Figure 1.
Maps of Sclerotinia sclerotiorum mating type alleles that differ by a 3.6-kb inversion.
Inv- (top) and Inv+ (bottom) MAT alleles are shown, the 3.6-kb MAT inversion region is indicated by a gray horizontal line. Genes are colored boxes, white and dotted boxes within genes correspond to alpha1 and HMG domains, respectively, flanking genes are white boxes, directions of transcription are indicated by arrows, gene names are inside or by the boxes. Dashed box and arrow represent MAT1-1-1 3’-fragment lacking an in frame start codon. Diagrams are to scale. The Inv+ alpha1 box is truncated after 45 bp and is not illustrated, for details see text.
Table 1.
Mating type gene statistics of Sclerotinia sclerotiorum strains 44Ba1, 44Ba12. and 44Ba18 in relation to S. sclerotiorum strain 1980 and Botrytis cinerea [17].
Figure 2.
Most parsimonious trees obtained from Sclerotinia sclerotiorum MAT1-1-1, MAT1-1-5, MAT1-2-1 and MAT1-2-4 single locus datasets (Alignments S6, S7, S8, S9).
The trees were rooted with B. cinerea which is not shown, numbers refer to alignment positions. Nucleotide substitutions along the branches are given for S. sclerotiorum. Accession numbers are shown for all sequences following the strain identifiers, for B. cinerea the sequence accession numbers are BC1G_15148 (MAT1-1-1), BC1G_15147 (MAT1-1-5), FQ790352 (MAT1-2-1), FQ790352 (MAT1-2-4).
Figure 3.
Inversion of the 3.6-kb MAT inversion region in Sclerotinia sclerotiorum by means of crossing over between the two 250-bp motifs.
Genes are colored boxes, white and dotted boxes within genes correspond to alpha1 and HMG domains, respectively, flanking genes are white boxes, directions of transcription are indicated by black arrows, gene names are inside or by the boxes, red arrows mark the orientations of the 250-bp motifs for which the 5’- and 3’-end sequence is given. Dashed box and arrow represent MAT1-1-1 3’-fragment lacking an in frame start codon. 3A) MAT region of S. sclerotiorum Inv- isolates. 3B) Crossing over between the two 250-bp motifs of a S. sclerotiorum Inv- isolate gives rise to 3C), the inversion of S. sclerotiorum Inv+ isolates. Figures are not to scale. The Inv+ alpha1 box is truncated after 45 bp and is not illustrated, for details see text.
Figure 4.
Evolutionary origin of the MAT locus in Sclerotinia sclerotiorum Inv- isolates from hypothetical ancestors by means of a double crossing over, an inversion and a single crossing over.
Genes are boxes, directions of transcription are indicated by arrows, gene names are inside the boxes, positions of the 250-bp motif are indicated by red horizontal lines, crossing overs are marked by ‘X’, the inversion by a curved arrow. 4A) A double crossing over between ancestral MAT1-1 (blue) and MAT1-2 (green) transferred a 250-bp fragment, the ‘250-bp motif’, from MAT1-1-1 to MAT1-2-1 and flanking region. 4B) A subsequent inversion (gray) in the ancestral MAT1-2 (green), followed by a crossing over, 4C) with an ancestral MAT1-1 (blue), results in the MAT arrangement in S. sclerotiorum Inv- isolates. The asterisk indicates the location of the DNA sequence alignment provided in Figure 5.
Figure 5.
Proposed MAT1-1 – MAT1-2 border in S. sclerotiorum MAT inferred from an alignment of S. sclerotiorum MAT, B. cinerea MAT1-1 and MAT1-2.
The location of the alignment reproduced above is indicated in Figure 4 by an asterisk, for entire alignment see Alignment S3. Sclerotinia sclerotiorum strain 44Ba1 MAT (GenBank Accession JQ815883) and B. cinerea MAT1-1 (AAID01003685) were alignable up to 248 bp downstream of S. sclerotiorum MAT1-1-1 as indicated by the top left arrow, homology ceases thereafter. The site where a crossover between ancestral MAT1-1 and MAT1-2 represented here by B. cinerea MAT1-1 and MAT1-2, might have occurred, is underlined. The border and crossover site positions are tentative, since S. sclerotiorum MAT and B. cinerea MAT1-2 (reverse complement of FQ790352) were too divergent to be aligned in the MAT1-2-4 downstream region (Figure 1), the top right arrow demarcates the MAT1-2 region in S. sclerotiorum based on lack of homology to B. cinerea MAT1-1, not based on homology to B. cinerea MAT1-2.
Figure 6.
Gene expression in Sclerotinia sclerotiorum Inv- and Inv+ isolates.
