A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB)

Fungi Journal of Article A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB) Sandra Garrigues †, Jose F. Marcos , Paloma Manzanares and Mónica Gandía * Food Biotechnology Department, Instituto de Agroqu í mica y Tecnolog í a de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, 46980 Valencia, Spain; [email protected].nl (S.G.); [email protected] (J.F.M.); [email protected] (P.M.) *Correspondence: [email protected]; Tel.: +34-963900022 † Present address: Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, 3584 CT Utrecht, The Netherlands. Received: 2 September 2020; Accepted: 30 September 2020; Published: 2 October 2020   Abstract: Antifungal proteins (AFPs) from ascomycete fungi could help the development of antimycotics. However, little is known about their biological role or functional interactions with other fungal biomolecules. We previously reported that AfpB from the postharvest pathogen Penicillium digitatum cannot be detected in the parental fungus yet is abundantly produced biotechnologically. While aiming to detect AfpB, we identified a conserved and novel small Secreted Cysteine-rich Anionic (Sca) protein, encoded by the gene PDIG_23520 from P. digitatum CECT 20796. The sca gene is expressed during culture and early during citrus fruit infection. Both null mutant ( ∆ sca) and Sca overproducer (Sca op ) strains show no phenotypic differences from the wild type. Sca is not antimicrobial but potentiates P. digitatum growth when added in high amounts and enhances the in vitro antifungal activity of AfpB. The Sca op strain shows increased incidence of infection in citrus fruit, similar to the addition of purified Sca to the wild-type inoculum. Sca compensates and overcomes the protective effect of AfpB and the antifungal protein PeAfpA from the apple pathogen Penicillium expansum in fruit inoculations. Our study shows that Sca is a novel protein that enhances the growth and virulence of its parental fungus and modulates the activity of AFPs. Keywords: antifungal protein (AFP); Penicillium digitatum; AfpB; postharvest decay; citrus fruit; virulence; cysteine-rich protein (CRP) 1. Introduction Infections caused by fungal pathogens pose a serious risk to human health, food production and security [ 1 , 2 ]. In agriculture, fungal plant pathogens are of great economic importance because they threaten the production of crops and can cause severe postharvest diseases, with an increasing incidence trend in the last decades [ 1 , 3 ]. To combat fungal infections, multiple chemical fungicide treatments are widely applied, which have negative effects on animal and human health and the environment. Currently, there are very few classes of fungicides available to treat (and prevent) fungal infections, leading to a rapid increase in resistance against the existing compounds [ 4 ]. Consequently, new antifungal strategies are urgently needed, and interest is focused in novel and sustainable antifungal agents with high efficacy, limited toxicity, low production costs and with different modes of action from the currently existing ones [5–10]. J. Fungi 2020,6, 203; doi:10.3390/jof6040203 www.mdpi.com/journal/jof J. Fungi 2020,6, 203 2 of 18 Antimicrobial peptides (AMPs) are a broad class of peptides and proteins with direct killing activity produced by organisms all along the phylogenetic scale [ 11 ], and serve as natural defenses against infections caused by microbial pathogens, mainly bacteria and fungi. AMPs can be classified as cationic or anionic, based on their net charge [ 12 ], and are gaining extensive attention as natural antibiotics among the scientific community. Antifungal proteins (AFPs) of fungal origin are a specific class of AMPs that have been considered as promising alternatives to chemical fungicides [ 13 ]. AFPs are small, secreted, cationic, cysteine-rich proteins (CRPs) that fold into compact structures stabilized by disulphide bonds [ 14 ], which makes them highly resistant to heat, proteolysis and extreme pH [ 15 , 16 ], and exhibit antifungal activity at micromolar