The Chestnut Blight Fungus for Studies on Virus/Host and Virus/Virus Interactions: from a Natural to a Model Host

Home , Chestnut blight, Rosellinia necatrix

Virology 477 (2015) 164–175

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The chestnut blight fungus for studies on virus/host and virus/virus interactions: From a natural to a model host

Ana Eusebio-Cope a, Liying Sun b, Toru Tanaka a, Sotaro Chiba a, Shin Kasahara c, Nobuhiro Suzuki a,n a Institute of Plant Science and Resources (IPSR), Okayama University, Chuou 2-20-1, Kurashiki, Okayama 710-0046, Japan b College of Plant Protection, Northwest A & F University, Yangling, Shananxi, China c Department of Environmental Sciences, Miyagi University, Sendai 982-215, Japan article info abstract

Article history: The chestnut blight fungus, Cryphonectria parasitica, is an important plant pathogenic ascomycete. The Received 16 August 2014 fungus hosts a wide range of viruses and now has been established as a model filamentous fungus for Returned to author for revisions studying virus/host and virus/virus interactions. This is based on the development of methods for 15 September 2014 artificial virus introduction and elimination, host genome manipulability, available host genome Accepted 26 September 2014 sequence with annotations, host mutant strains, and molecular tools. Molecular tools include sub- Available online 4 November 2014 cellular distribution markers, gene expression reporters, and vectors with regulatable promoters that Keywords: have been long available for unicellular organisms, cultured cells, individuals of animals and plants, and Cryphonectria parasitica certain filamentous fungi. A comparison with other filamentous fungi such as Neurospora crassa has been Chestnut blight fungus made to establish clear advantages and disadvantages of C. parasitica as a virus host. In addition, a few dsRNA recent studies on RNA silencing vs. viruses in this fungus are introduced. Mycovirus & Fungal virus 2014 Elsevier Inc. All rights reserved. Hypovirus RNA silencing Mycoreovirus Model organism Virus-host interaction

Introduction importance. Of them, only a few are studied as virus hosts one of which is the chestnut blight fungus, Cryphonectria parasitica (Murrill) It is believed that there exist at least 1.5 million fungal species on Barr. Chestnut blight attracts great attention mainly from intercon- the globe that include over 100,000 identified species and those nected factors as an important plant pathogen and as a virus host unidentified or unreported (Hawksworth, 1991). Viruses are found in (Hillman and Suzuki, 2004). Here, we overview properties of the all major groups of fungi (Ghabrial and Suzuki, 2009; Pearson et al., chestnut blight fungus as a model filamentousfungusforstudies 2009), and the International Committee on Taxonomy of Viruses now of virus/host and virus/virus interactions. Readers are also referred lists 80 definitive species (http://talk.ictvonline.org/files/ictv_docu to other related elegant reviews (Dawe and Nuss, 2001, 2013; ments/m/msl/4911.aspx). This number in reality should be far higher. Nuss, 2005; Turina and Rostagno, 2007; Wickner, 1996). Although It is reasonable to assume that a vast number of unrecognized fungal C. parasitica also has been a research subject in the biological control viruses (mycoviruses) make up a nano-world in the kingdom Fungi. of a plant pathogenic fungal disease involving fungal viruses (Heiniger In fact, viruses were found in a few to several tens of percent of field and Rigling, 1994; Milgroom and Cortesi, 2004), this is not the scope fungal isolates when they were screened (Arakawa et al., 2002; Ikeda of this paper. et al., 2004; Jiang et al., 2014). Among the enormous number of fungi, there are only a limited number of them are studied that are of Chestnut blight and C. parasitica: a brief overview economical, agricultural, medical, technological or purely scientific Chestnut trees occupied a large portion of the forest canopy. The trees provided shelter for a variety of wildlife, and were utilized for its n Corresponding author. Tel.: þ81 86 434 1230; fax: þ81 86 434 1232. hard-quality lumber, and sources for tannins, wood, fuel, and edible E-mail addresses: [email protected] (A. Eusebio-Cope), nuts. Common existence of chestnut trees was greatly involved in the [email protected] (L. Sun), [email protected] (T. Tanaka), life of people in North America and Europe, and had deeply taken root [email protected] (S. Chiba), [email protected] (S. Kasahara), [email protected] (N. Suzuki). into the culture. Thus, this crop was considered of great economic and URL: http://www.rib.okayama-u.ac.jp/pmi/index.html (N. Suzuki). ecological importance. However, the introduction of chestnut blight http://dx.doi.org/10.1016/j.virol.2014.09.024 0042-6822/& 2014 Elsevier Inc. All rights reserved. A. Eusebio-Cope et al. / Virology 477 (2015) 164–175 165

Fig. 1. Naturally occurring chestnut blight. (A) Virulent canker naturally induced by the chestnut blight fungus on a European chestnut tree (Castanea sativa). A European chestnut tree with die-back symptoms. These pictures are courtesy of Dr. Daniel Rigling at Swiss Federal Institute for Forest, Snow and Landscape Research WSL.

