Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum

Fungal Genetics and Biology 44 (2007) 521–530 www.elsevier.com/locate/yfgbi

Deletion of the adenylate cyclase (sac1) gene aVects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum Wayne M. Jurick II , JeVrey A. Rollins ¤ Department of Plant Pathology, University of Florida, Gainesville, FL 32611-0680, USA Received 2 August 2006; accepted 7 November 2006 Available online 18 December 2006

Abstract Sclerotinia sclerotiorum, a broad host range plant pathogen, produces pigmented, multihyphal sclerotia that are capable of long-term survival. Under favorable conditions, sclerotia carpogenically germinate to give rise to apothecia and forcibly discharged ascospores which serve as the primary source of inoculum in the disease cycle. The molecular regulator(s) of sclerotial development in Wlamentous fungi are largely unknown; however, pharmacological data has revealed that cyclic AMP (cAMP) negatively regulates sclerotial biogenesis in S. sclerotiorum. Based on this observation, we analyzed the role of cAMP by deleting the single copy adenylate cyclase (AC) sac1 gene from S. sclerotiorum. In culture, cyclic AMP levels in the knock-out (KO1) strain were greatly reduced compared to wild type, the hyphal branching pattern was altered, microconidia (spermatia) were more abundant, and aberrant sclerotia were produced in a concentric pattern. The KO1 strain was pathogenic on mechanically wounded tissues; however, virulence was severely attenuated. The pathogenicity defect on unwounded leaves is attributed to the absence of infection cushions and the attenuated virulence on wounded leaves correlates with the slow growth rate observed in culture. This study presents the Wrst description of an adenylate cyclase mutant that aVects both pathogenicity and sclerotial development in a broad host range necrotroph. © 2006 Elsevier Inc. All rights reserved. Keywords: Sclerotinia sclerotiorum; Adenylate cyclase; cAMP; Oxalic acid; Pathogenicity; Sclerotia; Hyphal branching pattern; Necrotroph; Attenuated virulence; Targeted gene deletion

1. Introduction Sclerotinia sclerotiorum does not produce viable mitotic spores, but is vegetatively propagated by myceliogenic germination of multihyphal, melanized, sclerotia. Sclerotia are capable of resisting physical, chemical and microbial degradation and can remain viable for up to 8 years in the soil (Adams and Ayers, 1979; Chet and Henis, 1975; Willets and Wong, 1980; Willets and Bullock, 1992). After appropriate conditioning (temperature, light, moisture, etc.), the sclerotia of this homothallic species can carpogenically germination to produce apothecia. A sclerotium can give rise to multiple apothecia that can individually produce as many as 3 £ 107 ascospores (Abawi and Grogan, 1979). These ascospores, which are forcibly discharged from the apothecium, are the primary source of inoculum in most *

Corresponding author. Fax: +1 352 392 6532. E-mail addresses: [email protected] (W.M. Jurick), [email protected] (J.A. Rollins). 1087-1845/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2006.11.005

Sclerotinia diseases and are critical for the maintenance and spread of the disease in the Weld (Steadman, 1979). Since sclerotia play a pivotal role in the disease cycle of S. sclerotiorum, previous studies have focused on determining the physiological factors that regulate sclerotial development (Chet and Henis, 1975; Le Tourneau, 1979; Willets and Bullock, 1992; Willets and Wong, 1980). However, few studies have been aimed at identifying the underlying molecular and biochemical mechanisms that signal and control sclerotial morphogenesis. Rollins and Dickman (1998) implemented a pharmacological screen to identify signaling components that aVected sclerotial development in S. sclerotiorum. They found that high intracellular cAMP levels inhibited sclerotial initiation and concomitantly raised oxalate levels which led them to hypothesize that a cAMP-dependent signaling pathway is involved in regulating the transition from mycelial growth to sclerotial initiation. The primary source of intracellular cAMP is adenylate cyclase (AC), which uses ATP to form cAMP and

