. 2012). However, each species technique, are likely to be the most informative apparently uses a different strategy to deconstruct on the transcriptional status of Colletotrichum host cell walls. More pectin-degrading enzymes biotrophic hyphae (Takahara et al. 2009; Klee- (51) are induced during necrotrophy in C. higgin- mann et al. 2012; O’Connell et al. 2012). sianum,whileC. graminicola deploys more cel- As observed in appressoria, many of the genes lulases and hemicelluloses at this stage. An expressed in C. higginsianum biotrophic hyphae example is provided by the GH61 monooxygena- encoded secreted effectors and SM enzymes ses, which act in concert with classical cellulases to (Kleemann et al. 2012; O’Connell et al. 2012). enhance lignocellulose hydrolysis (Beeson et al. Several LysM protein genes were expressed 2012; Quinlan et al. 2011). Twenty-two of 28 specifically in biotrophic primary hyphae, sug- GH61 monooxygenases were induced during C. gesting a role in ‘‘masking’’ of the hyphal wall graminicola necrotrophy, with 6 % of the most from host detection (O’Connell et al. 2012). highly induced genes during necrotrophy belong- Relatively few genes encoding lytic enzymes ing to this class (O’Connell et al. 2012). In contrast, were expressed, and in this respect the biotrophic only six out of 25 GH61 genes were expressed by hyphae resemble the haustoria of obligate bio- C. higginsianum during necrotrophy, and none trophs (O’Connell et al. 2012). However, unlike were highly induced (O’Connell et al. 2012). haustoria, there was no specific induction of Numerous nutrient uptake transporters are nutrient uptake transporters at this stage, which also induced at the switch to necrotrophy, sug- would be expected if the biotrophic hyphae of C. gesting that secondary hyphae provide the major 90 J. Crouch et al. organs for nutrient acquistion for Colletotrichum differences must be confirmed by manual anno- (Gan et al. 2013; O’Connell et al. 2012). In both tation before they can be fully accepted. C. higginsianum and C. orbiculare there was a The genus Colletotrichum includes species general decrease in the number of SM gene displaying a broad spectrum of pathogenic life- clusters expressed during necrotrophy, but in C. styles, providing many exciting opportunities for graminicola a large percentage of the genes comparative genomics and transcriptomics to induced at this stage were SM genes. Notably, study the molecular and evolutionary basis of the SM genes expressed by C. graminicola dur- these lifestyles. Comparisons of species dis- ing the necrotrophic phase differed from those playing ‘‘extreme’’ lifestyles, e.g., subcuticular expressed earlier in the interaction (Gan et al. necrotrophy or symptomless endophytism, 2013; O’Connell et al. 2012). Genes encoding should reveal how Colletotrichum fungi differ secreted effectors, including putative necrosis- from the better-characterized hemibiotrophic inducing proteins, were also induced at the nec- species, e.g., in gene repertoires dedicated to rotrophic stage of development in all three spe- host degradation (proteases, carbohydrate-active cies (Gan et al. 2013; O’Connell et al. 2012). enzymes) or ‘stealth’ (protein and secondary metabolite effectors), or in the timing with which those genes are deployed. Likewise, 3.7.3 How Can Genome Information comparisons of species with contrasting life- Help Us to Better Manage styles that infect the same host plant (endophytes and Exploit Colletotrichum? vs. pathogens, necrotrophs versus hemibio- trophs, or species preferentially infecting dif- We are now in the postgenomic era for Colleto- ferent organs of the same host (shoots or roots) trichum research. Numerous additional Colleto- could be especially informative. trichum genome-sequencing projects are Several major conclusions can be drawn from underway as we write, and sequencing technol- the comparative genome analyses described ogy has advanced to the point that the genome or herein, with implications for both applied and transcriptome of any strain of interest can be basic research disciplines. Remarkably, consid- sequenced quickly and cheaply. An important ering the extensive repertoire of pathogenicity- consideration emerging from the comparative related genes encoded in the four sequenced transcriptome analyses described here is the need Colletotrichum genomes and the patterns with to ensure that future analyses are performed at which those ‘weapons’ are deployed during equivalent infection stages, and under conditions infection, it is clear that there is far more held in promoting synchronous development, if they are common by these phylogenetically diverse spe- to be useful for a comparative