disease caused by the ascomycetous fungus, C. parasitica to the United sequence project of this fungus in 2007 and the Joint Genome States and Europe in the 20th century from East Asia (most likely from Institute (JGI, United States Department of Energy) released the Japan) resulted in destruction of the chestnut trees in those areas assembly version 2.0 of the whole genome shotgun reads of (Anagnostakis, 1982; Griffin and Elkins, 1986; Milgroom et al., 1996). C. parasitica for public use http://genome.jgi.doe.gov/Crypa2/Crypa2. The causal agent, C. parasitica, enters host trees from wound sites. The home.html. With its scaffold sequence bases total size of 43.9 Mb, a disease exhibits sunken canker in the stem of the affected tree that total of 11,609 genes were structurally and functionally annotated in continues to expand and penetrate, thus, demolishing the cambium the latest version. This number is close to the estimated 10,000 layer (Fig. 1A). Diseased trees often show die-back symptoms (Fig. 1B) protein-coding genes contained in the approximately 40-Mb in seven and are eventually killed. This fungus develops fan-shaped formation chromosomes of Neurospora crassa, orange bread mold, a relative of of mycelia in the cambium and bark of the host. Success of this the chestnut blight fungus and the best-characterized fungus among pathogen to perpetuate itself is through the multitude of conidia filamentous fungi (Galagan et al., 2003). Furthermore, a large-scale (asexual spores) and ascospores (sexual spores) loaded in cankers, transcriptome data set is available (Dawe et al., 2003; Shang et al., which act as effective propagules for dispersal and continuity of the 2008). disease cycle (Fig. 2). These propagules, dispersed by wind, rain, insects, or small animals, germinate and develop into a network of interconnected hyphae (mycelia). Matured mycelial strands then give C. parasitica as a model filamentous fungus for virological studies rise to new progenies (conidia) emanating either directly from the somatic hyphae or specialized conidiogenous cells, often borne on Early eukaryotic model organisms can be represented by Sacchar- hyphal branches (conidiophores). Regardless of tree age during the omyces cerevisiae (baker's yeast), Musa musculus (mouse), Drosophila onset of disease, the upper part of the infection site starts to melanogaster (fruit fly), Caenorhabditis elegans (nematode), and senescence showing wilted leaves and, at times, partial death of tree. Arabidopsis thaliana (thale cress). Properties common to these This is a typical symptom of blight, hencethenameofthedisease.Due include their small body and genome sizes, short generation time to the breadth of its destruction, chestnut blight is considered as one and easy propagation, available genome sequences with well-est- of the three most destructive tree diseases of the world along with ablished annotations, available techniques for gene manipulation, Dutchelmdiseaseandwhitepineblisterrust(Tainter and Baker, and useful biological resources and molecular tools. Findings with 1996). For the detailed pathology of this fungus, see previous papers model organisms can be applicable for more important organisms, (Anagnostakis, 1987; Roane et al., 1986). i.e., humans, livestock, and crops. During the past decade, the The fungal pathogen C. parasitica belongs to the phylum Ascomy- number of eukaryotes for which genome sequences were deter- cota, subphylum Pezizomycotina, class Sordariomycetes, order Dia- mined has grown, and many of them fulfill aforementioned criteria. porthales, and family Cryphonectriaceae (Gryzenhout et al., 2006). The chestnut blight fungus also meets most criteria expected of a As do most filamentous ascomycetes, each C. parasitica isolate has model virus-host organism as described below. C. parasitica is haploid nuclei with a single mating type either MAT-1 or MAT-2 reminescent of Nicotiana benthamiana in biological tractability as a (McGuire et al., 2004). Sexual crossing occurs only between the virus host to many viruses (Bombarely et al., 2012; Goodin et al., different mating types. Results of cytological and molecular karyotyp- 2008). As discussed below, like N. benthamiana, (tobacco plant) C. ing (Eusebio-Cope et al., 2009) of the standard wild-type (WT) strain, parasitica has some disadvantages over other better-established EP155, demonstrated the chromosome number of this fungus to be model organisms, but has great advantages as a virus host. Compar- nine (Fig. 3) and a rough estimate of its genome as 50 mega base pairs ison of the two organisms in this regard will be discussed in the (Mb).AgroupofresearchersledbyDonaldL.Nussinitiatedagenome forthcoming 60th Anniversary Special Issue of Virology. 166 A. Eusebio-Cope et al. / Virology 477 (2015) 164–175

Fig. 2. Asexual (A and B) and sexual (C–E) propagules produced by Cryphonectria parasitica strain EP155 used for its dispersal. Orange pustules (blue arrowhead) containing pycnidia (A) are composed of several single-celled conidia (B), which are easily produced in solid media under laboratory condition. (C) Perithecia embedded in chestnut bark clearly showing the exposed perithecial beaks (red arrows in C and D) where ascospores are released after being formed in the round-shaped structure (red arrowhead in D). Two-celled ascospores (red arrowhead in E), each cell with 14 nuclei are contained in an ascus; several asci containing eight ascospores each can be found in the same round-shaped structure in D. Panels D and E were prepared with the help of Prof. Masatoki Taga at Okayama University. A cale bar is included in each panel.

Fig. 3. Nine meiotic metaphase chromosomes of Cryphonectria parasitica strain EP155. Chromosome complements stained by 40,6-diamidino-2-phenylindole (DAPI) (A) and DAPI-propidium iodide (B) and were observed under fluorescent microscopy. The number and value below each chromosome indicate the identification number assigned to each chromosome and the relative chromosome size in the length of the longitudinal chromosome axis, respectively. Conspicuous knob-like intensely fluorescing segments and examples of constriction are indicated by arrows and arrowheads, respectively. This image was part of Fig. 2 of the Fungal Genet. Biol. 46, 342–351 (Eusebio-Cope et al., 2009).

Viruses infecting C. parasitica Partitiviridae (Hillman and Suzuki, 2004). Note that a member of the family Chrysoviridae was isolated from a close relative (sympa- Many natural viral infections of C. parasitica have been reported. tric species), Cryphonectria nitchkei (Kim et al., 2010; Liu Surveys of the chestnut blight have shown low incidences of et al., 2007). Unidentified viruses were also reported in this fungus double-stranded (ds) RNA in C. parasitica populations in China (Hillman and Suzuki, 2004; Liu et al., 2007). These viruses were and Japan (2% and 6%, respectively) and moderate incidences of isolated from different fungal strains with different genetic back- dsRNA in North American populations (28%) (Milgroom et al., 1996; grounds. In order to determine an etiological role for the virus, it is Park et al., 2008; Peever et al., 1997, 1998) which is in stark contrast necessary to compare phenotypic alterations of isogenic virus-free to the cases of the violet root rot fungus, Helicobasidium mompa and virus-infected fungal strains. Artificial virus introduction meth- (approximately 70%) and rice false smut fungus, Ustilaginoidea ods (see below) make it possible to compare phenotypic effects of virens (approximately 90%) (Ikeda et al., 2004; Jiang et al., 2014). some of these different viruses. Cryphonectria parasitica viruses reported thus far are classified into In addition, there are heterologous viruses that have proved to be four virus families, Hypoviridae, Reoviridae, Narnaviridae,and replication-competent in C. parasitica.TheseincludeFusarium A. Eusebio-Cope et al. / Virology 477 (2015) 164–175 167

Table 1 Viruses infecting Cryphonectria parasitica.

