A total of eight isolates of B. cinerea were used in this study (Table 1). Among these isolates, XN-1, S417, and T417 are OS isolates, whereas B05.10, HS016, S59, WXto2-2, and XN087 are BS isolates. All these fungal isolates were maintained in 20% glycerol (v/v) at −80°C for the long-term storage. Working cultures of each isolate were prepared by transferring the mycelia of that isolate in storage on potato dextrose agar (PDA) and the resulting cultures were incubated in the dark at 20°C for 5 days. Seven cultural media (MYA, PDA, PDB, PRM, YPDA, SD/-Trp, and SD/-Trp-His) were used in this study. The composition of these media was listed in Table S1.

Six B. cinerea isolates (XN-1, S417, T417, HS016, WXt02-2, XN087) were separately incubated on cellophane film (CF)-PDA at 20°C for 3 days (Zhou et al., 2018). The mycelial mats were collected from the cultures, and used for extraction of genomic DNA (gDNA) using the CTAB method (Zeng et al., 2014) and the total RNA using the Trizol® reagents (TaKaRa Biotechnol. Co. Ltd., Dalian, China). The gDNA was used as template to PCR-amplify the DNA sequences of bcsmr1 as well as bcpks12/13, bcbrn1/2, and bcscd1 with the specific primers and thermal programs (Tables S2, S3). The PCR product was separated by agarose gel electrophoresis. The target DNA band in the gel was purified and inserted into the pMD18-T vector (TaKaRa). The resulting re-combined plasmid was subsequently transformed into the competent cells of E. coli DH5α. The positive clones harboring the expected DNA insert were sent to AuGCT Biotechnological Company Ltd. (Beijing, China). The nucleotide (nt) sequences for bcsmr1 (including the promoter region) from different isolates and the amino acid (aa) sequences for BcSMR1 encoded by bcsmr1 were identified by BLAST searches in the public database (http://www.ncbi.nlm.nih.gov). The characteristic domains for BcSMR1 encoded by bcsmr1 were identified based on the annotation information of that protein in the reference isolate B05.10. Multiple alignments of the nt sequences of bcsmr1 or the aa sequences of BcSMR1 in the seven isolates as well as in B05.10 were done using DNAMAN version 5.2.2 (Lynnon Biosoft, Vaudreuil, Quebec, Canada).

The full-length cDNA of bcsmr1 was cloned by reverse transcription (RT)-PCR from the OS isolate XN-1 and the BS isolate B05.10 using the primer pair bcsmr1-etF/bcsmr1-etR (Table S2). The primers contain a Not I-restriction site. The resulting cDNA was separated by agarose gel electrophoresis and purified from the gel. Then, the cDNA was digested with Not I to release the target fragments of bcsmr1 from XN-1 (designated as bcsmr1^OS) or B05.10 (designated as bcsmr1^BS). The two cDNA fragments were separately inserted into the plasmid pET28a(+) (Beijing TransGen Biotechnol. Co. Ltd., Beijing China) at the Not I site and two new plasmids, namely pET28a-bcsmr1^OS and pET28a-bcsmr1^BS (both had a 6× His tag), were then regenerated (Figure S1). The two plasmids were separately transformed into the competent cells of E. coli BL21 (DE3) pLysS (Promega, Madison, WI, USA). Positive clones for the two plasmids were screened based on growth on LB containing kanamycin (50 μg/mL). They were shake-incubated and induced for production of the proteins BcSMR1^OS and BcSMR1^BS using the procedures described by Lou et al. (2015). The bacterial proteins were separated by 8% SDS-PAGE and visualized by staining with Coomassie brilliant blue G-250.