6A) Positions of RT-PCR primers on S. sclerotiorum Inv- (top) and Inv+ (bottom) MAT loci, the MAT inversion region is indicated by a gray horizontal line. Genes are colored boxes, white and dotted boxes within genes correspond to alpha1 and HMG domains, respectively, flanking genes are white boxes, directions of transcription are indicated by arrows, gene names are inside or by the boxes. Dashed box and arrow represent MAT1-1-1 3’-fragment lacking an in frame start codon. Diagrams are to scale. The Inv+ alpha1 box is truncated after 45 bp and is not illustrated, for details see text. 6B) Gels with RT-PCR results for all MAT genes, the MAT1-1-1 3’-fragment and an actin control, for all eight S. sclerotiorum strains 1B331-1 – 1B331-8 representing an ordered tetrad. Gene names are indicated above the gels. Lane numbers refer to S. sclerotiorum strain identifiers 1B331-1– 1B331-8. The letters M and N indicate size markers and negative controls, respectively. Band sizes are indicated on the left of each gel. For MAT1-1-1 and MAT1-2-1, the sizes of the transcript variants are indicated on the right of the gels.
Figure 7.
Alignment of MAT1-1-1 (top) and MAT1-2-1 (bottom) transcript variants (left) and deduced protein variants (right).
Transcript variants are colored boxes, black lines between the boxes represent alignment gaps with respect to other variants, the 5’- and 3’-ends are indicated, variant lengths are given underneath the boxes in base pairs, variant designation and presence in S. sclerotiorum Inv- and Inv+ isolates is indicated on the left. Protein variants are boxes, N and C terminals are indicated, white boxes mark alpha1 and dotted boxes mark HMG domains, black boxes represent unalignable regions, variant designation and presence in S. sclerotiorum Inv- and Inv+ isolates is indicated on the left. Lengths of protein variants are given underneath the boxes in residues. Protein variants were deduced conceptually taking transcript variants into account. Diagrams are not to scale. 7A) Schematic alignments of MAT1-1-1 transcript variants from Figure 6B and the corresponding inferred MAT1-1-1 variants. Only transcript variant 2 of S. sclerotiorum Inv- isolates implies the presence of a complete alpha1 box in MAT1-1-1. 7B) Schematic alignments of MAT1-2-1 transcript variants from Figure 6B and the corresponding inferred MAT1-2-1 variants. Only transcript variant 2 of S. sclerotiorum Inv- and Inv+ isolates contains an HMG box.
Figure 8.
Analysis of MAT1-2-1 copy number using Southern blotting.
8A) Gene diagrams of Sclerotinia sclerotiorum Inv- (left) and Inv+ (right) MAT loci illustrating the positions of the Southern probe with respect to the restriction sites used for Southern analyses. Boxes represent genes, white and dotted boxes correspond to alpha1 and HMG domains, respectively, dashed box represents MAT1-1-1 3’-fragment lacking an in frame start codon. Gene names are indicated above or within the boxes. The positions of the BsaHI restriction sites (black triangles) and the distances between the BsaHI sites are indicated above the boxes. The position of the Southern probe is marked by a horizontal black line beneath MAT1-2-1. The Inv+ alpha1 box is truncated after 45 bp and is not illustrated, for details see text. 8B) Southern blot of BsaHI-digested genomic DNA visualized with the digoxigenin-labeled MAT1-2-1 specific probe. Wells 1 - 8 correspond to S. sclerotiorum strains 1B331-1 – 1B331-8 that represent an ordered tetrad, band sizes are indicated on the right. Lanes 1, 2, 5 and 6 have the pattern reflective of an Inv+ MAT locus, lanes 3, 4, 7 and 8 have the pattern expected for an Inv- MAT locus.
Figure 9.
MAT locus distribution in the eight Sclerotinia sclerotiorum strains 1B331-1 – 1B331-8 representing an ordered tetrad.
PCR gels with primers specific to Inv- (top) and Inv+ (bottom) MAT loci are shown (Figure S1). Lane numbers refer to S. sclerotiorum strain identifiers 1B331-1 – 1B331-8. The letters M and N indicate size markers and negative controls, respectively.
Figure 10.
Orientation of MAT inversion region in Sclerotinia sclerotiorum isolates as evaluated by Inv+ and Inv- specific PCR reactions.