Virus family Virus Abbreviation Genome type Virion formation Reference

Natural infection Hypoviridae Cryphonectria hypovirus 1a CHV1 Undivided (þ)ssRNA Unable Hillman & Suzuki (2004) Hypoviridae Cryphonectria hypovirus 2 CHV2 Undivided (þ)ssRNA Unable Hillman & Suzuki (2004) Hypoviridae Cryphonectria hypovirus 3a CHV3 Undivided (þ)ssRNA Unable Hillman & Suzuki (2004) Hypoviridae Cryphonectria hypovirus 4a CHV4 Undivided (þ)ssRNA Unable Hillman & Suzuki (2004) Reoviridae Mycoreovirus 1a MyRV1 11-segmented dsRNA Spherical Hillman & Suzuki (2004) Reoviridae Mycoreovirus 2 MyRV2 11-segmented dsRNA Spherical Hillman & Suzuki (2004) Narnaviridae Cryphonectria mitovirus 1 CpMV1 Undivided (þ)ssRNA Unable Hillman & Suzuki (2004)

Artiﬁcial infection Reoviridae Mycoreovirus 3 MyRV3 12-segmented dsRNA Spherical Kanematsu et al. (2010) Partitiviridae Rosellinia necatrix partitivirus 1 RnPV1 Bi-segmented dsRNA Spherical Kanematsu et al. (2010) Partitiviridae Rosellinia necatrix partitivirus 2a RnPV2 Bi-segmented dsRNA Spherical Chiba et al. (2013b) Totiviridae Rosellinia necatrix victorivirus 1 RnVV1 Undivided dsRNA Spherical Chiba et al. (2013a) Megabirnaviridae Rosellinia necatrix megabirnavirus 1a RnMBV1 Bi-segmented dsRNA Spherical Salaipeth et al. (2014) Unassigned Fusarium graminearum virus 1 FgV1 Undivided (þ)ssRNA Unable Lee et al. (2011)

a Certain strains of these viruses carry satellite-like RNAs and/or defective RNAs. The table was adapted from Suzuki (2014). graminearum virus 1 (FgV1) (Lee et al., 2011) and members of the pairing with compatible mating types (sexual crossing). A very short family Reoviridae (mycoreovirus 3, MyRV3) (Kanematsu et al., 2010), diploid stage during the sexual cycle is followed by meiosis and Partitiviridae (Rosellinia necatrix partitivirus 1 and 2, RnPV1 and mitosis, leading to the production of eight ascospores (Fig. 2C–E). RnPV2) (Chiba et al., 2013a; Kanematsu et al., 2010), Totiviridae Thus these life style characteristics enable us to observe effects of (Rosellinia necatrix victorivirus 1, RnVV1) (Chiba et al., 2013b), and virus infection at different stages of growth/development although Megabirnaviridae (Rosellinia necatrix megabirnavirus 1, RnMBV1) such studies are limited somehow. (Salaipeth et al., 2014). Most of these viruses were originally isolated As mentioned earlier, the draft genome sequence is publicly from another phytopathogenic fungus, Rosellinia necatrix (white root available and expressed sequence tag (ESTs) of C. parasitica are fungus) that belongs to the order, Xylariales, different from Dia- registered; however, their annotations are not thorough. Because of porthales accommodating C. parasitica (Kondo et al., 2013b). Because monokaryon (monokaryosity) of conidia as well as haploidity, it is of this, viruses now can be maintained in the standard EP155 and easy to select homokaryotic clones of this fungus after transforma- other strains of C. parasitica. These allow for comparison of pheno- tion. The techniques for multiple transformation including multiple- typic effects of and host responses to distinct viruses, and exploration gene knockout were developed for C. parasitica (Choi et al., 2012; of viral gene expression strategies as demonstrated below. In fact, Faruk et al., 2008; Kasahara et al., 2000).Asinthecaseforsome these viruses incite host responses and cause distinct symptoms. other fungi, a mutant strain defective in non-homologous end- Table 1 shows viruses whose replication is supported by C. parasitica. joining DNA repair (cpku80)(Lan et al., 2008) increases levels of Importantly, there are a number of variants naturally occurring homologous recombination, thereby producing a large number of and generated in the laboratory for some of the aforementioned desired disruptants. This is based on the availability of multiple drug- viruses. Notably, reverse genetics was developed for several CHV1 resistance genes (see below) and ease of preparation of C. parasitica strains inducing distinct phenotypic changes in the host (Chen protoplasts that tolerate long-term storage at 80 1C without losing et al., 1994a; Lin et al., 2007). The mutants of hypovirus CHV1- competency in transfection and transformation. EP713 lacking some coding domains for ORFA-encoded p29 Most importantly, as a “model” virus host, virus elimination and (Δp29), p40 (Δp40), and both (Δp69), and ORFB-encoded p48 introduction must be achieved to establish an etiological role for (Δp48), were successfully used for mapping viral factors for virus-infected fungal colonies of interest, which often encounter symptom and virulence modulation, and virus replication (Chen technical difﬁculties. These are well-established for some viruses of et al., 2000; Craven et al., 1993; Deng and Nuss, 2008; Jensen and C. parasitica and other fungi as well (see below). Among viruses of Nuss, in Press; Suzuki et al., 1999, 2000, 2003; Suzuki and Nuss, C. parasitica, some viral strains can be introduced by transformation 2002). Variants generated in the laboratory also include a number with full-length virus cDNA clones (hypoviruses) (Choi and Nuss, of derivatives of MyRV1, RnPV2 and RnMBV1 with genome alter- 1992) or by transfection with synthetic virus RNA (hypoviruses) ations (Chiba et al., 2013a; Eusebio-Cope et al., 2010; Kanematsu (Chen et al., 1994b) or virions (mycoreoviruses, megabirnaviruses, et al., 2014; Salaipeth et al., 2014). These provided insights into partitiviruses, and victoriviruses) (Chiba et al., 2013a, b; Hillman functional roles of altered genome segments and rearrangement et al., 2004; Kanematsu et al., 2010; Salaipeth et al., 2014). Fig. 4 mechanics. Particularly, the MyRV1 rearrangements, produced in illustrates an example of biological comparison of CHV1 and MyRV1 the presence of a CHV1 infection or its RNA silencing suppressor in the standard C. parasitica EP155 that has been made possible by (RSS), CHV1 p29, have received great attention (Sun and Suzuki, MyRV1 virion transfection. Other methods available for virus 2008; Tanaka et al., 2011). introduction into C. parasiticaincludesprotoplastfusion(Lee et al., 2011) whereby hypoviruses (CHV2/NB58 and CHV3/GH2) and Tractability of C. parasitica mitovirus (NB631)-infected fungal strains were successfully trans- ferred to vegetatively incompatible fungal strain, EP155 (A. Eusebio- Cryphonectria parasitica is a biologically and genetically tract- Cope and N. Suzuki, unpublished results). In addition, the same able experimental organism. The fungus is phenotypically stable protocol allowed inter-species transmission of mycoreovirus relative to other ﬁlamentous fungi, and there are established (MyRV1/9B21) from C. parasitica to Valsa ceratosperma (A. Euse- measures for phenotypic assessment including virulence assays bio-Cope, S. Kanematsu and N. Suzuki, unpublished results). These (Hillman et al., 1990). It can complete its life cycle in a short period techniques are illustrated by Kondo et al. (2013b). One of the of time. Depending on growth condition, it produces asexual methods described by the authors and has yet to be examined in structures (uninucleated conidia or pycnidia [fruiting bodies]) in C. parasitica is the zinc ion-mediated approach originally taken for 12weeks(Fig. 2A and B). Sexual structures (ascospores and lateral virus transfer between vegetatively incompatible strains of R. perithicia [fruiting bodies]) are formed in a few months after necatrix (Ikeda et al., 2013; Zhang et al., 2014c). 168 A. Eusebio-Cope et al. / Virology 477 (2015) 164–175