analysis. For finer cies than there is unique. Thus, one can anticipate resolution of transcriptional differences, e.g., that conserved components and mechanisms will within intracellular primary hyphae occupying be discovered that could provide potential targets either living or dead host cells, it may be nec- for controlling many Colletotrichum diseases essary to use single-cell sampling methods such through chemical intervention or plant breeding. as FACS or LCM (Kleemann et al. 2012; Taka- Strong evidence for host specialization was also hara et al. 2009; Tang et al. 2006). Careful revealed through genome-scale studies of Col- cytological studies and bioassay development are letotrichum, including the apparent adaptation of essential for each new species that is analyzed. the CAZyme repertoire and its expression to Further considerations relate to aspects of bio- particular host cell wall composition, and the informatics and sequencing technologies. In large degree of diversity in SM and secreted particular, direct comparisons of genomes pro- protein effectors. Many Colletotrichum effectors duced using different sequencing techniques and are species-specific, while others are shared different assembly and annotation software must within the genus, or even with other fungal gen- be made with extreme caution, and perceived era, which may indicate conserved functions or 3 The Genomics of Colletotrichum 91 host targets. The priority now will be to identify stages and the infection strategies of obligately targets of both conserved and lineage-specific biotrophic or necrotrophic plant pathogens. effectors, and to determine the mechanisms by The primary hyphae of pathogenic IH Col- which effectors manipulate host cells to induce letotrichum are in some ways analogous to the compatibility. haustoria of obligate intracellular biotrophs Anthracnose diseases of field crops are gen- (Mendgen and Hahn 2002; O’Connell and erally managed by the use of resistant cultivars. Panstruga 2006; Perfect et al. 1999, 2001). These But resistance failures occur frequently, due to structures share certain morphological similari- the ability of pathogen populations to adapt ties, including the presence of a membrane that rapidly to traditional R-gene-mediated resistance differs in composition from the normal plant strategies. Ultimately, these boom-and-bust plasma membrane, and serves to separate the cycles lead to increased reliance on fungicides fungus from the living host cell (Shimada et al. (Parlevliet 2002; Thakur 2007). In particular, 2006). Obligate biotrophs cannot be cultured management of postharvest diseases caused by away from their plant hosts, and cannot be easily Colletotrichum requires the use of expensive and genetically manipulated, whereas hemibiotrophs toxic chemicals (Prusky 1996). Further genome/ are readily cultured and manipulated. This led to transcriptome-based insight into the conserved the idea that it might be possible to identify toolbox employed by members of the genus pathogen components required for biotrophic Colletotrichum could prove instrumental for the growth by studying the more experimentally design of durable strategies for disease control tractable hemibiotrophs (Mendgen and Hahn through resistance breeding. For example, 2002). However, genome analysis has suggested alternate breeding strategies, including the use that the appressoria and biotrophic hyphae of of mutant susceptibility (S) genes identified Colletotrichum function primarily as organs for using pathogen effectors as molecular probes, the synthesis and secretion of protein and sec- show promise in many pathosystems (Gawehns ondary metabolite effectors to host cells, and not et al. 2013). S genes encode host proteins that as organs of nutrient uptake like true haustoria. are co-opted by plant pathogens, resulting in Transcriptome analysis revealed that Collet- pathogen proliferation and ultimately leading to otrichum fungi are highly responsive to diseased host tissue. S gene inactivation reduces unknown plant signals, and that gene expression the pathogens’ ability to cause disease, provid- can differ remarkably between morphologically ing a durable form of resistance (Pavan et al. similar structures formed in vitro and in planta. 