The GATA-type IVb zinc-finger transcription factor

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The GATA-type IVb zinc-finger transcription factor SsNsd1 regulates asexual–sexual development and appressoria formation in Sclerotinia sclerotiorum J I N G T A O L I 1 , 2 , W E N H U I M U 1 , 2 , S E L V A K U M A R V E L U C H A M Y 1 †, Y A N Z H I L I U 2 , Y A N H U A Z H A N G 1 , 2 , HONGYU PAN2,* AND JEFFREY A. ROLLINS 1,* 1

Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA College of Plant Science, Jilin University, Changchun, Jilin Province, 130062, China

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INTRODUCTION SUMMARY The sclerotium, a multicellular structure composed of the compact aggregation of vegetative hyphae, is critical for the longterm survival and sexual reproduction of the plant-pathogenic fungus Sclerotinia sclerotiorum. The development and carpogenic germination of sclerotia are regulated by integrating signals from both environmental and endogenous processes. Here, we report the regulatory functions of the S. sclerotiorum GATA-type IVb zinc-finger transcription factor SsNsd1 in these processes. SsNsd1 is orthologous to the Aspergillus nidulans NsdD (never in sexual development) and the Neurospora crassa SUB-1 (submerged protoperithecia-1) proteins. Ssnsd1 gene transcript accumulation remains relatively low, but variable, during vegetative mycelial growth and multicellular development. Ssnsd1 deletion mutants (Dnsd1-KOs) produce phialides and phialospores (spermatia) excessively in vegetative hyphae and promiscuously within the interior medulla of sclerotia. In contrast, phialospore development occurs only on the sclerotium surface in the wild-type. Loss of SsNsd1 function affects sclerotium structural integrity and disrupts ascogonia formation during conditioning for carpogenic germination. As a consequence, apothecium development is abolished. The Ssnsd1 deletion mutants are also defective in the transition from hyphae to compound appressorium formation, resulting in a loss of pathogenicity on unwounded hosts. In sum, our results demonstrate that SsNsd1 functions in a regulatory role similar to its ascomycete orthologues in regulating sexual and asexual development. Further, SsNsd1 appears to have evolved as a regulator of pre-penetration infectious development required for the successful infection of its many hosts. Keywords: appressorium, asexual development, NsdD, sexual development, spermatia.

*Correspondence: Email: [email protected]; [email protected] †Present address: Mountain Horticultural Crops Research & Extension Center, North Carolina State University, Mills River, NC 28759, USA

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Sclerotinia sclerotiorum (Lib.) de Bary is a devastating necrotrophic fungal plant pathogen of many agricultural crops, including sunflower, soybean, oilseed rape, edible dry bean, chickpea, dry pea, lentils and non-commelinid monocots, such as onion and tulip (Boland and Hall, 1994). Taxonomically, this fungus is a homothallic ascomycete belonging to the family Sclerotiniaceae of the order Helotiales. Developmentally, a hallmark of this and related fungi is the production of hardened, multicellular sclerotia formed from the aggregation of vegetative hyphae enclosed by a melanized rind layer. These structures play a significant role in the survival and persistence of infectious propagules in agricultural fields. Sclerotia can survive under harsh biological and physical environments, including low temperature, microbially active soils and dry environments, for several years. Under suitable environmental conditions, sclerotia germinate into either vegetative hyphae (myceliogenic germination) or apothecia (carpogenic germination), with the latter releasing large quantities of ascospores that initiate new disease cycles (Bolton et al., 2005). With its adaptations for long-term survival and pathogenicity on a broad range of hosts, S. sclerotiorum is one of the most challenging agricultural pathogens to manage and causes large global economic losses annually (Bolton et al., 2005). In addition to its role in survival, the sclerotium of S. sclerotiorum represents a transitional structure spanning asexual and sexual development. During the development of sclerotia, phialospores are produced on the sclerotium surface and are presumed to function as spermatia in fertilization, as demonstrated for related heterothallic species (Faretra et al., 1988; Uhm and Fujii, 1983). Following fertilization, ascogonia within the sclerotium medulla differentiate into apothecial stipes which rupture through the rind and, under suitable light conditions, differentiate an apothecial disc at the stipe apex. Sclerotia development involves several distinct stages (Li and Rollins, 2009) and is affected by numerous exogenous factors, such as photoperiod, temperature, oxygen concentration, ambient pH, mechanical factors and nutrients (Chet and Henis, 1975). At the same time, this process is also tightly regulated by intrinsic genetic factors. Independent and cross-talking signalling 1

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pathways have been shown to be involved in sclerotium development (Chen et al., 2004; Harel et al., 2005, 2006; Li and Rollins, 2009, 2010); in particular, phosphorylative relays involving AMP cyclase, ERK-like mitogen-activated protein kinase and serine/threonine (Ser/Thr) phosphatases type 2A and 2B (Erental et al., 2007; Harel et al., 2005; Jurick and Rollins, 2007) play key roles in this process. However, the details of the signal transduction pathways and the transcriptional regulation mechanisms governing sclerotia development remain to be elucidated. The Ssnsd1 gene is predicted to encode a GATA-type transcription factor orthologous to the Aspergillus nidulans nsdD (never in sexual development) and the Neurospora crassa sub-1 (submerged protoperithecia-1) genes, which are positive regulators of sexual development (Han et al., 2001; Teakle and Gilmartin, 1998). We functionally characterized the Ssnsd1 gene through gene deletion and demonstrated that it functions similarly to its ascomycete orthologues in balancing development as a negative regulator of phialospore formation and a positive regulator of sclerotium, compound appressorium and apothecium development.