Virus-free CHV1 MyRV1

Fig. 4. Distinct symptoms induced by two representative viruses. Colony morphology of C. parasitica strain EP155 uninfectred (virus-free) or infected by CHV1 and MyRV1. Fungal strains were grown in PDA on the laboratory bench top for one week.

Resources and tools available for the C. parasitica/viruses contributed (Table S3). These should be useful biological resources for virus/host interactions studies. Natural variants and generated mutants of C. parasitica There are a number of mutants reported thus far that show phenotypes different from the parental WT involved in fungal Sub-cellular distribution markers physiology, virus replication, or virus symptom expression. Table S1 Eukaryotic cells are organized into various organelles and lists such mutants in which mutated genes responsible for the functionally associated compartments, which are often related to phenotype were identified. Many of them were created and analyzed virus replication and intercellular distribution. For example, many in the Nuss lab. It often makes it difficult to determine the direct positive senses (þ), single-stranded (ss) RNA viruses are known to effects of their mutations on virus symptom expression, because they utilize host subcellular membrane to anchor replication complexes cause phenotypic or physiological changes in the absence of viruses. in animal and plant systems (Ahlquist, 2006; Hyodo and Okuno, Nevertheless, some interesting mutants are known to show symp- 2014; Mackenzie, 2005), while mycovirus behaviors in general at toms different from those of the WT fungal strain, EP155. A putative the single cell level remain unclear. The determination of replica- Mg2þ transporter gene (nam1) is involved in normal symptom tion sites and sub-cellular localization of host and viral key induction by CHV1 in a strain- and species-specificway(Faruk et components certainly enhance our understanding of virus/host — al., 2008). Its mutant (namA) manifests an unusual “nami-gata” interactions. For this purpose, two methods biochemical co- fi (meaning wave-shaped in Japanese) phenotype upon infection by puri cation of organelles (Jacob-Wilk et al., 2006) and direct visualization of subcellular distribution with specific markers— the WT strain of CHV1, but not by CHV1-Δp69 or MyRV1. However, could be used. In this subdivision, we focus on the latter with the uninfected namA mutant is indistinguishable in colony morphol- some recent results from our laboratory using fluorescent proteins ogy from EP155. Other interesting mutants include Δcpst12,withits (FPs), as the FP-based approach is one of the most simple but deleted gene encoding CpST12, a homolog of the cellular transcrip- efficient methods for identifying subcellular distribution of given tion factor S. cerevisiae Ste12 (Deng et al., 2007b), and the two proteins in living cells. mutants (mutC and mutP) with a silenced gene encoding CpKex2, A suite of reliable organelle markers for the study on sub- a transmembrane kexin-like and calcium-dependent protease that cellular localization of proteins is available for well-established is down regulated upon CHV1-infection (Jacob-Wilk et al., 2009). model filamentous ascomycetes, such as N. crassa, Aspergillus Inactivation of the respective genes in those mutants affects nidulans, and some other fungi (Andrie et al., 2005; Freitag et al., ' C. parasitica s virulence when assayed on dormant chestnut stems. 2004; Maruyama and Kitamoto, 2007; Toews et al., 2004; Moreover, female sterility of the host fungus is attributed to CpST12 Westermann and Prokisch, 2002)(Table 2). However, their avail- while secreted proteins processed by CpKex2 are necessary for fungal ability in C. parasitica remains limited. We therefore generated a development. While the examples given above are all laboratory- number of markers for targeting C. parasitica organelles using FPs. derived mutants, a naturally occurring variant, NB58F (Polashock The FPs tested include enhanced green fluorescent protein (eGFP), et al., 1994), derived from the CHV2-infected NB58 culture, resists improved monomeric red fluorescent protein (mRFP), tdTomato, viral infection when paired with thesameparentalstrainwhere mCherry, and mOrange (Shaner et al., 2004) which were found it originated or other conversion-compatible, virus-containing str- suitable for use in C. parasitica. ains. Unlike in S. cerevesiae, few host factors associated with viral RNA Table 2 shows organelle-targeting proteins used as signal replication are identified in this fungus. In this regard, it should be peptides for eGFP-fusion proteins in other filamentous fungi, and noted that a component, HEX1, of the Woronin body (peroxisome- their orthologous genes in C. parasitica. For these markers, cDNA to derived organelle) was recently shown to enhance the replication of each orthologous gene was prepared from C. parasitica and fused FgV1 which is closely related to hypoviruses (Son et al., 2013). with eGFP at either its N- or C-terminus and cloned into an In addition, there are a number of naturally occurring and lab- expression vector, pCPXHY1 with a constitutive glyceraldehyde-3- oratory-derived mutant isolates (approximately 200), stored and phosphate dehydrogenase (GPD) promoter (Craven et al., 1993)or distributed by American Type Culture Collection (ATCC) (Table S2). its derivatives. All primers used in this study were designed based Some were characterized earlier by groups of Drs. S. Anagnostakis, on the draft sequence of the C. parasitica genome (Table S4). P. Cortesi, and E. Kulman and reported to harbor dsRNAs. Simil- Information on their cloning operations is available upon request. arly, the National Institute of Agrobiological Sciences (NIAS) Gene Tested markers included those for the nucleus, mitochondria, Bank (http://www.gene.affrc.go.jp/index_en.php)hasacollection, peroxisome, Golgi apparatus, and endoplasmic reticulum (ER). although relatively small, of C. parasitica field isolates available Transformants with each construct showed a green fluorescence to the community to which Drs. S. Kaneko and M. Kusunoki profile expected from its subcellular localization (Fig. 5). Unlike the A. Eusebio-Cope et al. / Virology 477 (2015) 164–175 169

Table 2 Fluorescent protein-tagged organelle markers for live-cell image in Cryphonectria parasitica.