concentrations [ 16 – 19 ]. AFPs are encoded as pre-pro-proteins with a signal peptide (SP) at the N-termini involved in protein secretion and a pro-sequence whose function is still unclear [ 13 , 20 ]. The proteins PAF from Penicillium chrysogenum and AFP from Aspergillus giganteus are the most studied and characterized AFPs to date [ 20 , 21 ]. However, the number of experimentally characterized AFPs and predicted AFP-like sequences in filamentous fungi is continuously rising given the increasing availability of fungal genome sequences. Based on phylogenetic analysis, our previous studies showed that a given fungal genome encodes up to three distinct AFPs and grouped fungal AFPs into three different classes (A, B and C) [ 22 ]. This classification expanded the two clusters previously reported [ 23 , 24 ], although a more recent group of AFPs has been described [ 18 ], suggesting the existence of new unidentified classes of AFPs yet to be characterized. Penicillium digitatum is the main citrus postharvest pathogen and causes green mold disease in citrus fruits, being responsible for important economic losses worldwide [ 25 – 27 ]. It only encodes one afp gene from class B, named afpB [ 22 ], and the corresponding protein AfpB could only be detected and produced when biotechnologically expressed in the yeast Pichia pastoris, the filamentous fungus P. digitatum [ 16 ] and in Nicotiana benthamiana plants [ 28 ]. AfpB shows potent in vitro inhibitory activity and was the first AFP protein for which self-inhibitory activity against its parental fungus was reported [ 16 ]. Additionally, AfpB has demonstrated high in vivo inhibition against Botrytis cinerea infection on tomato leaves [ 29 ], suggesting that AfpB could be a promising candidate as a bio-fungicide with no toxic effect on human red blood cells [ 16 ]. A detailed understanding of the mode of action is required for the potential application of AFPs as antifungal compounds. The most studied AFPs have similarities as well as differences in their mode of action against sensitive fungi, particularly in relation to whether they induce the cell wall integrity pathway, are internalized as part of their antifungal mechanism, induce disturbances in the intracellular Ca +2 concentration, induce the production of reactive oxygen species (ROS) or affect intracellular signaling (reviewed in [ 14 ]). In this context, we have initiated the study of the mode of action of AfpB from P. digitatum. From our previous work, we showed that this protein induces the phosphorylation of mitogen-activated protein kinases (MAPK) [ 30 ], establishing a connection between AfpB and cell wall stress. Additionally, we recently proposed that the AfpB killing activity occurs in three steps: (i) interaction with the cell wall; (ii) rapid cell internalization; (iii) ROS-mediated regulated cell death [ 31 ]. However, nothing is known about possible functional interactions between AFPs in general, and AfpB in particular, with other biomolecules produced by the parental fungi. In this study, we have identified a novel small Secreted Cysteine-rich and Anionic (Sca) protein in P. digitatum that modulates the activity of AfpB towards the parental fungus. In order to characterize this novel protein, we determined its gene expression pattern, generated Sca-null ( ∆ sca) and -overproducer (Sca op )P. digitatum strains and tested the putative activity of the protein and its effect on AfpB antifungal activity. Our data reveal that the Sca-encoding gene is expressed very early during citrus fruit infection. Additionally, Sca shows neither antifungal nor antibacterial activity but rather enhances antifungal activity of AfpB in vitro and increases incidence of P. digitatum infection in vivo , overcoming the protective effect of AFPs during citrus fruit infection. J. Fungi 2020,6, 203 3 of 18 2. Materials and Methods 2.1. Microorganisms, Media and Culture Conditions P. digitatum CECT 20796 (isolate PHI26) [ 25 ] and all transformant strains were cultured in potato dextrose agar (PDA; Difco, Sparks, MD, USA) for 7 days at 25 ◦ C. Growth