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pyrophosphate. Cyclic AMP is degraded into 5⬘ adenosine monophosphate (5⬘ AMP) by cAMP-speciWc phosphodiesterases (PDEs). AC’s and PDE’s work together to modulate the intercellular levels of cAMP. Mammalian ACs are membrane localized with two transmembrane domains and two catalytic domains (CycC) (Tang and Gilman, 1992). However, fungal adenylate cyclases share only the catalytic domain in common with mammalian isoforms and guanylate cyclases and are membrane associated via their aminoterminus. Fungal adenylate cyclases contain Wve highly conserved domains: an N-terminal Ras-association motif, multiple leucine rich repeats, a PP2C phosphatase, a catalytic region and 2 C-terminally located cAMP-associated protein binding sites (Klimpel et al., 2002). Recent molecular investigations involving adenylate cyclase mutants in phytopathogenic Wlamentous fungi have shown that cAMP and adenylate cyclase are required for normal vegetative growth, infection structure formation, and virulence. Choi and Dean (1997) demonstrated that strains of the ascomycete plant pathogen, Magnaporthe grisea, deWcient in adenylate cyclase (MAC1) were unable to produce appressoria and did not penetrate susceptible rice leaves. Deletion mutants of the adenylate cyclase gene, uac1, in the dimorphic basidiomycete plant pathogen Ustilago maydis were non-pathogenic and had a constitutive Wlamentous growth habit (Gold et al., 1994). In the postharvest necrotroph, Botrytis cinerea, replacement of the BAC adenylate cyclase gene resulted in reduced vegetative growth, low levels of intracellular cAMP, slow lesion development, and lack of sporulation in planta (Klimpel et al., 2002). The plant cuticle and cell wall provide an eVective physiochemical barrier against potential phytopathogens. This barrier is so eVective that some pathogens do not directly penetrate their hosts and only infect through natural openings (e.g. stomates and wounds) (Walton, 1994). However, a variety of taxonomically diverse fungal phytopathogens have evolved specialized infection structures, appressoria, to breach these barriers (Xu et al., 1998). The majority of molecular genetic investigations concerning appressorium development have been carried out in the Wlamentous, phytopathogenic, ascomycete, M. grisea (Thines et al., 2000; Choi and Dean, 1997; Xu and Hamer, 1996; Xu et al., 1998). This fungus produces a single-celled, dome-shaped, melanized appressorium at the end of a spore-derived germ tube. Several studies have shown that the MAP kinase and cAMP signal transduction pathways are responsible for regulating appressorium development in M. grisea (Xu et al., 1998; Xu and Hamer, 1996). In contrast, S. sclerotiorum produces a slightly melanized, multihyphal appresorium, commonly referred to as an infection cushion (Lumsden and Dow, 1973). These penetration structures are produced on hyphae that have grown saprophytically. The signaling pathway(s) that initiate and regulate infection cushion formation in S. sclerotiorum have not been determined. The main focus of this study was to determine the biological roles of cAMP in S. sclerotiorum through analysis of an adenylate cyclase loss-of-function mutant. A targeted

gene deletion approach was implemented and we hypothesized based on previous pharmacological data (Rollins and Dickman, 1998) and other Wlamentous fungal AC mutants that an adenylate cyclase deletion mutant in S. sclerotiorum may exhibit aberrations in sclerotial initiation, possess altered levels of oxalate, and may be non-pathogenic or reduced in virulence due to the lack of infection cushion formation. Results from this study provide new insights into the role that cAMP plays in the life cycle of a sclerotium-forming, broad host range necrotrophic plant pathogen. 2. Materials and methods 2.1. Fungal strains and culture conditions Wild-type (Wt) S. sclerotiorum isolate 1980 (Godoy et al., 1990) was used to derive all strains in this study. Cultures were routinely grown on potato dextrose agar (PDA) (Difco, Detroit, MI, USA). Transformants were cultured on PDA supplemented with either 100 g/ml hygromycin B (EMD Biosciences, USA) or 10 g/ml Bialaphos (PhytoTechnology Laboratories, Shawnee Mission, Kansas). Permanent stocks were maintained as desiccated mycelia-colonized Wlter paper and sclerotia at ¡20 °C. Liquid shake cultures containing 50 ml of YPSuc medium (4 g/L yeast extract [Difco] + 15 g/L sucrose + 1 g/L K2HPO4 + 0.5 g/L MgSO4, pH 6.5) were cultured as previously described (Rollins and Dickman, 2001). Growth and analysis of cultures on cAMP-amended PDA was executed as reported by Rollins and Dickman (1998). Apothecial induction was carried out using PDA culturederived sclerotia (Russo et al., 1982). 2.2. Isolation and analysis of nucleic acids Mycelia from liquid shake cultures were frozen in liquid nitrogen, lyophilized and stored at ¡80 °C. Lyophilized mycelia were used to isolate genomic DNA according to Yelton et al. (1984). Total RNA was extracted from lyophilized mycelia using Trizol® reagent (Gibco-BRL, Rockville, MD) according to the manufacturer’s instructions. Agarose gel fractionation of total RNA was conducted as described by Rollins and Dickman (2001). Escherichia coli strain JM109 was used to propagate all plasmid DNA. Plasmid isolations, agarose gel electrophoresis, DNA restriction digests, ligation reactions, and E. coli transformations were conducted using standard procedures (Sambrook and Russell, 2001). For Southern blot analyses, restriction enzyme-digested genomic DNA were transferred to MagnaGraph nylon membrane (Micron Separations Inc., Westborough, MA) by downward alkaline transfer (Chomczynski, 1992) and Wxed to the membrane using UV light. For Northern blot analyses, total RNA was transferred to MagnaGraph nylon membrane by standard procedures (Ausubel et al., 1991). Radioactive probes for all hybridizations were generated using the Random Primers DNA Labeling System (Invitrogen