Organelle Marker gene Protein ID References

ER SecE, translocates protein 38784 Maruyama and Kitamoto (2007) Mitochondria Mitochondria inner-membrane protein 295517 Westermann and Prokisch (2002) Nuclei Histone H1, DNA binding protein 235247 Freitag et al. (2004) Golgi-body Kex, endoprotease 62358 Jacob-Wilk et al. (2006) Peroxisome PEX, Peroxisomal- membrane protein-4 337479 Bartoszewska et al. (2011)

The first column indicates the marker-targeted organelle. Marker genes accession numbers (protein ID) and annotations of protein function were obtained from the portal for the C. parasitica genome sequence at the Department of Energy Joint Genome Institute (http://genomeportal.jgi-psf.org/Crypa2/Crypa2.home.html). Fluorescent proteins were fused at the C-termini of marker proteins. free-eGFP exhibiting a nucleo-cytoplasmic distribution pattern 2012). Moreover, a macroscopic, tissue-printing-based immunolo- (Fig. 5A), the C. parasitica histoneH1genefusedwitheGFPallowed gical detection of viral spread in colonies was achieved by Yaegashi nuclear detection as shown in Fig. 5B. Note that nuclear numbers in et al. (2011), as viral dsRNAs were targeted by commercially single cells varied in this fungus (Fig. 5B).AneGFP-tagged,mitochon- available antibody. These techniques are also applicable to the drial inner membrane protein provided small thread-like organelles C. parasitica/mycovirus system (L. Salaipeth and N. Suzuki, unpub- (Fig. 5C), which is known as typical morphology of mitochondria in lished results). However, immuno-fluorescent observation using filamentous fungi (Westermann and Prokisch, 2002). While the Golgi C. parasitica hyphae requires further improvements in protocols. apparatus consists of a large number of small stacks, the Kex protease from C. parasitica is reported to be present in the trans Golgi network Other molecular tools (TGN) (Jacob-Wilk et al., 2006). The eGFP-Kex fusion protein showed Many transformation vectors with a variety of selectable mar- punctate distribution pattern representing Golgi-derived vesicles kers are available that include blasticidin S (Kimura et al., 1994; (Fig. 5D). C. parasitica peroxisomal matrix protein (PER4) fused with S. Kasahara, unpublished results), bialaphos (Bradley I. Hillman, eGFP (PER4-eGFP) exhibited small punctate organelles that were personal communication), hygromycin B, benomyl, and G418 evenly distributed in the cells (Fig. 5E). As mentioned above, the (Churchill et al., 1990; Sun et al., 2009). Also important is that a Woronin body, which facilitates FgV1 replication (Son et al., 2013), is controllable promoter is available for this fungus (Willyerd et al., derived from peroxisomes. Thus, re-localization of PER4-eGFP might 2009) in addition to several constitutive ones (Churchill et al., 1990; be observed upon certain mycovirus infections. In filamentous fungi, Kwon et al., 2009). There are other potential, inducible promoters theER,asanextensivenetworkthroughoutthecytoplasm,ishighly such as the tannic acid-inducible promoter of a C. parasitica laccase differentiated in various regions of the hyphae (Maruyama and gene, lac3 (Chung et al., 2008). Table S6 shows such expression Kitamoto, 2007). The ER gradient is generally distributed from the vectors. apical region, localizes along the septum, and undergoes dynamic Recently, we also developed a luciferase reporter assay system morphological change during septum formation (Maruyama and in C. parasitica that was used to determine the reinitiation Kitamoto, 2007). Addition of ER retention signal peptide HDEL efficiency of ribosomes for translation of the downstream ORF B (His-Asp-Glu-Leu) or HEEL (His-Glu-Glu-Leu) at the GFP C-terminus of CHV1 (Guo et al., 2009). At present, we have developed a dual resulted in localization of GFP on the ER membrane of C. parasitica luciferase assay system with codons optimized for N. crassa (Gooch (L. Sun and N. Suzuki, unpublished results) and an indistinguishable et al., 2008) which should lead to identification of viral cis- pattern reported for A. niger (Derkx and Madrid, 2001). By contrast, a elements for non-canonical translation such as internal ribosome transitional ER (tER) protein SecE, was localized to ER continuous entry sites (IRESs). with the nuclear envelope when eGFP was tagged (Fig. 5F). Therefore, Suzuki et al. (2000) made a series of CHV1-based vector this nuclear-associated ER marker is considered useful for identifying constructs and one of them was shown to express GFP. The viral proteins involved in translation or their facilitation. problem with this vector is genetic instability that often occurs All FP-markers generated in this study should serve as localiza- with RNA virus-based vectors: foreign inserts are readily lost tion references when subcellular distributions of viral proteins and during short periods of time after transfection. A tobacco mosaic cytopathic effects of given mycovirus infections are explored in the virus-based expression and suppression (RNA silencing mediated) future. vector was developed in a phytopathogenic fungus, Colletotrichum In addition to FPs, the fluorescent dyes are also useful tools for acutatum (Mascia et al., 2014). The TMV-based vector is worth cytological studies in filamentous fungi, as shown in Table S5.Of testing in C. parasitica. those, membrane-selective dyes (FM-dyes such as FM-64 and FM1- 43) are widely used to stain the plasma membrane and most organelle membranes in a time-dependent manner; e.g., endocytic Recent research topic on C. parasitica as a virus host: defense and vesicles, endosomes, Spitzenkörper/apical vesicle cluster, and counter-defense vacuoles (Hickey et al., 2002). The 40,6-diamidino-2-phenylindole (DAPI) and mitoTracker staining properly acted in C. parasitica Plants and animals operate with several layers of antiviral protoplasts to visualize nuclei and mitochondria (Fig. 6; defense. Filamentous fungi have two known anti-viral defense A. Eusebio-Cope and N. Suzuki, unpublished results). The DAPI- mechanisms: one operates at the cellular level (RNA silencing) stained chromosomes and nuclei which are discharged from the (Segers et al., 2007; Sun et al., 2009) and the other at the population growing hyphae of C. parasitica are shown in Fig. 6A. Nuclei in level (vegetative incompatibility) (Choi et al., 2012; Zhang et al., conidia (Fig. 6B, arrowhead) and hyphae (Fig. 6C, arrowhead) could 2014b). The former is the subject of this subdivision. The RNA be clearly stained by DAPI. Other non-FP methods include immuno- silencing or RNA-interference (RNAi), conserved across eukaryotic fluorescent techniques which are less established in fungal cytology kingdoms, targets viral RNAs for sequence-specificdegradation compared to those in plants and animals. A notable report was (Billmyre et al., 2013; Nuss, 2011; Pumplin and Voinnet, 2013). recently made by Dr. Pearson's group in which the intracellular viral These regulatory pathways are involved not only in anti-viral distribution was visualized via immuno-fluorescence (Boine et al., defense but also in genome defense against transposons in fungi 170 A. Eusebio-Cope et al. / Virology 477 (2015) 164–175