in solid PDA medium was analyzed by depositing 5 µ L of conidial suspension (5 × 10 4 conidia/mL) in the center of PDA plates and daily measurement of growth diameter. Conidial growth in liquid medium was assessed in 100 mL of potato dextrose broth (PDB; Difco) at 25 ◦C with shaking. Different vectors used for fungal transformation were cloned and propagated in Escherichia coli JM109 cultured in Luria Bertani (LB) medium supplemented with the corresponding antibiotics (25 µ g/mL chloramphenicol; 50 µ g/mL kanamycin or 100 µ g/mL spectinomycin) at 37 ◦ C. Agrobacterium tumefaciens AGL-1 strain was cultured in LB medium with 20 µ g/mL rifampicin at 28 ◦ C. Yeast Saccharomyces cerevisiae BY4741 was incubated at 30 ◦ C. For antibacterial assays, E. coli JM109 and Bacillus subtilis CECT 498 were grown in LB medium with shaking at 37 ◦C. 2.2. Protein Identification, Structure and Functional Domain Prediction For Sca (PDIG_23520) identification, P. digitatum CECT 20796 cell-free supernatant was collected after 21 days of growth in PDB, centrifuged and tenfold concentrated (Speedvac, Concentrator plus, Eppendorf, Hamburg, Germany). Total proteins were separated by SDS-PAGE [ 32 ] using SDS-16% polyacrylamide gels calibrated with pre-stained protein size-standard SeeBlue ® (ThermoFischer Scientific, Waltham, MA, USA) and visualized by Coomassie blue staining. The ~12 kDa protein band was cut and analyzed in the proteomics facility of ‘Servei Central de Suport a la Investigaci ó Experimental’ (SCSIE) of University of Valencia (Spain). For protein identification, peptide mass fingerprinting (PMF) and N-terminal sequencing were performed (Supplementary Figure S1). For PMF, samples were subjected to trypsin digestion and the resulting mixtures were analyzed on a 5800 Matrix-assisted laser desorption/ionization (MALDI)-Time-of-flight (TOF)/TOF in positive reflectron mode (3000 shots at every position). Five of the most intense precursors (according to the threshold criteria: minimum signal-to-noise: 10, minimum cluster area: 500, maximum precursor gap: 200 parts per million (ppm), maximum fraction gap: 4) were selected for every position for the tandem mass spectrometry (MS/MS) analysis. MS/MS data were acquired using the default 1 kV MS/MS method. The MS and MS/MS information was sent to MASCOT via the Protein Pilot software (AB Sciex, Madrid, Spain). The N-terminal sequence of Sca was determined by the protein chemistry facility at the Margarita Salas Center for Biological Research (CIB-CSIC, Madrid, Spain) by N-terminal Edman degradation method [33]. Sequences from different Sca homologs among filamentous fungi were identified through BLASTP searches carried out at the National Center for Biotechnology Information server (https://blast.ncbi. nlm.nih.gov/Blast.cgi) (detailed in Supplementary Figure S2). A putative SP was predicted using the SignalP v4.0 server [ 34 ]. The theoretical molecular weight (MW) and isoelectric point (pI) of the mature Sca and Sca homologs were examined with the Compute pI/MW and ProtParam tools of the ExPASy Proteomics Server (https://www.expasy.org/). Sca putative Pfam domains were searched with the Pfam v33.1 online tool from the EMBL-EBI server [35] (http://pfam.xfam.org/). J. Fungi 2020,6, 203 4 of 18 The Sca secondary structure was predicted by the JPred4 server [ 36 ]. Accession numbers of protein sequences used in the alignments were obtained at the UniProt (http://www.uniprot.org) server. Amino acid sequence alignments were performed with the Clustal W algorithm [ 37 ] included in the MEGA v10 package [38], and alignments were further refined with minor adjustments. 2.3. Total RNA Isolation, Quantitative RT-PCR and Relative Expression Time course experiments in solid and liquid media to collect mycelia for RNA isolation were performed as previously described [39]. Total RNA from (i) fungal conidia, (ii) time course experiments of P. digitatum CECT 20796 grown in PDB or PDA and (iii) infected fruits was isolated following previously described procedures [ 39 , 40 ]. Treatment with RNase-free DNase(ThermoFischerScientific), synthesis of first-strand cDNA forquantitative RT-PCR assays, and determination of relative changes of gene expression between samples were conducted as described [ 39 , 41 ]. As independent housekeeping genes, P. digitatum β -tubulin [ 42 ], ribosomal protein L18a [43] and 18S rRNA [44] genes were simultaneously used (Supplementary Table S1). 