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Life Technologies, Carlsbad, CA.) as per the manufacturers’ instructions. A full-length 2.1 kb hph (hygromycin phosphotransferase) gene cassette (Redman and Rodriguez, 1994) and a 500 bp amplicon corresponding to the catalytic region of the sac1 gene were used to probe genomic DNA and RNA blots. 2.3. Cloning and identiWcation of sac1 A partial adenylate cyclase genomic clone was obtained from Drs. Changbin Chen and Martin. B. Dickman at the University of Nebraska, Lincoln and was used to obtain a full-length clone from a genomic cosmid library. Sequence information from the full-length sac1 (Sclerotinia adenylate cyclase 1) clone was obtained by primer walking (GenBank Accession No. DQ526020). Both strands were sequenced, and data were compiled into a contig using the Sequencher software (Gene Codes Corp, Ann Arbor, MI). 2.4. Construction of gene replacement and complementation vectors A pair of gene-speciWc primers FL-AC-5⬘-II (5⬘-TTA TCG ACG GCT TAT TAG AAC GTA CG-3⬘) and AC5⬘ + AscI-R (5⬘-AGG CGC GCC GCT AAA AAC CGT CCA TCC-3⬘) were used to amplify »2.0 kb of the 5⬘ untranslated region of the sac1 gene. To facilitate cloning an Asc I restriction enzyme recognition site was attached to AC-5⬘ + AscI-R (underlined). The 2.0 kb amplicon was gel puriWed and cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). This clone was designated AC-5⬘ and the clone was submitted for sequence analysis at the University of Florida ICBR sequencing core. A pair of genespeciWc primers AC-3⬘Flank-AscI (5⬘-AGG CGC GCC CCT GAA ACA GCA ATG CTT GAG-3⬘) and AC3⬘Flank-No AscI (5⬘-GGG CTG GTA AAT GGC GTA ATC-3⬘) were designed to amplify »2.2 kb of 3⬘ UTR and »0.5 kb of coding sequence corresponding to the 3⬘ end of the sac1 gene. This product was cloned using the TOPO TA cloning Kit and designated AC-3⬘. The identity of the insert was conWrmed by sequence analysis at the University of Florida ICBR sequencing core. The sac1 gene replacement vector was constructed in the following manner. Both AC5⬘ and AC-3⬘ were double digested with NotI/AscI and separated on a 0.8% agarose TBE gel. The linearized 6.0 kb AC-5⬘ and the 2.7 kb AC-3⬘ insert were gel puriWed and ligated together to form an 8.7 kb vector designated AC5⬘ + 3⬘. A hygromycin phosphotransferase (hph) cassette containing theTrpC promoter and terminator with Xanking AscI restriction enzyme recognition sites (Hutchens, 2005) was used in the following cloning reactions. The hph cassette and the AC-5⬘-3⬘ vectors were digested with AscI and separated on a 0.8% agarose gel. The 2.2 kb AscI-digested hph cassette was ligated into a linearized 8.7 kb plasmid vector which gave rise to a 10.9 kb sac1 gene deletion construct. The sac1 knock-out vector described above was linearized with NotI, gel puriWed and used to transform fungal

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protoplasts. This vector was also used as a template to produce two sac1-hph hybrid PCR products referred to as split marker fragments (Catlett et al., 2004). One primer set consisting of Split Marker-AC-5⬘ (5⬘-ATC CAG GGA CCT CGA ACG GCA TTT G-3⬘) annealed to the 5⬘-UTR of sac1 and Split Marker-HY (5⬘-AAA TTG CCG TCA ACC AAG CTC TGA TAG-3⬘) annealed internal to the hph gene cassette. The cDNA polymerase and primer set (Split Marker-AC-5⬘ + Split Marker-HY) was used according to the manufacturer’s instructions (BD Biosciences, Palo Alto, CA), to amplify a 3.2 kb amplicon that was designated AC5⬘-HY. A pair of gene-speciWc primers was designed to amplify the 3⬘ portion of the AC UTR using Split MarkerAC-3⬘ (5⬘-TGA CCT ACT TGC CGT CTT TCA GTG C3⬘) and an internal hph cassette primer Split Marker-YG (5⬘-TTT CAG CTT CGA TGT AGG AGG GCG-3⬘). Using cDNA polymerase and primer set (Split Marker-AC3⬘ + Split Marker-YG) a 4.3 kb amplicon was generated and designated AC-5⬘-YG. These two split marker fragments, AC-5⬘-HY and AC-3⬘-YG were gel puriWed, quantiWed and 5 g of each were used to transform wild-type fungal protoplasts. To complement the sac1 deletion mutant, the pBARKS1 vector containing the bar gene cassette (Pall and Brunelli, 1993) was digested with NotI restriction enzyme (New England Biolabs; USA) and treated with shrimp alkaline phosphatase according to the manufacturer’s instructions (Promega, Madison, Wisconsin). A pair of gene-speciWc primers AC-FL-5⬘-1.5 kb UTR (5⬘-TAC CCT GTG CTC TAA ATT TGG ATC ACC-3⬘) and AC-FL-3⬘-1.0 kb UTR (5⬘-AAT CCA AGC CAT CCA ACC TAT CTA ACC-3⬘) were designed to the 5⬘ and 3⬘ Xanking genomic regions of the sac1 gene and used to amplify 100 ng DNA from wildtype S. sclerotiorum. PCR was carried out using a high Wdelity polymerase (cDNA polymerase) according to the manufacturer’s instructions (BD Biosciences, Palo Alto, CA) and yielded a 9.5 kb product which contained the sac1 coding sequence and »1.5 kb of 5⬘ and »1 kb of 3⬘ untranslated region. The 9.5 kb sac1 amplicon was cloned into the pGEM-T easy vector (Promega, USA) and digested with NotI restriction enzyme. The NotI-digested sac1 amplicon was ligated into the 4.5 kb Not I-linearized pBARKS1 vector which gave rise to a 14 kb plasmid designated sac1pBARKS1. 2.5. Transformation and evaluation of transformant strains Sclerotinia sclerotiorum protoplasts from wild-type and sac1 deletion strains were prepared and transformed as described by Rollins (2003). The sac1 deletion strain was complemented with circular sac1-pBARKS1 plasmid and transformants were selected on RM medium containing 10 g/ml Bialophos. The non-complemented control strain (Nc1) was made by transforming protoplasts from the sac1 deletion strain with an intact pBARKS1 plasmid and were selected on RM medium containing 10 g/ml Bialophos.