Fig. 5. Subcellular distribution of marker proteins in Cryphonectria parasitica hyphae. The EP155 strains expressing enhanced green fluorescent protein (eGFP)-marker proteins were routinely grown with cover slips on potato dextrose agar (PDA) at room temperature. Glass-surface-grown hyphae and eGFP-tagged proteins expression were observed using a confocal laser scanning microscopy (VF1000, Olympus) at 3-days culture. (A) Cells expressing the free eGFP. Fluorescence was evenly diffused throughout the cell. (B) Cells expressing the nuclei-targeting H1-eGFP protein. The ‘N’ indicates the nuclei. (C) Cells expressing eGFP-tagged mitochondrial marker protein (mt-GFP). Fluorescence image showed thread-like architectures typical of fungal mitochondria. (D) Cells expressing eGFP-tagged trans-Golgi network marker protein (Kex-eGFP). Note that fluorescent protein localized at small punctate structures which are presumably Golgi-derived vesicles. (E) Cells expressing eGFP-peroxisomal matrix protein (PER4) tagged by eGFP. Fluorescence is present at small punctate organelles. (F) Cells expressing eGFP tagged by transitional ER (tER) protein, SecE. The ‘N’ indicates the nuclei that are surrounded by the tER marker. Left panels show green fluorescence images, while right panels are phase contrast overlay images (right panel). Bars represent 5 μm.

(Nakayashiki and Nguyen, 2008). The basic RNA silencing pathway into the Argonaute complex effector called RNA-induced silencing entails three key components: Dicer-like (DCL), Argonaute-like complex (RISC) with ssRNase activities, and guides the RISC to (AGL), and RNA-dependent RNA polymerase (RDR). Virally derived target mRNAs. It should be noted that studies of quelling (repeated dsRNAs are perceived and digested into small dsRNAs of 21–26 nt in DNA silencing) in N. crassa contributed to identification of the length (small interfering RNAs, siRNAs) by dsRNA-specificRNase, aforementioned RNA silencing key factors (see below). Neverthe- DCL. RDR is involved in amplification of this class of small RNAs by less, in contrast to the situations in plant or invertebrate, it was only synthesizing long dsRNAs. One of the siRNA strands is incorporated recently that RNA silencing as an antiviral defense was uncovered A. Eusebio-Cope et al. / Virology 477 (2015) 164–175 171

Fig. 6. Cryphonectria parasitica stained with 40,6-diamidino-2-phenylindole (DAPI). (A) Mitotic chromosomes and nuclei discharged from the growing hyphae of C. parasitica strain EP155. Specimens were prepared by the germ tube burst method and stained with DAPI. HN, hyphal nucleus; I, interphase nucleus; P, presumably prophase nucleus; M, early metaphase nucleus with slightly slender chromosomes; mt DNA, presumably scattered mitochondrial DNAs with thread- or particle-like appearance. Scale bar: 5 μm. With permission, Fig. 6A and its caption was adapted from Fig. 1 of the Eusebio-Cope et al. (Fungal Genet Biol. 46, 342–351, 2009). (B) Germinated and non-germinated conidia showing conidial nuclei/nucleus (arrowhead). (C) Actively growing hypha showing intact nuclei (arrowhead).