RESULTS Identification and developmental expression profiling of the Ssnsd1 gene From the genome of S. sclerotiorum (Amselem et al., 2011; Derbyshire et al., 2017), eight proteins containing GATA-type DNA domains are predicted on the basis of PFAM annotation. These include orthologues of the circadian and photoresponsive white collar proteins (Wc-1 and Wc-2), in addition to SsNsd1. SsNsd1 is predicted to contain a single type IV GATA zinc-finger domain (C-X2-C-X18-C-X2-C) at its C-terminus. Its orthologous relationship to the A. nidulans NsdD (GenBank accession XP_660756), N. crassa SUB-1 (GenBank accession XP_960576) and Botrytis cinerea BcLTF1 (GenBank accession ANQ80444.1) (Schumacher et al., 2014) proteins was determined on the basis of the best bidirectional BLAST results (E value: 0.0 2 1e244) and subsequent reciprocal smallest distance (RSD) analysis (RSD score: 2.3). To define the regulation of Ssnsd1 expression more precisely, we examined its transcript accumulation across developmental stages by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression profile revealed varied expression patterns for Ssnsd1 in response to different developmental stages. Overall, transcript accumulation of Ssnsd1 was significantly higher in vegetative hyphae than during sclerotium and apothecium developmental stages, but no clear tissue-specific pattern was observed (Fig. 1). Generation of Ssnsd1 knockout (KO) mutants and genetic complementation strains The strategy used to generate the Ssnsd1 KO mutants is shown in Fig. S1A (see Supporting Information). Two independent

Fig. 1 Ssnsd1 expression levels in different developmental stages of Sclerotinia sclerotiorum. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to profile Ssnsd1 expression at different developmental stages relative to undifferentiated hyphae: sclerotial stages (S1, S2–3 and S4–5); apothecial stages (A1, A2–4 and A5–6). Gene expression values are normalized relative to the constitutively expressed gene histone H3 and comparisons are made between expression at each individual developmental stage relative to expression in hyphae. Values represent the means 6 standard deviation (SD) (n 5 3), which were compared by analysis of variance and a multiple comparisons test. Values with adjusted P values below 0.001 (**P < 0.001) were considered to be significantly different from the wild-type.

homokaryotic KO mutants (Dnsd1-KO1 and Dnsd1-KO2) were obtained after several rounds of hyphal tip transfer on 100 mg/mL hygromycin plates. Homologous recombination events were verified by PCR (Fig. S1B). The absence of PCR amplicons indicated the absence of the Ssnsd1 gene in the KO mutants (Dnsd1-KOs) and their homokaryotic gene deletion status. As an additional form of control for pleotropic effects of transformation, the empty vector (EV) was also transformed into wild-type (WT) protoplasts, and a representative strain was purified by hyphal tip transfer for further analysis (Fig. S1B). Ssnsd1 gene complementation transformants were produced from Dnsd1-KO2 protoplasts following transformation with the full-length Ssnsd1 gene. Transformants were selected on RM medium with 100 mg/mL nourseothricin, and four PCR-positive transformants (Cnsd1 strains) were purified by several rounds of hyphal tip transfer and PCR verification. One recovered strain Cnsd1-2 (Fig. S1B) was chosen for more detailed phenotypic characterization. Deletion of Ssnsd1 leads to excessive production of phialides and phialospores in vegetative culture The WT, EV, two Ssnsd1 deletion strains (Dnsd1-KO1/Dnsd1-KO2) and Dnsd1-KO2 complementation strain (Cnsd1-2) showed similar vegetative growth and developmental phenotypes under standard culture conditions without antibiotic selection, and were used

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Fig. 2 Morphological observation of wildtype (WT), empty vector (EV), the two Ssnsd1 deletion strains (Dnsd1-KO1/Dnsd1-KO2) and one Dnsd1-KO2 complementation strain (Cnsd1-2). WT and mutant strains were inoculated on autoclaved smashed potato medium plates at 25 C for 12 days and colony phenotypes were photographed.