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Southern blot hybridization was used to screen integration events in all transformants. This was accomplished by digesting genomic DNA from hygromycinresistant strains with StuI and hybridizing with a 500 bp probe made from the catalytic region of the sac1 gene. A 2.1 kb fragment corresponding to the hygromycin resistance cassette was also used to probe genomic DNA blots. 2.6. Radial growth analysis, oxalate accumulation, and pathogenicity assays Radial growth analysis and oxalate accumulation kinetics were carried out as described by Rollins and Dickman (2001). Pathogenicity assays were conducted according to Rollins (2003) except that the percentage of leaf area colonized was quantiWed 7 days after inoculation using Spot Advanced Software program (Diagnostic Instruments, USA). 2.7. Infection cushion assay PDA plates were inoculated within 1 cm of the edge with 2.5 mm3 plugs using a cork borer from PDA-colonized wild-type (Wt), sac1 deletion (KO1), complemented (C1), ectopic (E1) and non-complemented control strains (Nc1). Two weeks after the colony had reached the side of the plate, small portions of that area were removed and examined using Cotton Blue stain and a compound light microscope (Leica model DM R HC, Germany). Pictures were taken with a digital camera (Diagnostic Instruments IncModel 3.2.0, USA) attached to the microscope using the Spot basic software program (Diagnostic Instruments, USA). 2.8. Extraction and quantiWcation of total cellular cAMP Lyophilized mycelium (15 mg) was ground to a Wne powder in a 1.5 ml Eppendorf tube using a metal spatula. Twenty microliters of lysis reagent 1A and 180 l of cAMP assay buVer (provided in the BIOTRAK cellular communication assay kit, GE Healthcare, USA) were added to the samples, mixed by inversion and were incubated at room temperature for 10 min. All samples were spun in a microcentrifuge for 5 min at 14 K rpm. The supernatant was removed, diluted with cAMP assay buVer and used in the ELISA-based BIOTRAK cellular communication assay (GE Healthcare, USA.) according to the manufacturer’s instructions. 3. Results 3.1. Isolation and characterization of the adenylate cyclase gene sac1 A full-length adenylate cyclase gene, designated sac1 (Sclerotinia adenylate cyclase 1), was obtained by screening