Fig. 7. Antiviral RNA silencing in Cryphonectria parasitica and its suppression. Schematic representation of interaction between CHV1 and Cryphonectria parasitica RNA silencing. Replication intermediate of CHV1, a dsRNA form, is targeted by host DCL2 and processed into small interfering RNAs (siRNAs). One strand of viral siRNAs (red) is incorporated into and guides RISC (RNA-induced silencing complex) to target mRNA and AGL2 in the RISC cleaves CHV1 mRNA. While host RNA-dependent RNA polymerases (RdRs) are most likely non-essential elements, at minimum DCL2 and AGL2 are necessary for defense against homologous (CHV1, MyRV1) and heterologous (RnPV2, RnVV1, and RnMBV1) viruses. Infections of C. parasitica by some viruses lead to transcriptional induction of dcl2 and agl2. However, CHV1 counteracts this enhanced transcription with the p29-RSS and elevates accumulation levels of itself and co-infecting viruses such as MyRV1 and RnVV1. in fungi including C. parasitica (Segers et al., 2007) and possibly animals. Because these inductions are consistently observed by A. nidulans (Hammond et al., 2008). transgene-derived dsRNA in N. crassa (Choudhary et al., 2007) and In C. parasitica, one of two Dicer homologs, Dicer-like 2 (DCL2), C. parasitica (Sun et al., 2009), and cytoplasmically replicating RNA and one of four Argonaute homologs, Argonaute-like 2 (AGL2), are viruses (Sun et al., 2009), it is anticipated that dsRNA molecules in involved in defense against CHV1 and MyRV1 (Segers et al., 2007; the cytoplasm of a fungal cell trigger this event. For more details, Sun et al., 2009)(Fig. 7). Disruption mutants, Δdcl2 and Δagl2 see a review article by Nuss (Nuss, 2011). manifest severely debilitated symptoms upon receipt of CHV1 and As stated earlier, many of R. necatrix mycoviruses that are distinct MyRV1 compared to WT EP155, serving as a convenient pheno- dsRNA viruses with spherical particles can replicate in C. parasitica typic marker for RNA silencing that acts against those viruses in (Table 1). Interestingly, some of those viruses (RnPV2, RnVV1 and C. parasitica. None of RDRs are essential for RNA silencing in C. RnMBV1) are targeted efficiently by C. parasitica antiviral RNA parasitica (Zhang et al., 2014a), which differs from the N. crassa silencing, similar to the cases of homologous C. parasitica viruses system. Of note is that the genes of proteins DCL2 and AGL2 (dcl2 CHV1 and MyRV1 (Chiba et al., 2013a, 2013b; Salaipeth et al., 2014). and agl2) are significantly induced upon viral infections in C. Comparative analyses showed that accumulation levels of the parasitica (40-fold), a unique feature of fungal RNA silencing heterologous viruses in the WT fungal host (RNA silencing-compe- (Fig. 7)(Sun et al., 2009). Such high-level transcriptional induction tent) are significantly lower than those in an RNA silencing-deficient of the key RNA silencing genes is not observed in plants or fungal strain Δdcl2 (a dcl2 disruptant) (e.g., 20-fold less for 172 A. Eusebio-Cope et al. / Virology 477 (2015) 164–175