Each isolate has one PCR band and is thus either Inv- or Inv+, weak bands in isolates with strong bands are false positives due to cross contamination. Isolates used are parental strains BS001, BS011, BS013, BS014, BS017 and BS028 (Table S1), and their progeny (Table S6), including the tetrads in Figure 10F. The top gel in each part figure shows the PCR bands obtained with primer pair Type-IIF / Type-IIR specific to Inv+, and the bottom gel the bands with primer pair MAT1-1-F / MAT1-1-R specific to Inv- (Figure S1). Lanes are numbered, lanes marked with asterisks contain negative controls, the first and last wells of each gel are DNA size standards, arrow heads in Figure 10A indicate positions of 1.2 kb and 0.6 kb bands, the remaining wells are as follows for S. sclerotiorum strains and negative controls in sequential order. 10A) BS011, BS011sa01, BS011sa02, BS011sa03, BS011sa04, BS011sa05, BS011sa06, BS011sa07, BS011sa08, BS011sa09, BS011sa10, BS011sa11, BS011sa12, BS011sa14, BS011sa15, BS011sa16, BS011sa17, BS011sa18. 10B) BS011sa19, BS011sa20, negative control, BS013, BS013sa01, BS013sa02, BS013sa04, BS013sa05, BS013sa06, BS013sa07, BS013sa08, BS013sa09, BS013sa10, BS013sa11, BS013sa12, BS013sa13, BS013sa14, BS013sa15. 10C) BS013sa16, BS013sa17, BS013sa18, BS013sa19, BS013sa20, negative control, BS017, BS017sa01, BS017sa02, BS017sa03, BS017sa04, BS017sa05, BS017sa06, BS017sa07, BS017sa08, BS017sa09, BS017sa10, BS017sa11. 10D) BS017sa12, BS017sa13, BS017sa14, BS017sa15, BS017sa16, BS017sa17, BS017sa18, BS017sa19, BS017sa20, negative control, BS028, BS028sa03, BS028sa04, BS028sa05, BS028sa06, BS028sa07, BS028sa08, BS028sa09. 10E) BS028sa10, BS028sa11, BS028sa12, BS028sa13, BS028sa14, BS028sa15, BS028sa16, BS028sa17, BS028sa18, BS028sa19, BS028sa20, negative control. 10F) BS001, BS014, 1B321-2, 1B321-4, 1B321-6, 1B321-8, 1B331-1, 1B331-2, 1B331-3, 1B331-4, 1B331-5, 1B331-6, 1B331-7, 1B331-8, negative control from upper gel, negative control from lower gel.
Table 2.
χ2 test results of observed versus expected Inv+ and Inv- frequencies among progeny from single apothecia of four parental strains, screening results are in Table S6.
Table 3.
Occurrence of Sclerotinia sclerotiorum Inv+ isolates in twelve states and on different hosts in the United States based on PCR screening of all isolates in Table S1.
Figure 11.
Phylogram inferred from 250-bp motifs of 36 S. sclerotiorum isolates.
The two 250-bp motifs of each of the 36 isolates in Alignment S4 were aligned after the MAT1-1-5 distal motifs were reverse complemented (Figure 3), resulting in a 72 taxa, 250 bp dataset (Alignment S5). Taxon names consist of strain identifier, inversion phenotype, and location of 250-bp motif with ‘left’ referring to MAT1-1-5 proximal, ‘right’ to MAT1-1-5 distal motifs (Figure 3). For sequences deposited or obtained from other sources, database accession numbers are also included. All isolates are listed in Table S1, except the isolates representing the random progeny of S. sclerotiorum strains BS011, BS013, BS017 and BS028 which are in Table S6. Groups of isolates are delimited on the right by vertical lines, numbers of isolates in each group are in parentheses. Included isolates were S. sclerotiorum strains 44Ba1, 44Ba12, 44Ba18 for which the entire MAT region was sequenced, S. sclerotiorum strains BS001 and BS014 that are in contention for parent of the two tetrads, S. sclerotiorum strains 1B321-2, 1B321-4, 1B321-6 and 1B321-8 of the incomplete tetrad, S. sclerotiorum strains 1B331-1 – 1B331-8 of the complete tetrad, S. sclerotiorum strains BS011, BS013, BS017 and BS028 with one Inv- and one Inv+ random progeny each, and the non-California isolates. There are no substitutions in the tree, illustrating that all 250-bp motifs have identical DNA sequences.
Table 4.
Apothecia production in 57 Sclerotinia sclerotiorum isolates from California.
Figure 12.
The S. sclerotiorum MAT inversion region changes orientation in every meiotic generation and segregates at the first or second division of meiosis.
Small ovals are ascospores, large ovals are asci, yellow ascospores are Inv+, red ascospores are Inv-. Single ascospores represent parental isolates, asci containing eight ascospores represent progeny. 12A) Regardless of whether the parent is Inv- or Inv+, 50% of the progeny is Inv+, suggesting a highly regulated process during the sexual cycle ensures the 1∶1 ratio. The transition of Inv- to Inv+ involves a change in orientation of the MAT inversion region. 12B) Meiotic segregation pattern of Inv- and Inv+ among ascospores in S. sclerotiorum. The pattern that is characteristic for first division segregation in Neurospora crassa is shown on the left, the pattern for second division segregation following recombination is shown on the right [42]. Figure 12 is based on PCR screens for absence and presence of the MAT inversion region in tetrad and random progeny and their parents with DNA from the mycelial phase (Figures 9, 10), DNA sequencing of inversion breakpoints (Figure 11) and the entire inversion regions and flanks in S. sclerotiorum strains 1B331-1 and 1B331-3 of the complete ordered tetrad (Alignment S1), and Southern blotting illustrating that S. sclerotiorum MAT is single copy (Figure 8).