2.4. Generation of Sca Null and Overproducer Strains The AccuPrime High Fidelity polymerase (Invitrogen, Eugene, OR, USA) was used for all PCR procedures, and the resulting DNA products were sequenced for verification. All primer sequences and their location are shown in Supplementary Table S2 and Supplementary Figures S3 and S4. The genetic construct to disrupt the sca gene by homologous recombination was generated by fusion PCR [ 45 ]. Previously described procedures were applied to obtain the vector pGKO2_ ∆ sca [ 22 , 41 ]. Briefly, the hygromycin-resistant cassette (hph) used as positive selection marker was flanked by fungal DNA fragments of 1035 bp (primers OJM449 and OJM450) and 991 bp (primers OJM451 and OJM452) amplified from P. digitatum CECT 20796 genomic DNA. The fusion PCR fragment obtained was purified and ligated into pGEM-T Easy Vector System I (Promega, Madison, WI, USA). SpeI and HindIII restriction sites were used to insert the construct into the binary vector pGKO2 [ 46 ], whose T-DNA also contains the Herpes simplex virus-1 thymidine kinase gene (HSVtk) used as negative selection marker, to obtain the plasmid pGKO2_∆sca. In parallel, to generate specific gene constructs for Sca overproduction, the FungalBraid (FB) modular cloning approach was applied (Supplementary Figure S4) [ 47 , 48 ]. A new FB element (FB034) was obtained by multipartite assembly using P. chrysogenum paf promoter (FB029), the transcriptional unit for sca expression (FB032) and the paf terminator (FB030). FB034 was assembled with the hph-resistant cassette (FB003) to obtain the binary vector FB038. Both binary vectors for sca disruption and overexpression were transformed into A. tumefaciens AGL-1 by electroporation. Fungal transformation of P. digitatum CECT 20796 with pGKO2_ ∆ sca and FB038 vectors was performed following the A. tumefaciens-mediated transformation (ATMT) protocol previously described [ 48 , 49 ]. Sca ectopic transformed strains were selected in 25 µ g/mL hygromycin B (Invivogen, San Diego, CA, USA). On the other hand, homologous transformants were initially pre-screened in 25 µ g/mL hygromycin B as a positive selection and subsequently in 25 µ M 5-fluoro-2 0 -deoxyuridine (F2dU) (Merck, Darmstadt, Germany) as a negative selection. All transformant strains were confirmed by PCR using genomic DNA as described previously [ 41 ] (Supplementary Figures S3 and S4). The size and presence of DNA amplicons were determined by 1% agarose gel electrophoresis. J. Fungi 2020,6, 203 5 of 18 2.5. Protein Production and Purification Sca was purified from the supernatant of P. digitatum Sca op strains (10 6 conidia/mL) grown in P. digitatum minimal medium (PdMM) [ 50 ] after 11 days of growth at 25 ◦ C with strong aeration. Cell-free supernatant was collected by centrifugation and dialyzed (2K MWCO, Sigma-Aldrich, St. Louis, MO, USA) against 20 mM phosphate buffer at pH 6.8. Given the predicted chemical properties of Sca (pI =4.54), the dialyzed solution was applied to an AKTA Purifier system equipped with a 6 mL RESOURCE Q column (GE Healthcare, Chicago, IL, USA) equilibrated in the phosphate buffer. Elution was set with a linear NaCl gradient from 0 to 1 M in the same buffer. Surprisingly, Sca protein was not adsorbed in the resin and was present (as the major protein) in the flow-through after chromatography (Supplementary Figure S5). Thus, the Sca-containing flow-through was collected, dialyzed against Milli-Q water and lyophilized. Protein concentration was determined spectrophotometrically (A 280 ) considering the Sca molar extinction coefficient ( E280 =2.43). The purity was monitored by SDS-PAGE using SDS-16% polyacrylamide gels calibrated with pre-stained protein size-standard SeeBlue ® and Coomassie blue staining. AfpB protein production, purification, and quantification were achieved as previously described [16]. 