for further morphological analyses (Fig. S2, see Supporting Information). When cultures were observed following 12 days of cultivation on smashed potato agar (SPA) medium, the Dnsd1-KOs exhibited excessive formation of phialides and phialospores over the entire colony surface (Fig. 2). To confirm this phenotype, WT and mutant strains were inoculated on different growth medium plates [5-cm-diameter potato dextrose agar (PDA), 9-cm-diameter SPA and 15-cm-diameter SPA plates at 25 C] for 12, 14 and 21 days, respectively (Fig. S3, see Supporting Information). Dramatic phialospore overproduction in DSsnsd1-KOs relative to WT and controls was observed on all media. The differentiation of phialides and phialospores from hyphae began after sclerotia formation (day 10 in cultivation) and continued for 2–3 weeks in the Dnsd1-KOs. Phialides and phialospores were clearly observed from Dnsd1-KOs, but only undifferentiated hyphae were observed from WT and Cnsd1-2 strains (Fig. 3A). Quantification indicated that the two Dnsd1-KOs produced up to 107 phialospores per 5-cm PDA plate, whereas the numbers of phialospores produced by the WT and Cnsd1 strains were negligible (Fig. 3B). Phialides and phialospores could only be observed on the sclerotia surface for the WT and complemented Cnsd1 strains (data not shown). These results demonstrate that SsNsd1 functions as a negative regulator of phialides and phialospore differentiation in WT S. sclerotiorum. Deletion of Ssnsd1 affects the structural integrity of sclerotia and disrupts ascogonia formation during carpogenic germination Although Dnsd1-KOs successfully formed sclerotia in culture, their surface, size and average mass differed from those produced by

the WT and Cnsd1 strains (Fig. 4). Dnsd1-KOs developed more hyphae on the surface of sclerotia than the WT and Cnsd1 strains on average (Fig. 4A), and the sclerotia were generally smaller (Fig. 4B), accounting for the reduction in total sclerotia dry weight of Dnsd1-KOs compared with WT and Cnsd1 strains (Fig. 4C). We tested the germination competence of Dnsd1-KO sclerotia. Mature (14-day) sclerotia of WT, EV, Dnsd1-KO1/Dnsd1-KO2 and Cnsd1-2 strains were induced for carpogenic germination using standard methods (see Experimental procedures). Germination was monitored for 90 days with incubation at 15 8C. Apothecium germination was only observed from the WT and EV strains, but not from the Dnsd1-KOs or Cnsd1 strains. Mature sclerotia from all strains were indistinguishable for myceliogenic germination (data not shown). We further compared the microscopic features of sclerotia between WT and Dnsd1-KOs during the carpogenic germination process. Thin sections of sclerotia were prepared and examined over time, beginning with mature sclerotia, through the conditioning period and ending when the WT produced apothecia. Microscopic observations indicated that the internal tissue of mature Dnsd1-KO sclerotia was disorganized, with an expanded rind layer and loosely organized medulla tissue (Fig. 5). During the carpogenic germination incubation period (WK3–4), the mutant medulla tissue became further disorganized with a loss of cell-to-cell adhesion, loss of cell integrity and a breakdown of tissue organization (Fig. 5). In contrast, following conditioning and 3 weeks of incubation at 15 8C, ascogonia were frequently observed within the medulla of WT sclerotia just inside the cortex layer (Fig. 5). By week five, apothecia began to emerge from the WT sclerotia, whereas the mutant sclerotia had a shrunken appearance and severe internal tissue disorganization was obvious. In the Dnsd1KO mutants, numerous pockets of developing phialides and

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Fig. 3 Production of phialospores (spermatia) on potato dextrose agar (PDA) medium following 3 weeks of cultivation of wild-type (WT), empty vector (EV), Dnsd1KO1/Dnsd1-KO2 and Cnsd1–2. (A) WT and Cnsd1-2 hyphae, phialides and phialospores from Dnsd1-KO2. a, phialides; b, phialospores. (B) Phialospore numbers were determined by haemocytometer quantification. The experiment was repeated three times and means 6 standard deviation (SD) are reported from one representative experiment.

phialospores were produced in the medulla at this same time point (Fig. 5). Deletion of Ssnsd1 abolishes compound appressorium development To determine whether SsNsd1 influences other multicellular developmental stages of S. sclerotiorum, we examined compound appressorium (infection cushion) development in the KO mutants. In the in vitro assay, WT and EV strains rapidly differentiated pigmented compound appressoria from vegetative hyphae on contact with parafilm. No pigmented compound appressoria were differentiated by the Dnsd1-KOs (Fig. 6A). This compound appressorium differentiation defect was significantly restored in the complementation strain Cnsd1-2 (Fig. 6A). We further tested whether the compound appressorium deficiency of Dnsd1-KOs occurred in planta using onion epidermal strips. The WT, EV and

complemented strains differentiated compound appressoria 1 day after inoculation, but no appressoria were observed in the KO mutants (Fig. 6B). Four days after inoculation, the WT, EV and Cnsd1-2 strains could penetrate onion cells, but no penetration was observed from the Dnsd1-KO mutants (data not shown). Deletion of Ssnsd1 results in a penetration-dependent loss of pathogenicity To determine whether the compound appressorium deficiency of Dnsd1-KOs causes a virulence defect on other unwounded/ wounded host tissues, infection assays were performed (Fig. 7). The WT, EV, Cnsd1-2 and Dnsd1-KO strains were inoculated on detached unwounded celery. By 2 days after inoculation (DAI), the WT and EV strains infected celery, developed visible lesions as soon as 24 h after inoculation and continued colonization of celery tissues; however, the Dnsd1-KOs failed to form lesions even by



