Molecular Dissection of Perithecial Mating Line Development in Colletotrichum fructicola, a Species with a Nontypical Mating System Featuring Plus-to-Minus Switch and Plus-Minus-Mediated Sexual Enhancement

Abstract

The genetic regulation of Colletotrichum (Glomerella) sexual reproduction does not strictly adhere to the Ascomycota paradigm and remains poorly understood. Morphologically different but sexually compatible strain types, termed plus and minus, have been recognized, but the biological and molecular distinctions between these strain types remain elusive. In this study, we characterized the sexual behaviors of a pair of plus and minus strains of C. fructicola with the aid of live-cell nucleus-localized fluorescent protein labeling, gene expression, and gene mutation analyses. We confirmed a genetically stable plus-to-minus switching phenomenon and demonstrated the presence of both cross-fertilized and self-fertilized perithecia within the mating line (perithecia cluster at the line of colony contact) between plus and minus strains. We demonstrated that pheromone signaling genes (a-factor-like and α-factor-like pheromones and their corresponding GPCR receptors) were differently expressed between vegetative hyphae of the two strains. Moreover, deletion of pmk1 (a FUS/KSS1 mitogen-activate protein kinase) in the minus strain severely limited mating line formation, whereas deletion of a GPCR (FGSG_05239 homolog) and two histone modification factors (hos2, snt2) in the minus strain did not affect mating line development but altered the ratio between cross-fertilization and self-fertilization within the mating line. We propose a model in which mating line formation in C. fructicola involves enhanced protoperithecium differentiation and enhanced perithecium maturation of the minus strain mediated by both cross-fertilization and diffusive effectors. This study provides insights into mechanisms underlying the mysterious phenomenon of plus-minus-mediated sexual enhancement being unique to Colletotrichum fungi. IMPORTANCE Plus-minus regulation of Colletotrichum sexual differentiation was reported in the early 1900s. Both plus and minus strains produce fertile perithecia in a homothallic but inefficient manner. However, when the two strain types encounter each other, efficient differentiation of fertile perithecia is triggered. The plus strain, by itself, can also generate minus ascospore progeny at high frequency. This nontypical mating system facilitates sexual reproduction and is Colletotrichum specific; the underlying molecular mechanisms, however, remain elusive. The current study revisits this longstanding mystery using C. fructicola as an experimental system. The presence of both cross-fertilized and self-fertilized perithecia within the mating line was directly evidenced by live-cell imaging with fluorescent markers. Based on further gene expression and gene mutation analysis, a model explaining mating line development (plus-minus-mediated sexual enhancement) is proposed. Data reported here have the potential to allow us to better understand Colletotrichum mating and filamentous ascomycete sexual regulation.

Keywords: Colletotrichum; mating type; perithecium; sexual reproduction.

FIG 1

Colony morphologies of 1104-7 (plus strain), 1104-6 (minus strain), and the mating line response. (A) Colony morphologies on PDA and OA. Red arrows point to examples of perithecial clusters. Scale bar, 1 mm. (B) Mating line formation induced by coculturing 1104-7 (left) and 1104-6 (right) on PDA and OA. dpi, days postinoculation.

FIG 2

Colony morphology phenotypes of 1104-7 ascospore progeny. (A) From left to right, squashed perithecial cluster (scale bar, 200 μm), maturing asci, and ascospores (scale bar, 10 μm), representative culture types of ascospore progeny on PDA medium. (B) Representative colony photos of the three types of 1104-7 ascospore progeny, type I resembles the 1104-7 plus parent, type II resembles the minus 1104-6 strain but with reduced melanization, and type III does not produce perithecia, is light yellow in colony color, and has poor viability. (C) Mating response of type I and type II strains with 1104-7 (P) and 1104-6 (M).

FIG 3

Low fertility of 1104-6 selfed perithecia on OA. (A) Classification of perithecia based on ascus and ascospore development. Form I, empty perithecium, no inner tissue; form II, abundant inner tissue, ascus initials may be observed (yellow arrowhead); form III, obvious ascus rosette; form IV, ascus dissolved but abundant ascospores. Scale bar, 50 μm. (B) Relative ratios of the four perithecia types at different days (d) postinoculation.

FIG 4

Mating line formation between 1104-7 and 1104-6 involves increased perithecial differentiation and elevated extent of perithecial maturation. (A) Relative perithecium density for the 1104-6 (minus, corresponding to region f in panel C) and the mating line (line, corresponding to region d in panel C). The values were normalized to the minus region at 5 days. (B) Perithecium diameter in the minus region and line region over time. (C) Example of a paired cross showing the sampled regions in panels A, B, and D. (D) Fertility of different regions (shown in panel C) at 11 days postinoculation. The number of perithecia examined is shown above individual columns. Form I, empty perithecium, no inner tissue; form II, abundant inner tissue, ascus initials may be observed; form III, obvious ascus rosette; form IV, ascus dissolved but abundant ascospores. Representative photos for different perithecial types are shown in Fig. 3A.

