Figure 1.
Chromosomal positions and expression levels ofMAT locus genes.
A. Diagram of four MAT locus genes. The thick black bars and arrows represent the exons and directions of MAT1-2-1, MAT1-1-1, MAT1-1-2, and MAT1-1-3 genes. I and T represent the initiation and termination sites of transcription. CTGTACAG and GAAAGCTTTC are palindromic sequences. B. Relative expression levels of individual MAT locus genes at 1, 2, 4, 6, and 10 days post-perithecial induction (dpi) were assayed by qRT-PCR. The expression level of individual genes in aerial hyphae collected from 7-day-old carrot agar cultures before perithecial induction (day 0 control) was arbitrarily set to 1. C. Relative expression levels of four MAT locus TF genes at 1, 2, 4, 6, and 10 dpi. For each time point, the expression level of MAT1-1-1 was set to 1. Mean and standard error were calculated from three independent biological replicates.
Figure 2.
Expression and subcellular localization ofMAT locus TF genes.
A. Weak GFP signals in vegetative hyphae (upper panels) and asci (lower panels) of the MAT1-1-3-GFP knock-in transformant. B. GFP signals in vegetative hyphae of the PTrpC-MAT1-1-3-GFP (90R) and PTrpC-MAT1-2-1-GFP (93R) transformants. The same field was observed by DIC or epifluorescence microscopy. Bar = 20 µm.
Table 1.
Strains used in this study.
Figure 3.
Defects of differentMAT locus gene deletion mutants in self-crosses.
A. Perithecia produced by 14-day-old carrot agar cultures of the wild type (PH-1) and different MAT locus gene deletion mutants. Arrows pointed to the cirrhi. Bar = 1 mm. B. Perithecia of PH-1 and the MAT locus gene deletion mutants were examined for ascus and ascospore development. Bar = 20 µm. C. Thick sections of representative perithecia produced by the wild type and mutant strains. Bar = 20 µm.
Figure 4.The.
mat1-1-1 and mat1-2-1 mutants were defective in corn stalk infection.
Corn stalks inoculated with PH-1 and four MAT locus gene deletion mutants. Stalk rot (discoloration) was restricted in plants inoculated with the mat1-1-1 and mat1-2-1 mutants.
Figure 5.
Yeast two-hybrid assays for the interactions among fourMAT locus TFs.
A. Different concentrations of yeast cells (cells/ml) of the transformants expressing the labeled bait and prey constructs were assayed for growth on SD-Leu-Trp-His plates. P and N were the positive and negative controls provided in the BD Matchmaker library construct kit. B. The same set of yeast transformants was assayed for β-galactosidase activities.
Figure 6.
Outcrossing defects in themat1-2-1 and various mat1-1 mutants.
A. The mat1-2-1 mutant was crossed as the male (M) or female (F) with the mat1-1-3, mat1-1-2, mat1-1-1, and mat1-1 mutants. B. The mat1-1-1 mutant was crossed as the male (M) or female (F) with the mat1-1-3 and mat1-1-2 mutants. The upper panels showed the size of representative perithecia and production of ascospore cirrhi (bar = 1 mm). The lower panels showed perithecia with or without fascicles of asci (bar = 20 µm).
Table 2.
Defects of different mutants in outcrossing.
Figure 7.
Mating defects of theFgso mutant.
A. The Fgso mutant was crossed as the male (M) or female (F) with the mat1-1-1 and mat1-2-1 mutants. Although it was female sterile, the Fgso mutant retained male fertility. Bar = 1 mm. B. Assays for hyphal fusion in carrot agar cultures of the wild type (PH-1) and the mat1-1-1, mat1-2-1, and Fgso mutants. C. The expression level of FgSO in the wild type and the mat1-1-1 and mat1-2-1 mutants.
Figure 8.
The expression of otherMAT locus genes in the mat1-2-1 and various mat1-1 mutants.
The expression of MAT1-1-1, MAT1-1-2, MAT1-1-3, and MAT1-2-1 in PH-1 (WT) or the mat1-1-1, mat1-1-2, mat1-1-3, and mat1-2-1 deletion mutants. The relative expression level of each gene in the wild type was arbitrarily set to 1. RNA samples were isolated from aerial hyphae harvested from 7-day-old carrot agar cultures. Mean and standard error were calculated from three independent biological replicates.
Figure 9.
Effects ofMAT locus gene deletion on the expression of pheromone and pheromone receptor genes.
The expression levels of PRE1, PRE2, PPG1, and PPG2 in PH-1 or the mat1-1-3, mat1-1-2, mat1-1-1, and mat1-2-1 deletion mutants were assayed by qRT-PCR. The relative expression level of each gene in the wild type was arbitrarily set to 1. RNA samples were isolated from aerial hyphae harvested from 7-day-old carrot agar cultures. Mean and standard error were calculated from three independent biological replicates.
Figure 10.
Vegetative growth and sexual reproduction in the PTrpC-MAT1-1-3 and PTrpC-MAT1-2-1 transformants.
A. Conidia and germ tubes of the PTrpC-MAT1-1-3 (OE90) and PTrpC-MAT1-2-1 (OE93) transformants. Bar = 20 µm. B. Carrot agar cultures (bar = 1 mm) and cracked perithecia (bar = 20 µm) of transformants OE90 and OE93. Sterile perithecia formed by the PTrpC-MAT1-2-1 transformant were smaller than those of the PTrpC-MAT1-1-3 transformant.