a S. sclerotiorum genomic DNA library with a PCR-derived fragment of the sac1 sequence. Multiple sequence alignment of the sac1 nucleotide sequence with other annotated Wlamentous fungal adenylate cyclase genes was used to determine the putative start and stop codons and the locations of intron/exon junctions. Based on this analysis, the sac1 gene contains 4 exons and the predicted joined open reading frame yields a 6.4 kb sequence that encodes 2157 amino acids. Although the nucleotide sequence we obtained was identical to the adenylate cyclase sequence (SS1G_07715.1) now annotated in the S. sclerotiorum genome project (Broad Institute of Harvard and MIT (http://www.broad.mit.edu)), the predicted 5⬘ exon–intron junction of the second intron diVered and is reXected in GenBank Accession No. DQ526020. Analysis of this fulllength sac1 nucleotide and encoded amino acid sequence using BLAST (Basic Local Alignment Search Tool, Altschul et al., 1997) revealed that it was most similar to the BAC adenylate cyclase gene from B. cinerea. The deduced 237 kDa polypeptide contains Wve domains typical of Wlamentous fungal AC proteins: an N-terminal Ras-association domain, multiple leucine-rich repeats, a PP2C phosphatase, a catalytic domain, and two cAMP-associated protein binding sites. Southern blot analysis at low and high stringency using the AC catalytic region as a probe to BglII, HindIII and SpeI-digested genomic DNA indicated that sac1 is a single copy gene (data not shown). 3.2. VeriWcation of sac1 gene replacement and control strains The strategy to delete the sac1 locus using a sac1-hph gene replacement construct is described in the materials and methods section and is shown in Fig. 1A. This construct was linearized and used to transform wild-type protoplasts. In addition, this same construct was used as a PCR template to produce split-marker fragments used to transform wild-type protoplasts. Approximately 25 transformants were isolated and analyzed by Southern blot analysis and three independent sac1-loss-of-function mutants were identiWed. Southern blot analysis of one wild-type (Wt), three deletions (KO1, 2, and 3), a complemented (C1), an ectopic (E1) and one non-complemented (Nc1) strain is shown in Fig. 1B which conWrmed random integration of the complemented, ectopic and non-complemented constructs in the S. sclerotiorum genome. Hybridization using the AC catalytic domain as a probe yielded a single 4.8 kb band for Wt and E1 strains and did not hybridize to the KO1/2/3 or Nc1 strain. DNA from all strains was probed with the full-length hph gene as a hybridization control and all strains except wild type possessed a 6.4 kb band corresponding to the hph gene replacement construct. All three AC-KO strains were genetically identical and morphologically indistinguishable, therefore one representative strain, KO1, was chosen for further characterization. Northern blot hybridization was used to determine if AC deletion mutants possessed residual wild-type sac1

W.M. Jurick II, J.A. Rollins / Fungal Genetics and Biology 44 (2007) 521–530

A

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S S

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Probe 1 Probe 2

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B

KO Wt 1 2 3 E1 C1 Nc1

KO Wt

1

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E1

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Nc1 6.4 kb

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hph

Fig. 1. Construction and analysis of sac1 gene replacement strains. (A) The sac1 locus and the gene replacement construct containing the hygromycin phosphotransferase (hph) cassette in place of the sac1 gene; S, StuI. The double cross-over homologous recombination event resulting in the replacement of the sac1 coding sequence with the hph sequence is illustrated. (B) Southern hybridization analysis of the sac1 gene replacement strains. Genomic DNAs from wild-type (Wt), sac1 deletion (KO1/2/3), ectopic (E1), complemented (C1), and non-complemented control (Nc1) strains were digested with StuI. The autoradiogram on the left was hybridized with probe#1 corresponding to a portion of the AC catalytic region. The autoradiogram on the right was hybridized with probe#2 corresponding to the entire hph gene cassette.

transcript. RNA blot hybridization using the AC catalytic region as a probe revealed that Wt, E1 and C1 strains possessed the 6.4 kb sac1 transcript, while the KO1 and Nc1 strains lacked the sac1 transcript (data not shown). 3.3. Morphological abnormalities exist in the sac1 mutant Figs. 2A and B illustrate a variety of morphological abnormalities observed in the KO1 and Nc1 strains. When grown on potato dextrose agar (PDA), these strains grew slowly, produced an abundance of dense, aerial hyphae, and produced sclerotia in concentric rings with clear zones devoid of aerial hyphae between each ring. In contrast, the wild-type, ectopic and complemented strains grew at a faster rate and produced sclerotia at or near the outer edge of the plate. The KO1 sclerotia were slightly larger than wild type and misshapen but produced a dark melanized rind similar to wild type. Sclerotia produced by the KO1 strain were capable of myceliogenic germination, however did not produce apothecia (data not shown) when conditioned and incubated for carpogenic germination using standard conditions. Microscopic examination of the hyphal branching pattern in PDA-grown expanding colonies revealed that hyphal branches were short and arose predominantly from the main hypha at 90° angles instead of acute angles found in wild type. The wild-type branching pattern was restored when the KO1 strain was grown on PDA supplemented with 10 mM cAMP, and the KO1 mutant remained cAMP-responsive as indicated by the lack of sclerotial

development in the presence of exogenous cAMP (data not shown). Growth of the KO1 strain on PDA supplemented with 10 mM cGMP did not complement the KO1 strain phenotype (data not shown). In addition to the aberrant concentric pattern of sclerotial development, the rate of radial expansion was »8£ slower than Wt (Fig. 3). The total cellular cAMP concentration was determined for Wt and KO1 strains. Based on this assay, mycelia from the KO1 strain contained (150 fMol/mg tissue) »78% less cAMP than Wt (683 fMol/mg tissue). 3.4. Pathogenicity is abolished in the sac1 mutant Inoculation of detached tomato leaXets revealed that the mock-inoculated, KO1, and non-complemented strains were non-pathogenic, as they were unable to produce disease symptoms or colonize host tissues (Fig. 4A). Conversely, wild-type, ectopic, and complemented strains completely colonized detached tomato leaXets within 5 days of inoculation. In very few instances, a brown spot of dead cells was observed directly under the inoculation plugs of KO1 and non-complemented strains. However, no internal colonization was observed when the area was examined microscopically utilizing trypan blue staining (data not shown). Exogenous application of oxalate to healthy tomato tissue replicated these symptoms (data not shown). Wounding of the tomato leaXets before inoculation allowed lesion formation with the KO1 mutant (Fig. 4B). However, the virulence of this mutant