FIG 5

Development of perithecia within the mating line between 1104-7-hisH1-GFP (P-His-GFP) and 1104-6-hisH1-mRFP (M-His-mRFP) on OA. (A) Perithecial fluorescence pattern at early (E), middle (M), and late (L) stages of mating line formation. Note that all perithecia initials emit red fluorescence, whereas from the middle stage onward, some perithecia start to emit double fluorescence (red arrowhead). Scale bars for E, M, and L represent 100 μm, 100 μm, and 1 mm, respectively. (B) A magnified view of the maturing mating line (late stage). Note the two types of perithecia differing in fluorescence pattern (red only and double fluorescence). Scale bar, 100 μm.

FIG 6

Mating line contains both cross-fertilized and self-fertilized perithecia. 1104-7-hisH1-GFP (P-His-GFP) and 1104-6-hisH1-mRFP (M-His-mRFP) were cocultured on cellophane overlaid on OA medium. Individual perithecia were squashed to examine ascospore fluorescence patterns. (Top) 4:4 segregation of green to red fluorescence of ascospores within individual asci, indicating cross-fertilization between the two strains. (Bottom) 0:8 segregation of green to red fluorescence for ascospores within individual asci, indicating self-fertilization of the minus strain; no 8:0 green to red fluorescence pattern was observed with over 90 examined perithecia. In top and bottom panels, merged photos were generated by combining views from the bright-field green (GFP) channel and red (mRFP) channel, respectively. Scale bar, 40 μm.

FIG 7

qRT-PCR expression analysis of genes related to fungal sexual development. Fungal tissues were sampled from the 0.5-cm OA colony edge of 1104-7 (plus vegetative hyphae, P-V), 1104-6 (minus vegetative hyphae, M-V), and mating line (L) at different days postinoculation (days 3, 5, 7, and 9). The C. fructicola alpha-tubulin gene (XP_031882639.1) was used as the internal control, and gene expression levels were normalized to plus vegetative hyphae (P-V). Numbers on the y axes represent relative expression fold change. The error bar represents standard deviations calculated from three independent replicates. An asterisk represents significant difference compared with P-V based on two-tailed t test at a significance level of 0.05.

FIG 8

Effects of target gene deletion on perithecial development. All cultures were grown on OA medium. (A) Perithecial morphology observed under dissecting microscope magnification. Scale bar, 500 μm. (B) Perithecial morphology observed under light microscope magnification (scale bar, 50 μm) and quantification of perithecial fertility at 11 days postinoculation (right bottom corner; the number of examined perithecia are shown above individual columns). (C, left) Relative perithecial density normalized to the WT (11 days). Means ± standard deviations (n = 9) were plotted. An asterisk represents significant difference (adjusted P < 0.05) compared with the WT based on Dunnett’s multiple-comparison test following one-way analysis of variance (ANOVA). (Right) Perithecial diameter (11 dpi). Means ± standard deviations were plotted as horizontal and vertical lines above individual values (dots; n ≥ 100). An asterisk represents significant difference (adjusted P < 0.05) compared with the WT based on Dunnett’s multiple-comparison test following one-way ANOVA.

FIG 9

Mating line formation phenotypes of 1104-6 gene deletion mutants when cocultured with 1104-7-hisH1-GFP on OA. Mycelial plugs of 1104-7-hisH1-GFP- and 1104-6-derived strains were inoculated on the left and right sides of the OA plate, respectively. Mating line formation phenotypes were photographed at 11 days postinoculation. Scale bar, 2 mm.

FIG 10

Fertilization events occurring within the mating line formed between 1104-7-hisH1-GFP and various 1104-6-derived strains. Based on ascospore fluorescence segregation patterns, each ascus or perithecium could be classified as minus selfing (fluorescent/nonfluorescent ascospore ratio, 0:8), cross fertilization (fluorescent/nonfluorescent ascospore ratio,  4:4), or plus selfing (fluorescent/nonfluorescent ascospore ratio,  8:0). The relative ratios of the three types of fertilization events (based on either perithecia or asci) are plotted. The total number of observations is given above individual columns.

FIG 11

Model describing the atypical mating system of C. fructicola. (A) The plus-to-minus switching phenomenon. The plus strain, via an unknown mechanism related to sexual reproduction, produces minus ascospore offspring at high frequency. The pairing of plus and minus strains boosts sexual reproduction, leading to the formation of a mating line between colonies. (B) Minus perithecium development (minus solo). The minus wild type forms abundant perithecia but many are infertile, indicating a defect in perithecium maturation (dash arrow). Deletion mutants of pmk1, gpcr1, hos2, and snt2 still formed abundant perithecia but were smaller, aberrant, and completely sterile, indicating their importance in both protoperithecium development and perithecium maturation (not shown). (C) Mating line development. When plus (orange) and minus (dark brown) strains come into contact, the minus strain is fertilized by the plus strain, which complements or derepresses its perithecium maturation defect; moreover, local diffused effectors (e.g., pheromones) may directly boost perithecium differentiation and development via autocrine signaling, leading to the development of abundant minus-selfed perithecia and, under certain conditions (e.g., minus selfing being suppressed), the development of fertile plus-selfed perithecia as well.