The proteins in E. coli transformed with pET28a-bcsmr1^OS or pET28a-bcsmr1^BS were prepared using the procedures described by Lou et al. (2015) and were separated by 8% SDS-PAGE. After electrophoresis, the proteins in the gel were transferred by electrophoresis blotting (20 V, 30 min) to a piece of Bio-Rad® polyvinylidene fluoride (PVDF) membrane (6 × 4 cm, length × width) in Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell. The membrane was washed in TBST buffer (20 mmol/L Tris-base, 150 mmol/L NaCl, 0.05% Tween 20), followed by soaking for 2 h in 5% (w/v) Difco^TM skim milk solution alone and then in 5% skim milk solution amended with 0.2% (w/v) anti-His tag antibody (Sangon Biotechnol. Co. Ltd., Shanghai, China) for another 2 h. After that, the membrane was washed again in the TBST buffer for three times, 10 min each time, followed by soaking for 2 h in 5% skim milk solution amended with 0.1% (w/v) alkaline phosphatase-conjugated goat anti-mouse IgG (Sangon Biotechnol. Co. Ltd., Shanghai, China). After another washing in the TBST buffer, the membrane was soaked in 10 mL Western Blue® Stablized Substrate for Alkaline Phosphatase (Promega) to visualize the specific protein bands on the membrane. Finally, the membrane was dipped in water to stop the reaction and photographed to show the hybridzation.

The transcription activity of bcsmr1^OS and bcsmr1^BS was determined using Matchmaker™ GAL4 two-hybrid system 3 (Clontech Laboratories, 2007). The cDNA sequences for bcsmr1^BS in the BS isolate B05.10 (2,080 bp long), bcsmr1^OS in the OS isolate XN-1 (2,079 bp long), and bcactA (coding for actin) in B05.10 (1,128 bp long) were separately amplified by RT-PCR with the total RNA extracts from two isolates as templates using the specific RT-PCR primer sets and the specific thermal programs (Tables S2, S3). Meanwhile, the DNA sequence containing the GAL4 activation domain (AD_GAL4, 408 bp long) was PCR amplified from the plasmid pGADT7 (TaKaRa) with the primer set GAL4-F/GAL4-R (Table S2). The resulting cDNA fragments for bcsmr1^OS, bcsmr1^BS, and bcactA, and the DNA fragment of AD_GAL4 were purified from a 1% agarose gel after electrophoresis. They were separately inserted into the plasmid pGBKT7 containing the DNA binding domain of GAL4 (BD_GAL4) using ClonExpress® II One Step Cloning Kit (Vazyme Biotechnol. Co. Ltd., Nanjing, China). The resulting plasmids pBD_GAL4-AD_GAL4 (positive control), pGBKT7 (negative control 1), pBD_GAL4-BcACTA (negative control 2), pBD_GAL4-BcSMR1^OS, and pBD_GAL4-BcSMR1^BS were separately transformed into the yeast cells of strain AH109 using the LiAc/SS carrier DNA/PEG transformation method (Clontech Laboratories, 2007). Selection of the yeast mutants harboring the plasmids and detection of their transcription activity were done using the procedures described by Lou et al. (2015).

The transcripts of bcsmr1, bcygh1, bcpks12/13, bcbrn1/2, and bcscd1 in the sclerotia of the B. cinerea isolates were determined using qRT-PCR with the specific primers and thermal cycles (Tables S2, S3). All the investigated isolates were incubated on PDA at 20°C. The sclerotial primordia, immature sclerotia, and mature sclerotia were collected from the 6-, 8-, and 15-day-old cultures of each isolate, respectively. The total RNA in the sclerotial samples was extracted using TaKaRa RNAiso Reagent Kit (TaKaRa) and was subjected to treatment with DNase I (TaKaRa) to eliminate DNA contamination in the extracts. The treated RNA was then used as templates in qRT-PCR to determine the transcripts of bcactA (reference), bcsmr1, bcpks12/13, bcygh1, bcbrn1/2, and bcscd1. The transcript levels of each gene were calculated by the ^ΔΔCt method (Zeng et al., 2014). For normalization of the data, the transcript level of each gene in the sclerotial primordia of isolate B05.10 was considered as 1.0. The scale was used to calibrate the transcript level of that gene in the immature and mature sclerotia of B05.10, as well as in the sclerotial primordia, immature sclerotia, and mature sclerotia of other isolates. The qRT-PCR assay was independently repeated three times.