2.6. Antimicrobial Activity Assays The antifungal activity of Sca was evaluated with two different growth inhibition assays. A final concentration of 10 5 conidia/mL from P. digitatum CECT 20796 was inoculated in 100 mL flasks containing 25 mL 1/4 diluted PDB supplemented with different concentrations of Sca (6 µ g/mL) or AfpB (0.032 and 0.065 µ g/mL) proteins. To evaluate potential synergism between Sca and AfpB, combinations of both proteins were added simultaneously (6 and 0.032 µ g/mL or 6 and 0.065 µ g/mL of Sca and AfpB, respectively). Cultures were grown under strong aeration at 25 ◦ C for 48 h. After this, mycelia were recovered, filtered, washed and paper-dried, and (wet weight) biomass was measured. Additional growth inhibition assays with Sca, AfpB and PeAfpA against the chosen fungi (Supplementary Table S3) were performed in 96-well flat bottom microtiter plates (Nunc, Roskilde, Denmark) in a total volume of 100 µ L as described previously [ 29 ]. Synergy assays in 96-well microtiter plates were conducted as follows: 25 µ L of two different 4 × concentrated proteins were mixed in the same well with 50 µ L of P. digitatum conidia (5 × 10 4 conidia/mL) in 1/10 diluted PDB containing 0.02% (w/v) chloramphenicol to avoid bacterial contamination. Both protein concentrations in these experiments were: 2 and 32 µ g/mL for Sca; from 0.03 to 16 µ g/mL for AfpB. In all cases, plates were statically incubated for 4 days at 25 ◦ C, and growth was determined daily by measuring the optical density at 600 nm (OD 600 ) using a FLUOstar Omega plate spectrophotometer (BMG labtech, Orlenberg, Germany). Data are expressed as OD 600 means ± standard deviation (SD) of three replicates and dose–response curves were generated from measurements after 72 h. Antibacterial assays were carried out as described previously [ 51 ]. Different experiments were repeated at least twice. The minimum inhibitory concentration (MIC) is defined as the peptide concentration that completely inhibited growth in all experiments. J. Fungi 2020,6, 203 6 of 18 2.7. Fruit Infection Assays In vivo analyses were performed by infecting non-treated, mature, freshly-harvested orange fruits (Citrus sinensis L. Osbeck cv Navel and Lanelate) with different P. digitatum strains following previous protocols [ 43 ]. Three replicates of five fruits were inoculated at four wounds around the equator with 5 µ L of conidial suspensions (10 4 conidia/mL). At different days post-inoculation (dpi), each inoculated wound was scored for specific green mold infection symptoms. Data were calculated as the means and SD of the percentage of infected wounds. Moreover, tissue samples (discs of 5 mm in diameter around the inoculation site) at 1, 2, 3, 4 or 7 dpi for quantitative RT-PCR were collected, crushed and frozen at −80 ◦C to be used for RNA extraction. 2.8. Statistical Analysis Differences in protein activities were determined using the one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test. Statistical significance was referred for pvalue <0.05. Analyses were done using STATGRAPHICS Centurion XVI Version 16.1.17 and Microsoft Excel 2016 software (Real Statistics Resource Pack, http://www.real-statistics.com/). 3. Results 3.1. A Novel Small Cysteine-Rich and Anionic (Sca) Protein Is Abundantly Produced and Secreted by P. digitatum In an attempt to detect the antifungal protein AfpB in the culture supernatant of P. digitatum CECT 20796, a faint band of a protein of about 12 kDa was detected by SDS-PAGE after 11 days of growth in PDB (Figure 1a), which was more evident after tenfold concentration of the supernatant samples (Figure 1b). In order to identify this protein, a MALDI-TOF/TOF analysis was performed and the results revealed that this protein was encoded by the PDIG_23520 gene. We named it Sca (Secreted Cysteine-rich and Anionic), an anionic protein (pI =4.54) with a molecular mass (MM) of 12,205.4, 117 amino acids and 4 cysteines (Supplementary Figure S1). A SP of 19 