2010). In some cases, S gene-based immunity Understanding the role of plant signals in tran- has provided broad spectrum resistance against scriptional reprogramming and the mechanisms pathogens for several decades (Gawehns et al. by which those signals are sensed and trans- 2013). Although S genes provide highly effec- duced by the fungus could lead to new oppor- tive sources of resistance, few have been iden- tunities to alter those signals by manipulation of tified to date and even fewer are commercially the plant genome or to interfere with their per- viable. But with the power of genomics, the ception by the pathogen. Transcriptomics also discovery of new candidates may be accelerated demonstrated that massive shifts in gene through Colletotrichum effector-target screens. expression underlie the developmental transi- tions that occur in planta, from spore germina- tion to necrotrophy. Thus, the lifestyle switch to 3.7.4 The Genomics of Hemibiotrophy necrotrophy is characterized by a massive shift in fungal gene expression, with the activation of Some of the most important results gleaned from large numbers of genes encoding lytic enzymes genomics-enabled research of Colletotrichum are and membrane transporters. It will be crucial to those that provide insight into the hemibiotrophic understand the signals that trigger this switch lifestyle, and the parallels between distinct life and the transcriptional/epigenetic regulators and 92 J. Crouch et al. signaling pathways that underlie it. It is possible (Crouch et al. 2006, 2009a, b; Crouch and that these differ between hemibiotrophic patho- Tomaso-Peterson 2012; Crouch 2013). Simi- systems, but comparative transcriptome analyses lar resolution has resulted from the study of focused on the key transitional phase will help C. gloeosporioides, C. acutatum and several clarify this. other taxa (Damm et al. 2009, 2012a, b; Hyde et al. 2009; Weir et al. 2012). With increasingly precise demarcations of species boundaries, and 3.7.5 Genomics Applied to Elucidating the insight into host association that such data the Systematics provides, accurate biological, epidemiological, of Colletotrichum and mechanistic interpretation of genome and transcriptome data become possible. Concurrent with genomics-enabled research of A very broad pattern of host association is Colletotrichum pathology, the genus is undergo- evident across the Colletotrichum phylogeny, ing a taxonomic renaissance, enabled by the with older, basal lineages uniquely associated application of molecular phylogenetic approa- with dicots and non-graminicolous monocots ches (Crouch et al. 2006, 2009a, b, c; Damm et al. (Fig. 3.1). Colletotrichum pathogenic to grasses 2009, 2012a, b; Hyde et al. 2009; Rojas et al. form a cohesive, monophyletic group, the gra- 2010; Weir et al. 2012). Continued synergy minicola aggregate, originating from ancestral between genomics, transcriptomics and molecu- lines of non-graminicolous Colletotrichum. lar phylogenetic research is beginning to provide Thus, the pathogenic association of Colletotri- us with a long-awaited glimpse into the forces chum with grass hosts appears to be a derived impacting the evolution of Colletotrichum spe- trait, of relatively recent origin. Taken together cies. Importantly, molecular phylogeny-based with data from genome and transcriptome anal- diagnosis of species boundaries will enable more yses, this evolutionary trajectory suggests that accurate predictions about genome evolution, the expanded gene cohorts held in common by mechanisms of host adaptation, the evolution of dicot infecting Colletotrichum, particularly key pathogenicity traits and other topics. One genes encoding pectinases, most closely reflect illustration of this point involves the host range of the ancestral state for the genus, and the reduced Colletotrichum species. Molecular phylogenetic cohort characterized from C. graminicola is studies are confirming long-held suspicions that potentially due to the loss of these genes as they twentieth century species concepts and host range became unnecessary. Additional research is assumptions made using classical morphology needed to investigate these possibilities. are overly broad and often inaccurate (Sutton Phylogenomic analysis and divergence dating 1980; Cannon et al. 2013). Increasingly well- from a sample of C. graminicola, C. higginsia- resolved species diagnoses are now incorporated num and seventeen other fungi estimated a recent across the entire genus (Cannon et al. 2013). For divergence between these two species, just instance, until recently, C. graminicola was rec- 47 million years ago (O’Connell et al. 2012). ognized as a broad host range generalist pathogen Notably, the divergence between C. graminicola of nearly every grass and cereal in the Poaceae and C. higginsianum occurred approximately family, despite considerable evidence of physi- 100 million years after the divergence between ological specialization and distinctive appresso- their respective host groups, monocots and dicots rial structures (Sutton 1968; Sherriff et al. 1995; (Chaw et al. 2004). As such, it is unlikely