The complementation plasmid pKS1004-BS was constructed based on the plasmid pKS1004 containing the hygromycin-resistance gene (hph) (Zeng et al., 2014). The full-length cDNA of bcsmr1^BS from B05.10 was ligated at the downstream of the Aspergillus nidulans promoter Polic by fusion PCR. The resulting DNA fragment was inserted into the plasmid pKS1004 using the protocol and the reagents in ClonExpress® II One Step Cloning Kit (Vazyme Biotechnol. Co. Ltd., Nanjing, China). The plasmid was then transformed into the protoplasts of XN-1 (Zeng et al., 2014). The transformed protoplasts were regenerated at 20°C on the PRM medium (Table S1) amended with 50 μg/mL hygromycin B. The emerging colonies (isolates) were individually transferred to PDA. Meanwhile, isolates B05.10 and XN-1 were inoculated on PDA as two controls. The cultures (isolates) were incubated at 20°C for 35 days for production of sclerotia. Six complementation mutants were selected based on formation of brown- to black-colored sclerotia (indicating sclerotial melanogenesis). Integration of bcsmr1^BS in the genomes of the complementation mutants was confirmed by PCR using the primer set Olic-F/bcsmr1R (Table S2). XN-1 was used as reference in the specific PCR assay. Two representative complementation mutants, namely XN-BS11 and XN-BS14, were selected for determination of the copy number of bcsmr1 by Southern blotting also with XN-1 as a reference. The bcsmr1-specific DNA probe was generated by PCR from the genomic DNA of B05.10 with the primer set bcsmr1-jcF/bcsmr1R (Table S2). In Southern blotting, the gDNA of XN-1, XN-BS11, and XN-BS14 was extracted and digested with EcoRI. The resulting DNA fragments were separated by electrophoresis and detected by the DNA probe, which was labeled with the reagents in DIG High Prime DNA Labeling and Detection Starter Kit II (Roche®, Mannheim, Germany).

Expression the six melanogenic genes (bcpks12/13, bcygh1, bcbrn1/2, bcscd1) in sclerotia produced by XN-1 (negative control), XN-BS11, XN-BS14, and B05.10 (positive control) was determined using qRT-PCR. The procedures for isolation of the total RNA from the sclerotial samples and qRT-PCR were similar to those described above.

The procedure UNIVARIATE in the SAS software (SAS Institute, Cary, NC, USA, v. 8.0, 1999) was used to analyze the data on relative expression levels of each gene. Average values of each gene between the OS (3 isolates) and BS (4 isolates) groups, and between each complementation mutant and XN-1/B05.10 were compared using Student's t-test at P < 0.05 or P < 0.01.

To analyze the melanin-biosynthesis ability in orange-colored sclerotia of B. cinerea, expression of six melanogenic enzymes-coding genes, including bcpks12/13, bcygh1, bcbrn1/2, and bcscd1, in the sclerotia of seven isolates of B. cinerea (Table 1) was determined by qRT-PCR using the specific primers and thermal programs (Tables S2, S3). The results showed that all the seven investigated isolates (OS isolates: XN-1, S417, T417; BS isolates: B05.10, HS016, XN087, WXt02-2) had low expression levels of bcpks13 at all sclerotial developmental stages (Figure 1). Thus, bcpks13 did not seem to be involved in the formation of the orange-colored sclerotia.

However, significant differences were found between the OS and BS isolates in expression levels of the polyketide synthase gene bcpks12 (Figure 1). Although all the seven investigated isolates showed the increased expression pattern in maturation of sclerotia, the expression levels were significantly lower in the OS isolates than in the BS isolates. In the sclerotial primordia (white-colored) from the 6-day-old PDA cultures (20°C), the expression level of bcpks12 was low with the relative expression values (REVs) ranging from 0.3 to 1.4 for the OS isolates and from 1.0 to 22.6 for the BS isolates. In the immature sclerotia (gray-colored) from the 8-day-old PDA cultures and in the mature sclerotia (black- and orange-colored in the BS and OS isolates, respectively) from 15-day-old PDA cultures, the bcpks12 expression levels slightly increased by 2.6 and 7.8-folds, respectively, in the OS isolates, whereas were largely increased by 40.4 and 121.8-folds, respectively, in the BS isolates (Figure 1).