residues was identified at the N-terminus, indicating its processing and secretion, followed by a pro-sequence of 11 amino acids which is missing in the mature protein (Figure 1c and Supplementary Figure S1). BLASTP analyses revealed the presence of Sca homologs in a wide range of filamentous ascomycetes. Based on protein sequence alignments, two clear domains can be distinguished in the mature Sca protein: (i) a more variable N-terminal domain that contains four conserved cysteines and two likely disulphide bridges, and (ii) a much more conserved C-terminal domain, which is rich in aromatic residues (Figure 1d, Supplementary Figures S1 and S2). In the N-terminal domain, four cysteines were highly conserved in all protein sequences except for the ones from Metarhizium rileyi and Pochonia chlamydosporia (Figure 1d and Supplementary Figure S2). Secondary structure prediction based on the full sequence alignment showed that Sca is a beta-stranded protein with eight β -sheet structural motifs (Figure 1d). Finally, Pfam domain searches did not reveal any known functional motif for Sca. Our results revealed the identification of a novel and highly-conserved protein not described to date and with an unknown function. J. Fungi 2020,6, 203 7 of 18 Figure 1. Production and identification of the Secreted Cysteine-rich Anionic (Sca) protein from P. digitatum CECT 20796 strain. ( a ) SDS-PAGE of non-concentrated supernatants of P. digitatum grown in potato dextrose broth (PDB) for 3, 6 and 11 days. Sca is marked by an arrow. ( b ) SDS-PAGE of the same supernatants 10 × concentrated. M: SeeBlue ® pre-stained protein standard. ( c ) Amino acid sequence of the Sca protein. Predicted signal peptide (SP, framed) and small pro-sequence are absent in the mature protein. Highly conserved and conserved amino acids are shadowed in black and in gray, respectively, and are concluded from the alignment in Supplementary Figure S2. ( d ) Amino acid sequence alignment of chosen Sca homologs from different filamentous fungi. Arrows represent predicted secondary structural elements of Sca from P. digitatum. Conserved cysteine residues are shadowed in red. Extremely conserved amino acids are shadowed in blue in the consensus sequence. Highly conserved amino acids are shadowed in black and conserved amino acids are shadowed in gray. Abbreviations: PENDIG, Penicillium digitatum; PENRUB: Penicillium rubens; FUSOXY, Fusarium oxysporum; GIBFUJ, Gibberella fujikuroi; TRIHAR, Trichoderma harzianum; EMENID, Emericella nidulans; ROSNEC, Rosellinia necatrix; MAGORY, Magnaporthe oryzae; METRIL, Metarhizium rileyi. An extended version of this alignment can be seen in Supplementary Figure S2. J. Fungi 2020,6, 203 8 of 18 3.2. Gene Expression of Sca (PDIG_23520) and Its Comparison with afpB (PDIG_68840) In order to further characterize this novel protein, the relative expression pattern of its encoding gene PDIG_23520 was determined during fungal axenic growth and infection, and compared to that of PDIG_68840, which encodes the previously reported AfpB (Figure 2). The expression pattern of both genes was similar during submerged growth in PDB, reaching the highest value of induction after 3-4 days of growth (Figure 2a). In contrast, afpB is much more induced than sca during aerial growth on PDA plates, concomitant with conidiogenesis, and the highest amount of afpB mRNA is found in quiescent conidia with levels more than 10 times above the reference condition (Figure 2b), as previously described [ 22 ]. In contrast, the amount of sca mRNA in conidia is more than 10 times below the reference condition. Remarkably, upon infection (Figure 2c), the sca expression pattern indicates a very early induction (from the first dpi) and a later decline, which qualitatively differs from the gene expression pattern of afpB which was not detected until 3 dpi, and from this point onwards, the relative level of mRNA remained approximately constant. It must be noted that maceration symptoms appeared from 3 dpi. These results would suggest a relevant role of Sca during fruit infection in its initial stages. Figure 