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Fig. 4 Effect of Ssnsd1 gene deletion on sclerotium development in culture. (A) Sclerotium morphology of the wild-type (WT), empty vector (EV), the two Ssnsd1 deletion strains (Dnsd1-KO1/Dnsd1-KO2) and the complementation strain Cnsd1-2. Sclerotia were collected from 15-cm smashed potato agar at 12 days after inoculation (DAI). (B) Comparison of sclerotia size amongst WT, EV, Dnsd1-KO1/DSsnsd1-KO2 and Cnsd1-2 strains. (C) Average sclerotium dry weight collected from 9-cm culture plates. All experiments were repeated three times and means 6 standard deviation (SD) are reported from one representative experiment. Dry weights determined to be statistically different from the wild-type by analysis of variance with a multiple comparisons adjusted P value of less than 0.001 are indicated (**P < 0.001).

4 DAI (Fig. 7A). The WT fully colonized celery by 3 DAI, but the Cnsd1-2 strain was delayed by 3–4 days (Fig. 7B). On wounded celery, both the WT and mutants formed visible lesions, and no difference in virulence was observed (Fig. 7C). On detached unwounded tomato leaves (Fig. 7D), the WT infected plants by 1 DAI, similar to celery, the Cnsd1 strains formed visible lesions by 3 DAI, and no visible lesions were formed on the leaves inoculated with Dnsd1-KOs even up to 6 DAI (Fig. 7D). On wounded tomato leaves, the Dnsd1-KO mutants formed lesions similarly to the WT and EV strains.

DISCUSSION The Ssnsd1 orthologue sub-1 was first identified as a positive regulator of sexual development from a high-throughput screen of transcription factor mutants in N. crassa (Colot et al., 2006). Phenotypically, loss-of-function sub-1 mutants are arrested at the stage of protoperithecia development. In subsequent transcription profiling studies, sub-1 was identified as a late light response

gene. In addition, nearly all late light response genes are misregulated in the loss-of-function sub-1 mutant in a white collar complex-dependent manner (Chen et al., 2009). This role in N. crassa is also consistent with the reported function of the A. nidulans NsdD orthologue as a positive regulator of sexual development and component of the endogenous regulatory circuit including brlA, rosA, nosA and nsdB functioning to balance asexual and sexual development (Han et al., 2001; Lee et al., 2014, 2016). Given the positive roles of Sub-1 orthologues in light-responsive sexual development, we hypothesized that loss-of-function DSsnsd1 mutants would exhibit phenotypes associated with lightdependent apothecium development. Instead, phenotypes were observed at earlier stages of the life cycle, specifically in sclerotium development and the transition from the quiescent asexual sclerotium to the developing sexual apothecium. Additional regulatory roles in apothecium disc development may exist, but the loss-of-function phenotype pre-empted our ability to assess the regulatory role of SsNsd1 on post-germination stages of apothecial development. These observed phenotypes in sclerotium


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Fig. 5 Sclerotial thin sections from wild-type (WT) and Ssnsd1 gene deletion mutant Dnsd1-KO2. Mature sclerotia were cycled at 220 8C for 24 h and at room temperature for 24 h three times, and then placed at 15 8C under constant cool white fluorescent lights for 3 weeks (WK3), 4 weeks (WK4) and 5 weeks (WK5). Thin section samples were stained with cotton blue and examined by light microscopy. Representative photographs are presented.

Fig. 6 Morphological features of in vitro/in vivo-produced compound appressoria of wildtype (WT), empty vector (EV), Dnsd1-KO1/Dnsd1-KO2 and Cnsd1-2 strains. (A) Pigmented compound appressoria were observed on parafilm at 4 days after inoculation (DAI) using 5-mm-diameter mycelial plugs. (B) Compound appressoria on onion epidermal strips were observed by light microscopy at 1 DAI. The expanded image shows the restored compound appressorium of the Cnsd1-2 strain.