RnMBV1). In the course of such investigations, we made an intriguing Comparison of the C. parasitica/virus vs. other fungus/virus systems observation that the RnVV1-infection in the WT fungus resulted in no obvious induction of dcl2 (Chiba et al., 2013b). This is remarkably As a model system for genetics, biochemistry or developmental different from behaviors of CHV1 or MyRV1/C. parasitica and suggests biology, N. crassa (Dunlap et al., 2007) has greater advantages over C. different responsiveness of the RNA silencing genes to different virus parasitica due to its even shorter life cycle time, ease of crossing, and infections. Our recent experiment showed up-regulation of dcl2 well-established tools and resources represented by a number of expression by RnMBV1-infection (S. Chiba and N. Suzuki, unpub- gene knockout strains available from the Fungal Genetics Stock lished results), under the condition that the virus conferred hypo- Center (http://www.fgsc.net)(McCluskey et al., 2010). Other advan- virulence to C. parasitica (Salaipeth et al., 2014). By contrast, RnVV1 tages of N. crassa includes accessibility of tools and resources induced very mild symptoms in C. parasitica.Somemycoviruses, (expression vectors with regulatable promoters and many other while targeted by host RNA silencing, may have a counter-defense mutants) to the community, complete annotated genome sequence, mechanism to circumvent host surveillance. Alternatively, viral RSS and a detailed genetic map. In fact, the first RNA silencing compo- may inhibit transcriptional up-regulation of dcl2 and agl2 as does nents were identified in N. crassa, i.e., an RNA-dependent RNA CHV1 p29. polymerase QDE1 (quelling defective 1) (Cogoni and Macino, Counteraction of viruses to the host RNA silencing is well 1999a), an Argonaute protein QDE2 (Catalanotto et al., 2000), a RecQ documented in other eukaryotic kingdoms. Viruses have acquired helicase protein QDE3 (Cogoni and Macino, 1999b), and two Dicer- evolutionarily diverse RSSs for their survival (Burgyan and like proteins DCL1 and DCL2 (Catalanotto et al., 2004). In Havelda, 2011; Wu et al., 2010). Characterization of RSSs revealed N. crassa,therearetwoDCLs,threeRDRs,andthreeAGLs(Naka- various strategies for interfering with antiviral RNA silencing. yashiki and Nguyen, 2008). Most of them are shown to be involved in Importantly, these characterizations largely contributed to under- either of two RNA silencing pathways: quelling and meiotic silencing standing of the RNA silencing machinery itself. In mycoviruses, by unpaired DNA (MSUD). In C. parasitica,whilecomparative there are only two reported RSSs; a multifunctional cysteine genomics showed the presence of two DCLs, four AGL and four protease p29 encoded by CHV1 ORFA (Segers et al., 2006, 2007; RDR genes, only DCL2 and AGL2 were shown in RNA silencing- Suzuki et al., 2003) and VP10 encoded by MyRV3 segment 10 mediated antiviral defense. No MSUD phenomenon has been rep- fi (Yaegashi et al., 2013). This number is in stark contrast to the great orted for C. parasitica. However, the fatal de ciency of N. crassa as a number of RSSs from plant viruses. The exact mode of MyRV3 model host for studying fungal virology is the lack of viruses infecting it. Virus-like particles have been reported (Küntzel et al., 1973; VP10 is still unclear; however, p29 has an aforementioned, known Tuveson and Peterson, 1972); however, there are no reports on unique function: the p29-carrying (WT) CHV1 decreases induction viruses established as infectious agents in or research materials for levels of both genes for DCL2 and AGL2 relative to those induced this fungus. Once viruses infecting N. crassa arefoundinthefuture, by the p29-lacking counterpart virus (Segers et al., 2007; Sun et al., comparative functional genomics analyses would present interesting 2009)(Fig. 7). It is likely that through this action of p29 CHV1 findings. For example, it would be of great interest to examine which causes synergistic effects on RnVV1 and MyRV1 multiplications by genes are required for antiviral defense in N. crassa.Thedcl2 and agl2 co-infection and also transgenic expression of p29 (Chiba et al., of C. parasitica,albeitnotdcl1 or agl1, are up-regulated greatly by 2013b; Sun et al., 2006). Interestingly, CHV1-p29 and MyRV3-VP10 virus infection or transgenic expression of dsRNA. In N. crassa,dsRNA are functional in plant tissue as demonstrated by conventional induces some key genes for RNA silencing components, such as dcl2 fi agro-in ltration (Segers et al., 2006; Yaegashi et al., 2013). This and qde2 (Choudhary et al., 2007). It would be interesting to address encouraged us to investigate a reverse situation, but none of tested whether virus-infection responsiveness of RNA silencing key RNA plant viral RSSs, potyviral HC-Pro (N. Suzuki, unpublished results), silencing genes is conserved in N. crassa. cucumoviral 2b and tombusviral p19 were functional at least using There are some other important “model fungi” for other research transgenic expressions in C. parasitica (Chiba et al., 2013b). To our areas such as A. nidulans (fungal development and morphogenesis) knowledge, no RSSs from plant or animal viruses function in the (Wortman et al., 2009)andMagnaporthe oryzae (fungus/plant kingdom Fungi. These may imply some differences in key step(s) interactions) (Li et al., 2012). Diverse mycoviruses increasingly have of antiviral RNA silencing between plants and fungi, e.g., massive been reported in these fungi (e.g., Hammond et al., 2008; Urayama production of DCL2 and AGL2. et al., 2014). Unfortunately, methods for artificial virus introduction RNA silencing, particularly in C. parasitica, also attracts great into most of these fungi have not yet been developed. In this reg- attention not only as an antiviral defense but also as an integral ard, it is important that the transfection technique developed in player in viral RNA recombination. The CHV1 defective-interfering R. necatrix (Kondo et al., 2013b)andSclerotinia sclerotiorum (Jiang (DI) RNA molecules are often produced in WT, RNA silencing- et al., 2013) have redounded emerging hosts for studies on virus/ competent C. parasitica strains. However, CHV1 DI-RNA is not virus and virus/host interactions. generated or maintained in RNA silencing-defective strains, Δdcl2 or Δagl2. Nuss and colleagues suggest that production, horizontal Limitations of the C. parasitica as a virus host transfer and/or maintenance of CHV1 DI-RNA require RNA silencing components (Sun et al., 2009; Zhang and Nuss, 2008). The Unlike N. crassa resources and tools, those of C. parasitica are same mechanism operates for the instability of CHV1-based not systematically prepared for their public use, although they vectors (Sun et al., 2009). Interesting differences were found may be available upon direct request to respective researchers among DI-RNAs of dsRNA viruses that were different from the who prepared them. However, it should be noted that Dr. Chen's (þ) ssRNA virus, CHV1. A naturally occurring DI-RNA of a bi- group generated a mutant defective in non-homologous recombi- segmented dsRNA virus, RnPV2 (Table 1), is stably maintained nation that enhances homologous recombination for targeted gene both in WT and Δdcl2 of C. parasitica (Chiba et al., 2013a). disruptions (Lan et al., 2008). Also note that the development of a Moreover, genome rearrangements (recombinants) of a hetero- Cre/loxP-based gateway system for this fungus that can make logous dsRNA virus, MyRV1, were shown to be generated fre- multiple transformation by deletion and reuse of drug-resistant quently in C. parasitica by transgenic expression of CHV1-RSS p29 marker genes (Zhang et al., 2013). Thus, multiple-gene knockout (Sun and Suzuki, 2008; Tanaka et al., 2011). There may be a strains can be instantly prepared upon one's desire in C. parasitica. missing link between RNA silencing and RNA recombination that While the majority of fungal viruses are dsRNA viruses and is being explored in the Suzuki lab. (þ)ssRNA viruses, ()ssRNA viruses and gemini-like ssDNA A. Eusebio-Cope et al. / Virology 477 (2015) 164–175 173 viruses were recently discovered in filamentous ascomycetes Anagnostakis, S.L., 1987. Chestnut blight: the classical problem of an introduced (Kondo et al., 2013a; Liu et al., 2014; Yu et al., 2010). Neverthless, pathogen. Mycologia 79, 23–37. Andrie, R.M., Martinez, J.P., Ciuffetti, L.M., 2005. Development of ToxA and ToxB there are no reports on viruses with such genome types, i.e., promoter-driven fluorescent protein expression vectors for use in filamentous ()ssRNA viruses or ssDNA viruses, from C. parasitica. ascomycetes. Mycologia 97, 1152–1161. Other drawbacks include the lack of a versatile synchronized Arakawa, M., Nakamura, H., Uetake, Y., Matsumoto, N., 2002. Presence and infection system useful for time-course analysis of virus replication, distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix. Mycoscience 43, 21–26. and a simple and dependable system for RNA silencing suppressors, Bartoszewska, M., Opalinski, L., Veenhuis, M., van der Klei, I.J., 2011. 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Science 264, 1762–1764. fi plant and animal viruses, these research areas in fungal virology Chen, B., Geletka, L.M., Nuss, D.L., 2000. Using chimeric hypoviruses to ne-tune the fi interaction between a pathogenic fungus and its plant host. J. Virol. 74, are less unexplored partly due to the technical dif culties encoun- 7562–7567. tered by genetic and biochemical studies. However, some viral Chen, B.S., Choi, G.H., Nuss, D.L., 1994b. Attenuation of fungal virulence by synthetic factors associated with the replication cycle of C. parasitica infectious hypovirus transcripts. Science 264, 1762–1764. Chiba, S., Lin, Y.H., Kondo, H., Kanematsu, S., Suzuki, N., 2013a. Effects of defective- mycoviruses, for which transfection protocols are available, were interfering RNA on symptom induction by, and replication of a novel partiti- identified successfully. Moreover, transcriptome analyses showed virus from a phytopathogenic fungus Rosellinia necatrix. J. 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A series of studies related to this paper was financially supported in Churchill, A.C.L., Ciuffetti, L.M., Hansen, D.R., Vanetten, H.D., Van Alfen, N.K., 1990. part by Yomogi Inc. and the Science and Technology Research Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Curr. Genet. 17, 25–31. Promotion Program for Agriculture, Forestry, Fisheries and Food Cogoni, C., Macino, G., 1999a. Gene silencing in Neurospora crassa requires a protein Industry. The authors are grateful to Drs. Hideki Kondo, Masatoki homologous to RNA-dependent RNA polymerase. Nature 399, 166–169. Taga,Ken-IchiIkeda,andSatokoKanematsu,andcurrentandprevious Cogoni, C., Macino, G., 1999b. Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase. Science 286, 2342–2344. lab members of the Suzuki lab for fruitful discussion. We would also Craven, M.G., Pawlyk, D.M., Choi, G.H., Nuss, D.L., 1993. Papain-like protease p29 as like to thank Drs. 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