2. Relative expression of afpB and sca genes. Relative expression of P. digitatum afpB (black bars) and sca genes (gray bars) over different times of growth in: ( a ) liquid medium (PDB), ( b ) solid medium (potato dextrose agar (PDA)) and conidia and ( c ) during infection of citrus fruits. Gene expression for each condition was normalized independently to the expression at day 6 in PDB, marked by a black arrow. n.d, expression of afpB not detected in these infection samples. dpi, days post-inoculation. Bars show the means ± standard error (SE) of three technical replicates. Note the logarithmic scale for Y axis. 3.3. Null Mutants and Overproducers Do Not Show Phenotypic Differences with Parental Strain during Axenic Growth Null ( ∆ sca) and overproducer (Sca op ) mutants were generated in order to study the biological role(s) of sca gene in P. digitatum. To obtain the null mutants, sca was replaced with the hph cassette as a positive selection marker for hygromycin resistance by homologous recombination (Supplementary Figure S3). The binary vector pGKO2_ ∆ sca obtained to delete the sca gene also contains the HSVtk gene used as a negative selection marker to discard ectopic insertions, as previously described [ 22 , 41 ] (Supplementary Figure S3a). Six independent ∆ sca transformants were obtained and confirmed by PCR using a set of distinct primer combinations located inside and outside the constructs (Supplementary Figure S3b and Supplementary Table S2). The FB modular cloning technology recently described [ 47 , 48 ] was applied to generate Sca op strains aimed at overproducing J. Fungi 2020,6, 203 9 of 18 the Sca protein, as described in Materials and Methods and Supplementary Figure S4. We aimed to generate Sca op strains by using the paf-based expression system [ 50 ], which contains the paf promoter and terminator sequences from P. chrysogenum, and has been demonstrated to work efficiently for the biotechnological production of small CRPs with antifungal activity, including AfpB [ 16 , 29 , 50 ]. Molecular characterization of six independent overproducers was confirmed by PCR using different primer sets (Supplementary Figure S4b,c and Supplementary Table S2). Two independent strains of ∆ sca and Sca op transformants were selected to characterize their phenotypic behavior, and results showed no major phenotypical differences under axenic culture on PDA plates (Figure 3a,b), indicating that gene deletion or protein overproduction has no influence on P. digitatum growth ability. SDS-PAGE analysis of tenfold concentrated supernatants obtained from CECT 20796 and the two ∆ sca strains phenotypically characterized (PDSG241 and PDSG253) confirmed the lack of Sca protein band in the mutants grown on PDB (Figure 3c). In contrast, Sca op transformants produced large amounts of Sca after 8–10 days of growth in PdMM [ 50 ], which were clearly visible and highly abundant even in the non-concentrated culture supernatants (Figure 3d). Figure 3. Characterization of different Penicillium digitatum sca null and overproducer strains. ( a ) Images of PDA plates after 5 days of growth of P. digitatum CECT 20796, null ∆ sca strains (PDSG241 and PDSG253, in red) and Sca op strains (PDSG31 and PDSG42, in green). ( b ) Colony diameter on PDA plates from 3 to 7 days of the same strains represented as the mean ± SD of three replicates. ( c ) SDS-PAGE of 10 × supernatants of P. digitatum CECT 20796 and null ∆ sca strains (PDSG241 and PDSG253) grown in PDB for 11 days. Pure Sca protein (1 µ g) was used as control. Note that the band corresponding to the Sca protein disappeared in these mutants. ( d ) SDS-PAGE of 8-day non-concentrated P. digitatum minimal medium (PdMM) supernatants of different P. digitatum Sca op strains compared with 1 µ g of pure Sca protein. M: SeeBlue ®pre-stained protein standard. 3.4. The Purified Sca Does Not Have Antimicrobial Activity In Vitro but Enhances the Antifungal Activity of AfpB Protein Sca was purified from culture supernatant of Sca op PDSG31 grown for 11 days in PdMM. 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