For the other sclerotial melanogenesis genes (bcygh1, bcbrn1/2, bcscd1), the OS isolates differed from the BS isolates both in expression pattern and in expression level (Figure 1). While the BS isolates showed an increased expression pattern, the OS isolates showed a decreased expression pattern. In the sclerotial primordia of the OS isolates, the REVs ranged from 0.01 to 0.03 for bcygh1, 1.5 to 2.1 for bcbrn1, 1.3 to 2.6 for bcbrn2, and 0.4 to 0.8 for bcscd1. In the sclerotial primordia of the BS isolates, the REVs ranged from 0.1 to 1.0 for bcygh1, 1.0 to 3.4 for bcbrn1, 1.0 to 6.9 for bcbrn2, and 0.3 to 1.1 for bcscd1. In the immature sclerotia and mature sclerotia, expression of these genes was decreased by 43.2–88.5% in the OS isolates, whereas it was increased by 8 to 90-folds in the BS isolates, compared the corresponding REVs of these genes in the sclerotial primordia of B05.10.

To rule out the possibility that suppressed expression of bcpks12, bcygh1, bcbrn1/2, and bcscd1 in the sclerotia of the OS isolates is caused by mutations of these genes, the DNA sequences of these genes were cloned from the BS and OS isolates mentioned above (Table S4). Alignment analysis showed that the identity of the DNA sequences of each gene and the amino acid sequences of each protein in the seven above-mentioned isolates of B. cinerea was higher than 98% (Table S5). Single-amino acid polymorphisms were detected in the amino acid sequences of each protein (Figure S2). However, none of the polymorphic amino acids was found to be associated with formation of the orange-colored sclerotia. Therefore, suppressed expression of bcpks12, bcygh1, bcbrn1/2, and bcscd1 in the sclerotia of the OS isolates of B. cinerea is probably not caused by the point mutations in these genes. It might be caused by some regulatory genes for the sclerotial melanogenesis.

To confirm whether the abnormal expression pattern of the melanin biosynthesis genes was caused by some regulatory malfunction, the transcription factor (TF) gene bcsmr1 related to the melanin biosynthesis was cloned from three OS isolates (XN-1, S417, T417) and four BS isolates (HS016, S59, WXt02-2, XN087) by PCR using the specific primers and specific thermal cycles (Tables S2, S3). The DNA sequences for bcsmr1 in these isolates were submitted to GenBank and were assigned with the accession numbers from KU743104 to KU743109, and KX098785 (Table S4). They were aligned with the DNA sequence of bcsmr1 in the reference isolate B05.10 (Accession Number: Bcin02g08760 in the website http://fungi.ensembl.org/Botrytis_cinerea). The results showed that bcsmr1 in the OS isolates (designated as bcsmr1^OS) differed from bcsmr1 in other five BS isolates (designated as bcsmr1^BS) in length of the open reading frame (ORF), 3,096 bp long for bcsmr1^BS, whereas 3,095 bp long for bcsmr1^OS (Figure 2A). The single-nucleotide deletion in bcsmr1^OS is located at the position of nt 1,524, where guanine (G) is present in bcsmr1^BS, but absent in bcsmr1^OS. Thus, the single-nucleotide deletion was designated as G1524X. In spite of the single-nucleotide deletion, the identity of the DNA sequences in the ORF region of bcsmr1^OS and bcsmr1^BS in the seven isolates was higher than 98% (Table S5).