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Fig. 2. Morphology of wild-type and transformant strains. (A) Colony morphology of wild-type (Wt), sac1 deletion (KO1), ectopic (E1), complemented (C1), and non-complemented control (Nc1) strains grown on PDA (potato dextrose agar) for 14 days. (B) Branching pattern of Wt, KO1, and C1 strains.

Radial Growth Analysis 10

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assaying oxalic acid accumulation kinetics and infection cushion development. Interestingly, the KO1 strain produced as much oxalate as the Wt and E1 and accumulation kinetics for all strains were virtually indistinguishable (Fig. 5A). The production of infection cushions was investigated in Wt, KO1 and control strains (Fig. 5B). The Wt, E1 and C1 strains developed typical multihyphal infection cushions in the Petri plate assay. Infection cushions were never observed in the KO1 or Nc1 strains but unlike the wild type, abundant microconidia production was observed in these strains (Fig. 5B).

0 0

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Fig. 3. Radial growth of wild type and transformants. Analysis of wildtype (Wt), sac1 deletion (KO1), ectopic (E1), complemented (C1) and noncomplemented control (Nc1) strains radial growth. Colony diameters were measured in 12 h intervals. Each point represents the mean and standard deviation from three independent cultures from a single experiment. This experiment was repeated three times and one representative experiment is shown.

remained severely attenuated as the percent of lesion area colonized by the KO1 strain was 32.7% § 17.9 compared to 100% by the wild type. Based on this observation, we sought to investigate the source of the pathogenicity defect observed in the KO1 mutant by

4. Discussion A number of physiological and functional-genetic studies have revealed that cAMP controls diverse biological processes in Wlamentous fungal plant pathogens (Choi and Dean, 1997; Gold et al., 1994; Hall and Gurr, 2000; Klimpel et al., 2002; Lee and Dean, 1993; Robson et al., 1991). Based on data from AC-deWcient fungal phytopathogens and Wndings from a previous pharmacological investigation in S. sclerotiorum (Rollins and Dickman, 1998), we hypothesized that a mutant impaired in its ability to synthesize cAMP would exhibit defects in sclerotial development and would be non-pathogenic or reduced in virulence. We tested this hypothesis by

W.M. Jurick II, J.A. Rollins / Fungal Genetics and Biology 44 (2007) 521–530

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Fig. 5. Oxalate accumulation, infection cushion, and microconidia formation. (A) Oxalic acid accumulation kinetics from 0.5 M MOPS-buVered YPSU cultures pH 7.0 of wild-type (Wt), sac1 deletion (KO1), ectopic (E1) strains. Data points represent the means and standard deviation from three independent cultures from one representative experiment that was repeated 3 times. (B) Infection cushion formation without microconidia development in the Wt and microconidia development without infection cushion formation in the KO1 strain. Bar equals 10 m. Fig. 4. Pathogenicity assays using intact and wounded detached tomato leaXets. (A) Tomato leaXets (cv. Bonnie Best) were mock inoculated with an uncolonized potato dextrose agar (PDA) plug (M), a PDA plug colonized with wild-type (Wt), sac1 deletion (KO1), ectopic (E1), complemented (C1), and non-complemented control strain (Nc1). Photographs were taken 7 days post-inoculation. (B) Tomato leaXets (c.v. Bonnie Best) were wounded with a dissecting needle and an uncolonized potato dextrose agar plug (M), wild-type (Wt), and sac1 deletion strain (KO1) were placed directly over the wound. One representative replication from four experiments is shown.

cloning the single copy adenylate cyclase gene, sac1, and deleting it from the genome to create an adenylate cyclase loss-of-function strain. Three independent sac1 mutants were generated and all displayed virtually identical phenotypes in culture. The representative deletion strain (KO1) grew 1/8 the rate of wild type, possessed dense aerial hyphae, and exhibited rightangle branching on rich solid, deWned solid, and rich liquid medium. Slow growing, dense, compact phenotypes have been previously described for other adenylate cyclase mutants like cr-1 in Neurospora crassa (Lindegren, 1936), MAC1 in M. grisea (Choi and Dean, 1997) and the BAC mutant in B. cinerea (Klimpel et al., 2002). However, the observed branching defect is novel, and has not been reported for other fungal AC-deWcient mutants. The wild-type branching pattern could not be restored with the