that Hsiang and Goodwin 2001; Du et al. 2005; divergence in this part of the Colletotrichum Crouch et al. 2006). Molecular data showed this phylogeny was temporally associated with host broad circumscription as false, with C. gramini- evolution. However, the divergence of species cola limited to maize, while more than sixteen within the graminicola aggregate may have distinct Colletotrichum species are now descri- involved a coevolutionary process with the Poa- bed as pathogens and endophytes of the Poaceae ceae family. The most basal species in the 3 The Genomics of Colletotrichum 93 graminicola aggregate, C. cereale, is uniquely strategies yield a biased view of evolutionary associated with cool-season (C3 physiology) relationships or gene-specific noise. There is an Pooideae grasses such as , oats, , increasing body of work that shows many of these bluegrass, etc. (Fig. 3.1). C. cereale is the pro- borrowed markers lack the power to fully resolve genitor of numerous Colletotrichum species distinct organisms or provide incongruous results adapted to warm-season (C4 physiology) cereals (Aguileta et al. 2008; Rokas et al. 2003; Town- and grasses in the Panicoideae subfamily, send 2007; Townsend and Lopez-Giraldez 2010). including maize, sorghum and sugarcane. The To overcome these biases, researchers are divergence between wheat (Pooideae; C3) and increasingly developing datasets from larger maize (Panicoideae; C4) is estimated at numbers of orthologous genes, targeted genes or 50–60 million years ago (Chaw et al. 2004), even whole genomes (e.g., Aguileta et al. 2008; generally compatible with the estimated timing Du et al. 2005; Crouch et al. 2009a, b, c; Fitz- of the graminicola/higginsianum divergence, patrick et al. 2006; Rokas et al. 2003; Wang et al. particularly given the margin for error associated 2009). Comparative genome analysis of 52 Col- with calibration of divergence times (O’Connell letotrichum isolates from the graminicola and et al. 2012). This temporal correspondence sug- acutatum aggregates using Illumina sequenced gests that speciation and host adaptation in the restriction-associated DNA tagged (RAD-Seq) graminicola aggregate may have mirrored host SNP datasets demonstrated that a subset of diversification, or may even have been driven by commonly used Sanger-sequenced PCR ampli- the evolutionary radiation of the Poaceae family. con-derived single locus molecular markers— Genome and transcriptome assessments of addi- Apn2 and Sod2—individually provided reliable tional Colletotrichum species that are either identification of Colletotrichum species compa- members of, or closely related to, the graminicola rable to a 1,723 locus genome-wide dataset and destructivum aggregates, especially those (Crouch et al. 2013). These findings led to the associated with non-graminicolous monocots or development of real-time PCR diagnostic assay members of the Pooideae-infecting C. cereale, based on the Apn2 marker capable of population- could be particularly informative. specific detection of C. cereale from infected host tissue, and herbarium specimens up to 100 years old (Beirn et al. 2013). Similar development and 3.7.6 Genomics Tools for Studying application of genome data for the identification the Population Genetics of other economically important Colletotrichum of Colletotrichum could provide pathologists with tools that help mitigate losses due to disease. Phylogenetic and population genetic investiga- tions are benefiting from the increased avail- ability of genome-scale datasets. One of the 3.7.7 Genomics and the Commercial major obstacles facing phylogenetic and popu- Exploitation of Colletotrichum lation researchers is the identification of appro- priate and informative molecular markers to Colletotrichum have long been utilized for bio- gauge diversity, relationships, and evolutionary technology applications, with many species traits. Standard methodology uses ‘‘borrowed’’ yielding a diversity of compounds and secondary markers: a handful of conserved loci that are used metabolites with commercially valuable biologi- primarily because primers exist that are capable cal activity (García-Pajón and Collado 2003). of amplifying a broad range of organisms with Recent notable applications include the purifica- relative ease (e.g., Carbone and Kohn 1999; tion of large quantities of the alkaloid compound White et al. 1990). These markers have been in huperzine A used for treatment of Alzheimer’s common use for almost two decades in some disease from C. gloeosporioides (Zhao et al. cases. However it is unknown whether such 2013), the use of lipid-accumulating 94 J. Crouch et al.

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