The single-nucleotide deletion in bcsmr1^OS caused change of the triplet codon at the position nt 1,522 to 1,524 from ATG (for methionine or M) in bcsmr1^BS to ATA (for isoleucine or I) in bcsmr1^OS. The subsequent triplet codon at the position nt 1,525 to 1,527 was also changed from ATA (for isoleucine or I) in bcsmr1^BS to TAG (stop) in bcsmr1^OS (Figure 2A). As a consequence, the ORF of bcsmr1^OS was deduced to encode a protein with 465 amino acid (aa) residues (designated as BcSMR1^OS). It is a truncated protein of the 935-aa protein encoded by the ORF of bcsmr1^BS (designated as BcSMR1^BS). Alignment analysis showed that both BcSMR1⁴⁶⁵ and BcSMR1⁹³⁵ have the DNA binding domain at the N-terminus, consisting of two Cys₂His₂-zinc finger motifs and a Zn₂Cys₆-binuclear motif (Figure 2B). BcSMR1^OS has a small portion (32 aa long) of the fungal specific transcription factor domain at the C-terminus without the nuclear localization signaling, whereas BcSMR1^BS has the full fungal specific TF domain (298 aa long) with the nuclear localization signaling region. Alignment analysis also showed that there are six point mutations (T125I, A174D, V181I, L223S, S232P, M465I) in BcSMR1^BS and BcSMR1^OS (Figure 2B) and five of these mutations (T125I, A174D, V181I, L223S, S232P) were found in both BcSMR1^BS and BcSMR1^OS. Therefore, they are probably not associated with formation of the orange-colored sclerotia. The other mutation, namely M465I, was detected only in BcSMR1^OS, but it was not detected in BcSMR1^BS. This mutation is caused by the single-nucleotide-deletion-mediated one-nucleotide shift (Figure 2A). Therefore, the single-nucleotide deletion is probably important for formation of the orange-colored sclerotia by the OS isolates of B. cinerea. In spite of the point mutations, the identity of bcsmr1^OS and bcsmr1^BS is higher than 99% (Table S5).

Prokaryotic expression of bcsmr1^OS and bcsmr1^BS in E. coli was done to confirm formation of BcSMR1^OS through the single-nucleotide deletion-mediated truncation of BcSMR1^BS. The full-length cDNA of bcsmr1^OS in XN-1 and bcsmr1^BS in B05.10 were separately cloned by RT-PCR using the specific primers and specific thermal program (Tables S2, S3). The resulting cDNA fragments were separately introduced into the expression vector pET28a(+) (Figure S1). The resulting two plasmids, namely pET28a-bcsmr1^OS and pET28a-bcsmr1^BS, were then constructed and separately transformed into E. coli BL21 (DE3) pLysS. The transformed bacterial clones were selected, followed by shake-incubation in LB medium for production of BcSMR1^OS and BcSMR1^BS under induction by isopropyl β-D-1-thiogalactopyranoside (IPTG). The bacterial proteins extracted from different E. coli cultures were detected by 8% SDS-PAGE. Results showed that two differentially-expressed proteins were consistently detected in the positive transformants of E. coli (data not show). The molecular weight was ~66 and 120 kD (both had a 6× His tag) in the transformants with pET28a-bcsmr1^OS and pET28a-bcsmr1^BS, respectively. The 54-kD difference in molecular weight between the two proteins is close to the 53.2-kD difference in molecular weight between the putative BcSMR1^OS and BcSMR1^BS, which had the predicted molecular weight of 51.3 and 104.5 kD, respectively. Western blotting analysis using the anti-His tag antibody further confirmed production of the 66- and 120-kD proteins by bcsmr1^OS and bcsmr1^BS, respectively, in E. coli (Figure 3).