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Fig. 7 Pathogenicity assays with the wild-type (WT), empty vector (EV), two Ssnsd1 deletion strains (Dnsd1-KO1/Dnsd1-KO2) and complementation strain Cnsd1-2 on unwounded/wounded celery stems and tomato leaflets. Mycelia-colonized potato dextrose agar (PDA) plugs were used for inoculation. (A) WT, EV, Dnsd1-KO1/ Dnsd1-KO2 and Cnsd1-2 strains on unwounded celery stems at 0, 2 and 4 days after inoculation (DAI). (B) Lesion diameters were measured on unwounded celery stems daily until 6 DAI. Lesion diameters determined to be statistically different from WT by a two-way analysis of variance, and diameters at each time point considered to be different from WT with a multiple comparisons adjusted P value of less than 0.001, are indicated (*P < 0.001). (C) WT, EV, Dnsd1-KO1/Dnsd1KO2 and Cnsd1-2 strains on wounded celery stems at 0, 2 and 3 DAI. (D, E) WT, EV, Dnsd1-KO1/Dnsd1-KO2 and Cnsd1-2 strains infecting unwounded/wounded tomato leaflets. Photographs were taken on the indicated days following inoculation.

development are still of interest for a better understanding of sexual development, as the sclerotium represents a transitional stage connecting the mitotic and meiotic cycles in Sclerotiniaceae species. In B. cinerea, loss-of-function mutants and overexpression mutants for the SsNsd1 orthologue BcLTF1 exhibited conidiation phenotypes consistent with the role of BcLTF1 as a negative regulator of conidiation, whilst completely abolishing sclerotium development (Schumacher et al., 2014). Although S. sclerotiorum does not produce a propagative conidial stage, it does produce phialospores whose ontogeny, although not borne on conidiophores, is similar to the conidia of Aspergillus spp. Our results showed that the Ssnsd1 deletion mutants produce many thousand-fold more phialospores compared with the WT and in a developmental context not observed in the WT, i.e. in vegetative culture and within the interior of sclerotia. Smaller sized mature sclerotia with expanded rind and cortex layers are also prominent features of the DSsnsd1 mutants. One hypothesis concerning these

phenotypes is that tissues functioning in the sexual cycle are specified early during sclerotium development and not strictly during the ‘conditioning’ period coincident with ascogonium differentiation. In support of this hypothesis, phialospore development in the WT occurs external of the rind during sclerotium maturation; likewise, compatible receptive cells in the rind and cortex may also begin their specification during this developmental period. The proliferation of these tissues during sclerotium development in DSsnsd1 mutants may represent a void in meiotic sexual tissue specification that is filled by mitotic development. Although phialospores function as spermatia in the sexual cycle (Faretra et al., 1988; Uhm and Fujii, 1983), they are mitotic spores and, given the dual asexual–sexual nature of the sclerotium, the phenotypic effects of DSsnsd1 mutation, leading to a block in apothecial development and the overproduction of phialospores, are consistent with the functions of Sub1 and NsdD in providing regulatory balance of asexual and sexual development


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in ascomycete fungi (Han et al., 2001; Tsitsigiannis et al., 2004). The lack of sclerotium development in the B. cinerea loss-offunction mutant relative to the misregulation of tissue specificity in the analogous S. sclerotiorum mutant may have its basis in the function of these proteins as regulators of photoresponses. Although B. cinerea exhibits photoregulation of sclerotium development, with B. cinerea sclerotia only produced in the absence of light, S. sclerotiorum forms sclerotia in the light and in the dark. BcLTF1 loss-of-function mutants may be blocked at a darkdependent initiation stage before tissue specification, akin to the lack of dark-dependent cleistothecia observed in the A. nidulans loss-of-function nsdD mutants (Han et al., 2001). Although the Ssnsd1-overexpressing complementation strain (Cnsd1–2) phenotype is not fully WT, in that compound appressoria and apothecia production are not completely restored, the reduction in phialospore production and the number, size and structural development of sclerotia are restored. Our interpretation of these results is that precise expression balance is required for a full restoration of positively regulated phenotypes not achieved by ectopic overexpression complementation. Examination of the Ssnsd1 upstream sequence [National Center of Biotechnology Information (NCBI) Assembly accession GCA_001857865.1] reveals an upstream intergenic region of greater than 12 kb. This large non-protein coding region may function as a complex regulatory sequence subject to multiple competing factors and dynamics to properly balance the expression of Ssnsd1 and the temporal and spatial activity of the SsNsd1 protein. Of particular interest concerning phenotypic variation between orthologous mutants in B. cinerea and S. sclerotiorum are infection defects. Although B. cinerea Dbclft1 mutants exhibited a post-penetration virulence defect, but no significant defect in compound appressorium (infection cushion) development, the S. sclerotiorum DSsnsd1 mutant was essentially reversed. In this study, the DSsnsd1 mutant failed to produce compound appressoria, and wounding the plant tissue prior to inoculation completely rescued the virulence defects of Dnsd1-KO strains. This result demonstrates that penetration failure as a result of a lack of compound appressorium development is solely responsible for the virulence defects of Dnsd1-KOs. SsNsd1 appears to strictly regulate development and plays no discernible role in regulating pathogenicity post-penetration, whereas, in B. cinerea, the orthologous gene appears to only affect post-penetration virulence. Phenotypic effects on both sclerotium and compound appressorium development are a common finding amongst developmental mutants of B. cinerea and S. sclerotiorum. For example, the adenylate cyclase (sac1) gene, which regulates cyclic AMP levels, negatively impacts compound appressorium development in S. sclerotiorum, whilst reducing growth and producing sclerotia that fail to carpogenically germinate (Jurick and Rollins, 2007). Similarly, the g-glutamyl transpeptidase (Ss-ggt1), through apparent redox