addition of 10 mM cGMP, but was restored in the presence of 10 mM cAMP or by complementation with an intact copy of the wild-type sac1 gene. Precedent for cAMP-modulated control of branching in Wlamentous fungi (N. crassa) has been previously described as low intracellular cAMPlevels have been correlated with an increased branching rate and a decreased rate of radial expansion (Terenzi et al., 1974; Scott and Solomon, 1975; Flawia et al., 1977; Rosenberg and Pall, 1979.) Typically, S. sclerotiorum forms sclerotia at the outer edge of plates where nutrient conditions may be limiting and polar elongation is inhibited. The abnormal appearance and the concentric pattern of sclerotial development in culture that is exhibited by the AC KO strain has not been reported in other AC-deWcient sclerotial forming fungi. This unique cultural phenotype is consistent with previous pharmacological data (Rollins and Dickman, 1998) that invokes an inhibitory role for cAMP in regulating sclerotial initiation. When intracellular cAMP levels are elevated, the mycelial transition from vegetative growth to sclerotial morphogenesis is blocked. We hypothesized that when intracellular cAMP levels are low, initiation of sclerotia will increase as compared to wild type. Our observations with the sac1 knock-out strains support this hypothesis.

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Accumulation of staling compounds, inhibition of polar elongation, nutrient limitation, light, pH, temperature, mobilization of nutrients/metabolism, and oxidative stress are all factors that have been shown to regulate sclerotial development (Chet and Henis, 1975). The concentric pattern of sclerotia development observed with the sac1 KO gives the appearance of rhythmic control. The periodicity of sclerotia initiation, approximately one initiation every three days, however, is not suggestive of a circadian clock-controlled process. Alternatively, the slow growth rate of KO strains may lead to more frequent initiation of sclerotia due to physical sensing of the decreased polar elongation or the accumulation of small diVusible stalling or quorum sensing molecules. Low levels of intracellular cAMP may also have a more direct role in initiating sclerotial development by mediating the regulation of glycogen metabolism. Several studies have demonstrated a link between intracellular cAMP levels and glycogen metabolism in Wlamentous fungi including: Coprinus cinerea, Sclerotium rolfsii, M. grisea, and Saccobolus platensis (Kuhad et al., 1987; Shapira et al., 1986; Thines et al., 2000; Galvagno et al., 1984). With this in mind, we hypothesize that the low intracellular cAMP levels in the sac1 mutant are not suYcient to inhibit glycogen mobilization and utilization. Accordingly, nutrients may be constantly translocated to the colony periphery triggering more frequent sclerotial initiation and giving rise to the observed concentric pattern of sclerotial development. This hypothesis could be evaluated by several means, one which includes the cloning, identiWcation and functional analysis of genes and enzymes involved in glycogen metabolism (e.g. glycogen synthase and glycogen phosphorylase). Functional analysis of these two biosynthetic enzymes singly, and in the context of the AC mutant may further clarify the link between nutrient metabolism and cAMP in sclerotial development. Deletion of nearly the entire sac1 coding sequence, did not abolish cAMP accumulation in the sac1 deletion strain (KO1). There was a »4-fold reduction in cAMP in the KO1 strain compared to wild type. In other AC mutant strains, N. crassa (cr-1) and the Aspergillus nidulans (cya), “no detectable” amounts of cAMP were reported (Fillinger et al., 2002; Terenzi et al., 1974). The levels of intracellular cAMP in other Wlamentous fungal AC mutants: M. grisea (MAC1), and P. anserina (Pa AC) have not been published (Klimpel et al., 2002). Low levels of intracellular cAMP were detected, however, in the B. cinerea BAC and in the Ustilago maydis uac1 mutants using Enzyme Immunoassays for cAMP quantiWcation (Klimpel et al., 2002 and Martínez-Espinoza et al., 2004). The BAC mutant possessed a »5-fold reduction in cAMP compared to wild-type and the uac1 mutant contained very low levels of cAMP that varied slightly under diVerent growth conditions. As cyclic AMP has been shown to be involved in numerous biological processes, it is reasonable to assume that this important signaling molecule may be required for viability. Since all of the AC-deWcient