Both the OS isolates and the BS isolates had an increased expression pattern of bcsmr1 in sclerotia (Figure 4). We hypothesized that compared to bcsmr1^BS, bcsmr1^OS might be suppressed for activating transcription of the downstream genes for sclerotial melanogenesis. To test this hypothesis, the transcription activity of bcsmr1^OS and bcsmr1^BS was determined in Saccharomyces cerevisiae AH109 using Matchmaker™ GAL4 Two Hybrid System 3 (Clontech Laboratories, 2007). Five plasmids (pBD_GAL4, pBD_GAL4-BcACTA, pBD_GAL4-AD_GAL4, pBD_GAL4-BcSMR1^OS, and pBD_GAL4-BcSMR1^BS) were constructed. Four plasmids (pBD_GAL4-BcACTA, pBD_GAL4-AD_GAL4, pBD_GAL4-BcSMR1^OS, and pBD_GAL4-BcSMR1^BS) contain two domains, namely the DNA-binding domain (e.g., BD_GAL4) and the activation domain (e.g., AD_GAL4, BcACTA, BcSMR1^OS, BcSMR1^BS). The remaining plasmid pBD_GAL4 contains BD_GAL4 alone. The plasmids were separately transformed into the yeast cells and the resulting transformants were screened on two auxotrophic media SD/-Trp and SD/Trp-His. On SD/-Trp, all the mutants showed growth. On SD/-Trp-His, however, the mutants differed in growth (Figure 5). While the transformants harboring either pBD_GAL4 or pBD_GAL4-BcACTA failed to grow on SD/-Trp-His, the transformants of pBD_GAL4-AD_GAL4, pBD_GAL4-BcSMR1^OS, and pBD_GAL4-BcSMR1^BS grew on this medium and produced blue color on the colonies when X-α-gal was added. This result indicates that AD_GAL4, bcsmr1^BS, and bcsmr1^OS can activate transcription of the MEL1 gene coding for α-galactosidase. Relatively, the transformants of pBD_GAL4-AD_GAL4 and pBD_GAL4-BcSMR1^BS grew better than the transformant harboring pBD_GAL4-BcSMR1^OS. The blue color on the colonies of the transformants of pBD_GAL4-AD_GAL4 and pBD_GAL4-BcSMR1^BS was darker than that on the colonies of the transformant of pBD_GAL4-BcSMR1^OS. Therefore, compared to bcsmr1^BS, bcsmr1^OS is greatly suppressed for transcription of MEL1.

To confirm responsibility of the single-nucleotide deletion in bcsmr1^OS for sclerotial melanogenesis deficiency, the full-length cDNA of bcsmr1^BS from B05.10 was used to complement the function of bcsmr1^OS in XN-1. A complementation plasmid pKS1004-BS containing the cDNA of bcsmr1^BS under the control of the constitutive A. nidulans PoliC promoter was successfully constructed on the basis of the plasmid pKS1004 harboring the hygromycin-resistance gene hph (Zeng et al., 2014). The plasmid was introduced into XN-1 by protoplast-mediated transformation. Ten transformants were obtained based on hygromycin resistance. After incubation at 20°C on PDA for 35 days, XN-1 formed orange-colored sclerotia, as it has defect in sclerotial melanogenesis, whereas B05.10 formed black-colored sclerotia, as it has normal sclerotial melanogenesis (Figure 6). Four of these transformants formed orange-colored sclerotia like those formed by XN-1, indicating that the sclerotial melanogenesis in these transformants was not restored at all. In contrast, six other transformants (XN-BS1, XN-BS2, XN-BS11, XN-BS12, XN-BS14, XN-BS16) formed partially-melanized sclerotia, which were characterized by formation of color mosaic on the sclerotial surface (Figure 6). These six transformants could be classified into two groups based on the color mosaic on the sclerotia. Three transformants (XN-BS11, XN-BS12, XN-BS16) showed a brown-gray mosaic pattern, whereas the other three transformants (XN-BS1, XN-BS2, XN-BS14) showed a gray-black mosaic pattern. This result indicates that the sclerotial melanogenesis in these transformants was at least partially restored, compared to the complete sclerotial melanogenesis in B05.10 and the defective sclerotial melanogenesis in XN-1. Meanwhile, sporogenic germination (indicating lack of dormancy) was observed on the orange sclerotia produced by XN-1 and on the brown-grayish sclerotia produced by the transformants XN-BS11, XN-BS12, and XN-BS16 (Figure 6). The sporulation appeared to be vigorous on the sclerotia of XN-1, whereas was sparse on the sclerotia of the three transformants. In contrast, the black-colored sclerotia produced by B05.10 and the gray-colored sclerotia produced by the transformants XN-BS1, XN-BS2, and XN-BS14 did not germinate at all. This result indicates that the dormancy deficiency of the orange sclerotia (germination-active) of XN-1 due to lack of melanin was partially restored in the partially-melanized sclerotia produced by the complementation mutants.