imbalance, produces defective compound appressoria and sclerotia (Li et al., 2012). In addition, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase encoding nox genes are involved in the regulation of development through reactive oxygen species generation. RNAi-Ssnox1 strains are unable to produce sclerotia and are reduced in virulence, whereas RNAi-Ssnox2 strains produce only limited numbers of sclerotia (Ayarpadikannan et al., 2011). In B. cinerea, both bcnoxA and bcnoxB gene deletion mutants are unable to form sclerotia and bcnoxB mutants are further impaired in appressorium development (Ayarpadikannan et al., 2011; Segmuller et al., 2008). Although the regulatory connections between sclerotia and compound appressoria are not understood and variation exists between S. sclerotiorum and B. cinerea, unique regulators and links amongst signalling pathways may be elucidated as we gain more information on the suite of regulators affecting these developmental stages, such as SsNsd1 and their downstream regulated genes. In conclusion, the Ssnsd1 gene from S. sclerotiorum is characterized as an orthologue of the nsdD and sub-1 GATA-type zinc-finger transcription factor encoding genes. This study provides evidence consistent with other fungal NsdD orthologues that SsNsd1 plays a critical role in balancing sexual and asexual development, as loss of SsNsd1 function leads to smaller sized sclerotia and increased phialospore production, whilst blocking ascogonium and subsequent sexual fruiting body development. The Ssnsd1 deletion mutants were also defective in compound appressorium formation and lost pathogenicity on unwounded hosts. The difference in infection phenotype compared with the related broad-host-range pathogen B. cinerea exemplifies how these closely related species have evolved unique regulatory means to achieve similar host compatibility outcomes. Further studies linking signalling pathways and downstream genes have the potential to increase our understanding of the common and unique pathways adopted for specifying multicellular sexual and asexual development in this and other filamentous fungi.

EXPERIMENTAL PROCEDURES Fungal strains, culture conditions and plant materials The fully genome-sequenced WT S. sclerotiorum isolate 1980 (Amselem et al., 2011; Derbyshire et al., 2017) was used to derive all strains in this study. Cultures were routinely grown on PDA (Difco, Detroit, MI, USA) at room temperature. Transformation mutants were cultured on PDA medium supplemented with either 100 mg/mL hygromycin B (EMD Biosciences, La Jolla, CA, USA) or 100 mg/mL nourseothricin (Phyto Technology Laboratories, Shawnee Mission, KS, USA). Hyphal stocks were maintained as desiccated mycelia-colonized filter paper at 220 8C and as dry sclerotia at 4 8C.

Isolation and manipulation of nucleic acids Genomic DNA was isolated based on the A. nidulans DNA mini-preparation protocol, as described previously (Yelton et al., 1984). Total RNA was extracted from lyophilized mycelia, sclerotia and apothecia tissues using Trizol



reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA electrophoresis was conducted as described previously (Rollins and Dickman, 2001). The Ssnsd1 gene sequence is annotated as locus Sscle16g109570 (GenBank accession CP017829) in the completed, gapless (excluding the rRNA repeat) version of the S. sclerotiorum genome (Derbyshire et al., 2017), and is identical to GenBank accession ANQ80447 deposited as S. sclerotiorum light responsive transcription factor 1. Orthologous gene analysis with the predicted S. sclerotiorum proteome (ASM185786v1) was conducted using the RSD algorithm (Wall and DeLuca, 2007) queried to the genomes of N. crassa (GenBank accession AABX00000000), A. nidulans (GenBank accession NZ_AACD00000000) and B. cinerea (GenBank accession GCA_000832945.1) with the following parameters: divergence 5 0.5, threshold 5 1e-10, number of top blast hits 5 3, fix alpha 5 1 and alpha 5 1.53.

Ssnsd1 gene expression qRT-PCR analysis of Ssnsd1 transcript accumulation in WT tissue from several developmental stages of the life cycle was performed using total RNA isolated from hyphae cultured on cellophane PDA plates, sclerotial stages S1, S2–3 and S4–5, as defined previously (Li and Rollins, 2009), and apothecial stages 1 (dark etiolated stipes), 2–4 (early expanding discs) and 5–6 (late expanding discs) (Veluchamy and Rollins, 2008). RNA was extracted and used for reverse transcription with a PrimeScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa, Mountain View, CA, USA). The cDNA was diluted 20-fold, and quantitative expression assays were performed using a TaKaRa SYBRV Green Reagent Kit with an Applied Biosystems (Foster, CA, USA) 7500 real-time PCR detection system according to the manufacturer’s protocol (TaKaRa). Primer pairs used for qRT-PCR are listed in Table S1 (see Supporting Information). qRT-PCR experiments were repeated with RNA from three biological replicates. The relative quantification method (22DDCt ) was used to quantify differences in Ssnsd1 transcript levels between the different developmental stages relative to vegetative hyphae. Data were normalized against housekeeping gene histone H3 (Sscle15g103590; GenBank accession CP017828.1). Statistical analysis was performed by comparing 2–DDCt data between each of the developmental stages and hyphae. The data were first tested and found to pass (alpha 5 0.05) the D’Agostino and Pearson normality test and the Brown–Forsythe test for equal variance. Subsequently, analysis of variance, followed by Dunnett’s multiple comparisons test, was utilized to determine the significance of the differences in Ssnsd1 transcript accumulation between each stage of development compared with hyphae. Samples with adjusted P values below 0.01 were considered to be significant. Data are presented as a histogram of expression levels normalized relative to hyphae (Li et al., 2016). These and subsequent statistical analyses were performed using Graphpad Prism version 7.0c for Mac (Graphpad Software, Inc., La Jolla, CA, USA). R