mutants characterized to date have not been lethal, a basal level of intracellular cAMP in these mutants might be expected. The source of intracellular cAMP in the cell is adenylate cyclase, however the catalytic domain of adenylate cyclase and guanylate cyclase are very similar at the amino acid level (Roelofs et al., 2001a). Therefore it is possible that a guanylate cyclase is capable of producing the low level of intracellular cAMP observed in the adenylate cyclase-deWcient sac1 mutant. A possible explanation for the presence of low intracellular cAMP levels in the AC-KO mutant whereas the undetectable levels of cAMP reported for A. nidulans and N. crassa AC mutants may be due to the lack of sensitivity of the detection methods used to assay cAMP levels in those species. Consequently, a basal level of intracellular cAMP may also persist in the A. nidulans and N. crassa AC mutant strains. The ELISA-based assay that was used in this study and the one by Klimpel et al. (2002), are highly sensitive and capable of detecting femtomolar amounts of cAMP. This alternative hypothesis may be investigated by assaying intracellular cAMP levels from various Wlamentous fungal AC-deWcient mutants using the ELISA-based assay and making comparisons of the determined intracellular cAMP levels for each strain. Pathogenicity assays involving detached tomato leaves revealed that sac1 mutants were non-pathogenic (Fig. 4A). However, these mutants were able to partially colonize mechanically wounded tomato leaXets (Fig. 4B). We hypothesized based on these Wndings that sac1 mutants may possess undetectable or reduced levels of oxalate that would render the sac1 deletion mutants non-pathogenic (Godoy et al., 1990). Previous pharmacological data also supported this hypothesis as treatment with exogenous cAMP resulted in increased oxalate levels compared to wild type (Rollins and Dickman, 1998). However, production and accumulation of oxalate was no diVerent in the KO1 relative to wild-type and control strains (Fig. 6A). Perhaps cAMP is not an important regulator of oxalate biosynthesis, or the basal level of cAMP observed for the KO mutant may be at a threshold concentration that allows the synthesis and accumulation of oxalate in culture. Reduction in pathogenicity and virulence in AC-deWcient mutants has been shown in B. cinerea (Klimpel et al., 2002), U. maydis (Gold et al., 1994) and M. grisea (Choi and Dean, 1997). Defects in infection structure formation were documented in MAC1 mutants as they did not produce appressoria and were non-pathogenic on susceptible rice cultivars (Choi and Dean, 1997). However, appressorium formation could be restored with application of exogenous cAMP and mutant strains were able to colonize wounded rice leaves. We sought to determine if the defect in S. sclerotiorum pathogenicity was due to a lack of infection cushion (appressorium) formation in sac1 deletion strains. Since we were unable to visualize infection cushions in Petri dish assays with the KO1 strain, we hypothesize that the

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lack of infection cushion formation accounts for the loss of pathogenicity on detached tomato leaXets. In addition, the reduced growth rate of the KO1 mutant appears to be the primary factor responsible for the decrease in lesion expansion rate observed in mechanically wounded tomato leaXets. Data presented in this work have demonstrated that cAMP is involved in several diVerent biological processes in S. sclerotiorum including: sclerotial development, microconidia production, infection cushion formation, mycelial growth rate, and hyphal branching pattern. However, much remains to be known about the upstream and downstream components in the cAMP-signaling pathway that signal and control these processes. With the availability of the S. sclerotiorum draft genome sequence, it will now be possible to eYciently target known members of the cAMP-signal transduction pathway, both upstream and downstream, for functional analyses to further dissect this signaling pathway. Acknowledgments The authors acknowledge Drs. Changbin Chen and M.B. Dickman at the University of Nebraska, Lincoln for the adenylate cyclase (sac1) cosmid clone. We would like to thank Ulla K. Benny for her technical assistance and KuangRen Chung and Daryl R. Pring for critically reviewing this manuscript. Wayne M. Jurick II was supported through an Alumni Fellowship awarded by the University of Florida. References Abawi, G.S., Grogan, R.G., 1979. Epidemiology of diseases caused by Sclerotinia species. Phytopathology. 69, 899–904. Adams, P.B., Ayers, W.A., 1979. Sclerotinia sclerotiorum: ecology of Sclerotinia species. Phytopathology 69, 896–899. Altschul, S.F., Madden, T.L., SchaVer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1991. Current Protocols in Molecular Biology. John Wiley, New York. Catlett, N.L., Lee, B.N., Yoder, O.C., Turgeon, G., 2004. Split-marker recombination for eYcient targeted deletion of fungal genes. Fungal Genet. Newslett. 50, 9–11. Chet, I., Henis, Y., 1975. Sclerotial morphogenesis in fungi. Ann. Rev. Phytopathol. 13, 169–192. Choi, W., Dean, R.A., 1997. The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9, 1973–1983. Chomczynski, P., 1992. One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 134–139. Fillinger, S., Chaveroche, M.K., Shimizu, K., Keller, N., d’Enfert, C., 2002. cAMP and Ras signaling independently control spore germination in the Wlamentous fungus Aspergillus nidulans. Mol. Microbiol. 44, 1001–1016. Flawia, M.M., Terenzi, H.F., Torres, H.N., 1977. Characterization of Neurospora crassa mutant strains deWcient in adenylate cyclase activity. Arch. Biochem. Biophys. 180, 334–342. Galvagno, M.A., Forchiassin, F., Cantore, M.L., Passeron, S., 1984. The eVect of light and cyclic AMP metabolism on fruiting body formation in Saccobolus platensis. Exp. Mycol. 8, 334–341.

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