The six complementation transformants were confirmed for integration of bcsmr1^BS in their genomes by PCR using the primer set Olic-F/bcsmr1R (Table S2) (data not shown). Two of these transformants (XN-BS11, XN-BS14) were further determined for bcsmr1^BS integration in their genomes by Southern blotting using the bcsmr1-specific DNA probe (Figure S3A). XN-1 was used as the reference isolate in Southern blotting. The result showed that the three isolates had one to three hybridization bands, one band in XN-1, two bands in XN-BS11, and three bands in XN-BS14 (Figure S3B). This result indicates that the genomes of XN-1, XN-BS11, and XN-BS14 harbor one (bcsmr1^OS), two (bcsmr1^OS + bcsmr1^BS), and three (bcsmr1^OS + bcsmr1^BS + bcsmr1^BS) copies of bcsmr1, respectively.

Expression of the six melanogenic genes (bcpks12/13, bcygh1, bcbrn1/2, bcscd1) in the sclerotia of isolates XN-1, XN-BS11, and XN-BS14 was determined by qRT-PCR. The two transformants were similar to XN-1 both in expression pattern and in expression level of bcpks13, which is not required for sclerotial melanogenesis according to Schumacher (2016). All the three isolates showed a decreased expression pattern and low expression levels of bcpks13 in sclerotial maturation.

In contrast, the two transformants differed greatly from XN-1 either in expression pattern or in expression level of the other five sclerotial melanogenesis genes (bcpks12, bcygh1, bcbrn1/2, bcscd1). For bcpks12 and bcygh1, the two transformants were similar to XN-1 in expression pattern, but differed from XN-1 in expression level (Figure 7). The two genes showed an increased expression pattern in maturation of the sclerotia produced by the three isolates. In the sclerotial primordia, the REVs ranged from 1.0 to 49.8 for bcpks12 and from 1.0 to 10.0 for bcygh1. In the immature sclerotia and mature sclerotia, the REVs of the two genes were largely increased by 12 to 350-folds in the two transformants, whereas were slightly increased by 3 to 12-folds in XN-1.

For bcbrn1, the two transformants differed from XN-1 both in expression pattern and in expression level (Figure 7). In XN-1, the REV was reduced from 1.0 in the sclerotial primordia to < 0.3 in the immature sclerotia/mature sclerotia. In XN-BS11 and XN-BS14, however, the REVs were increased in maturation of the sclerotia. XN-BS11 had the REV increase from 0.6 in the sclerotial primordia to 2.6 in the immature sclerotia, and 7.4 in mature sclerotia. XN-BS14 had a REV increase from 2.9 in the sclerotial primordia to 10.8 in the immature sclerotia, and 13.1 in the mature sclerotia.

For bcbrn2 and bcscd1, the two transformants were also similar to XN-1 in expression pattern, but differed from XN-1 in expression level (Figure 7). All the three isolates showed a decreased expression pattern in maturation of the sclerotia. In the sclerotial primordia, the three isolates had the REVs of 0.9–2.3 and 0.9–1.8 for bcbrn2 and bcscd1, respectively. In the immature sclerotia and mature sclerotia, while XN-1 had negligible expression of both genes, XN-BS11 had the REVs of 0.1–0.6 for bcbrn2 and 0.4–1.7 for bcscd1. In general, XN-BS14 had higher REVs of bcpks12, bcygh1, bcbrn1/2, and bcscd1 than the corresponding REVs for XN-BS11 (Figure 7). Meanwhile, the expression pattern of bcpks12, bcygh1, and bcbrn1/2 in the mutants XN-BS11 and XN-BS14 resembled that in the BS isolate B05.10, although the expression levels of these genes at each stage were significantly (P < 0.05 or P < 0.01) lower than those of B05.10 (Figure 7). This result further confirmed that the single-nucleotide deletion in bcsmr1 is the major reason for formation of the orange-colored sclerotia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.