Gene replacement and complementation The Ssnsd1 gene replacement vector construction strategy has been described previously (Jurick et al., 2004). The gene-specific primers used to amplify the 1.1-kb 50 -untranslated region (50 -UTR) of the Ssnsd1 gene with an added AscI restriction enzyme site (underlined) and the 1.2-kb 30 UTR of the Ssnsd1 gene sequence with an added AscI restriction enzyme site (underlined) from the S. sclerotiorum genomic DNA are shown in

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Table S1: 50 -SsNsd1-F, 50 -SsNsd1-R, 30 -SsNsd1-F and 30 -SsNsd1-R. The two fragments were cloned into independent pGEM-T vectors (Promega, Madison, WI, USA) and named pNSD1–50 and pNSD1–30 , respectively. pNSD1–50 was double digested with NotI-AscI and the 1.2-kb Ssnsd1-50 fragment was ligated into NotI and AscI sites upstream of the Ssnsd1-30 sequence on pNSD1–30 . The resulting vector was named pNSD1–50 130 . A hygromycin phosphotransferase (hph) cassette containing the trpC promoter and terminator released from the pGEM-HPH vector by AscI digestion was ligated into pNSD1–50 130 to obtain a 7.5-kb Ssnsd1 gene replacement vector pNSD1–50 1hph130 . Primers (50 -SsNsd1-F/Hy and yg/ 50 -SsNsd1-R) were used to PCR amplify two overlapping Ssnsd1-hph fragments from the pNSD1–50 1hph130 template (Fig. S1 and Table S1). These fragments were used with the split marker strategy to replace the Ssnsd1 coding sequence with the hygromycin resistance cassette (Hyg) during transformation. The S. sclerotiorum protoplast transformation method was used, as described previously by Rollins (2003). For genetic complementation of the DSsnsd1 KO mutant, primers (C-SsNsd1-F and CSsNsd1-R) were used to amplify a 1.5-kb fragment with added NocI and NotI restriction enzyme sites (underlined) containing the full-length Ssnsd1 open reading frame sequences (Table S1). This 1.5-kb fragment was cloned into pNDN-OGG vector (Schumacher, 2012) under the oliC promoter for transformation with 100 mg/mL nourseothricin selection on RM medium (Sucrose 239.6g/L; Yeast Extract 0.5 g/L; Agar 15g/L for bottom agar and 8g/L for top agar). The restriction enzyme digestions, gel electrophoresis, DNA fragment purifications and ligations were performed using standard procedures (Sambrook and Russell, 2001).

Morphological characteristics and phenotypic analysis The WT, DSsnsd1 gene deletion and complementation strains were cultured on PDA medium at room temperature. Onion epidermal strips were used for inoculation with a colonized PDA agar plug (5 mm in diameter) for observations of hyphae and compound appressorium development using light microscopy (Leica model DM R HC, Wetzlar, Germany). Mature sclerotia from each strain, including the WT, EV (pGEM-HPH) transformant, Ssnsd1 KO strains (Dnsd1-KO1/Dnsd1-KO2) and Dnsd1-KO2 complementation strain (Cnsd1-2), were produced on autoclaved SPA at room temperature. These sclerotia were used for size comparisons and weight measurements, and also to assess carpogenic germination. Sclerotia were picked from individual plates, dried in a desiccator at room temperature for 6 days, photographed with a Nikon P100 digital camera (Nikon, Melville, NY, USA) and weighed. The average sclerotium weight was found to be normally distributed by the D’Agostino and Pearson normality test, and each strain was compared with WT by a one-way analysis of variance followed by Dunnett’s multiple comparisons test. The experiment was repeated three times and results from one representative experiment are presented. To induce apothecia, mature sclerotia were surface sterilized with 6% household bleach for 5 min and rinsed for 5 min with sterile water three times; sclerotia were covered with 70% ethanol for 5 min and then dried in a laminar flow hood on sterile paper towels overnight. Approximately 50 dry sclerotia were uniformly spread on a glass Petri dish (10 cm in diameter) containing autoclaved, water-saturated vermiculite. Plates were cycled between 220 8C for 24 h and room temperature for 24 h three times, and then placed in a 15 8C incubator with constant cool white fluorescent lights. Sclerotia were sampled at each step of this


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process and at weekly intervals during the 15 8C incubation, fixed and embedded in paraffin using the method described previously by Kladnik et al. (2004). Embedded samples were sectioned at 3 mm with a rotary microtome HM325 (Richard-Allan Scientific, Kalamazoo, MI, USA), stained with cotton blue and examined by light microscopy, as described previously (Li and Rollins, 2009). Each strain was cultured on three independent 9-cm PDA plates to assess phialospore production. Following 3 weeks of incubation at room temperature, 5 mL of sterile water were spread on the surface of each culture plate using a cotton swab. The resulting suspensions were quantified for the total number of phialospores per plate using a haemocytometer, and the mean value 6 standard deviation was calculated for each strain. Phialospore production for each strain was compared with WT, but not statistically analysed as the WT values were always zero. The experiment was repeated three times and results from one representative experiment are presented.

Pathogenicity assays Tomato (Lycopersicon solanum cv. Bonnie Best) plants were grown in the glasshouse under natural sunlight, with temperature in the 16–25 8C range. Celery (Apium graveolens) was purchased from a local grocery store and the stalks were washed with running water and cut into 3-cm pieces for inoculation. Fresh tomato leaflets and celery stems were inoculated with a PDA-colonized agar plug of WT, EV, DSsnsd1 gene KO and complementation strains at room temperature; three leaflets/stems were inoculated with each strain and the experiment was repeated three times. For celery stem inoculations, lesion diameters, including the 5-mm inoculation plug, were measured from the day of inoculation to day 6 after inoculation (0–6 DAI) from three independent stem pieces. Average lesion diameters 6 standard deviation of one representative experiment and accompanying photographs are presented. Lesion diameters were determined to be normally distributed by the D’Agostino and Pearson normality test, and lesion diameter values for each strain at each time point were compared with WT by a two-way analysis of variance followed by Dunnett’s multiple comparisons test. Photographs were taken every day after inoculation with a Nikon P100 digital camera and representative photographs are presented.

ACKNOWLEDGEMENTS We gratefully acknowledge Ulla K. Benny for her excellent technical assistance and Nicholas Dufault for advice on statistical analysis. This work was financially supported by the Florida Agriculture Experiment Station project FLA-PLP-005374 and the National Natural Science Foundation of China (31471730, 31271991, 31772108). Jingtao Li was supported by the China Scholarship Council for 2 years of study at the University of Florida. All authors declare that there are no conflicts of interest.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s website: Fig. S1 Gene replacement strategy used to create Ssnsd1 knockout mutants and polymerase chain reaction (PCR) analysis to identify and confirm knockout and overexpression complementation transformants. (A) The Ssnsd1 locus and homologous

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recombination-based gene replacement of the Ssnsd1 coding sequence. (B) PCR analysis to identify the Ssnsd1 gene replacement, determine the genetic purity of the Dnsd1-KO strain and confirm the Ssnsd1 complementation (Cnsd1) strain. All templates were genomic DNA and the primer pairs are given in Table S1 (see Supporting Information). nsd1, open reading frame (ORF) of the nsd1 gene amplified with the primers CSsNsd1-F and C-SsNsd1-R; OE-nsd1, nsd1 gene under the oliC promoter amplified with the primers Polic-F and C-SsNsd1-R; Hyg, partial hygromycin resistance cassette amplified with the primers yg and Hy. Fig. S2 In vitro early hyphal growth phenotypes of wild-type (WT) and mutant strains on potato dextrose agar (PDA) plates. (A) Each strain [WT, Ssnsd1 deletion strain (Dnsd1-KO2) and Dnsd1-KO2 complementation strain (Cnsd1-2)] was cultured on PDA medium without/with antibiotic [100 mg/mL hygromycin (HygB) or 100 mg/mL nourseothricin (Nour)]. Photographs were taken 1 day after inoculation (DAI). (B) Mycelial radial growth on PDA without selection was measured at 0, 6, 12, 24 and 36 h after inoculation (HAI). (C) Mycelial radial growth on PDA with selection (for Dnsd1-KO1, Dnsd1-KO2 and Cnsd1-2) or without selection [WT and empty complementation vector (EV)] measured at 0, 6, 12, 24 and 36 HAI. Fig. S3 Morphological observations of wild-type (WT), empty complementation vector (EV), the two Ssnsd1 deletion strains (Dnsd1-KO1/Dnsd1-KO2) and one Dnsd1-KO2 complementation strain (Cnsd1-2) on different growth medium plates. WT and mutant strains were inoculated on 5-cm-diameter potato dextrose agar (PDA), 9-cm-diameter potato medium and 15-cmdiameter potato medium plates at 25 8C for 12, 14 and 21 days, respectively. DAI, days after inoculation. Table S1 Primers used for Ssnsd1 gene replacement and complementation.
