Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea

Abstract

Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction.IMPORTANCE Fungal fruiting bodies are formed by sexual reproduction. We studied the development of fruiting bodies ("apothecia") of the ubiquitous plant-pathogenic ascomycete Botrytis cinerea The role of mating type genes in apothecium development was investigated by targeted mutation. Two genes are essential for the initiation of sexual development; mutants in these genes are sterile. Two other genes were not essential for development of stipes; however, they were essential for stipes to develop a disk and produce sexual ascospores. We examined gene expression profiles during apothecium development, as well as in ascospores sampled from apothecia. We provide the first study ever of the transcriptome of pure ascospores in a filamentous fungus. The expression of numerous genes involved in plant infection was induced in the ascospores, implying that ascospores are developmentally primed for infection before their release from apothecia.

Keywords: ascospore; epigenetic regulation; plant disease; sexual reproduction; transcriptome.

FIG 1

Different stages of sexual reproduction in Botrytis cinerea. Following fertilization of asexual resting structures (sclerotia), apothecium development is divided into six stages: primordia emerging (stage 1), primordia extending (stage 2), extended stipes before tip swelling (stage 3), stipes with swollen tips (stage 4), immature apothecium (stage 5), and mature apothecium with asci and ascospores (stage 6). Pure ascospores sampled from mature apothecial disks are shown in the right-hand image. The five samples used for transcriptome analyses are indicated at the top.

FIG 2

Results of crosses with B. cinerea mutants in MAT genes. The maternal parent (sclerotia) is mentioned first, and the paternal parent (microconidia) is second. The six images below the dashed line show the results for reciprocal crosses of the six images above. For each mutated target gene, results of one mutant are shown; identical results were obtained with two additional, independent deletion mutants.

FIG 3

Closeup of defective stipes formed in a cross between wild-type sclerotia of SAS405 and a ΔMAT1-1-5 mutant (A) and of a fully developed wild-type apothecium (B). The mutant stipe is blocked in transition to the apothecial disk and forms only a lobed, indented structure at the tip of the stipe. Crosses between wild-type sclerotia of SAS56 and MAT1-2-10 deletion mutants, as well as between a MAT1-1-5 deletion mutant and a MAT1-2-10 deletion mutant, result in very similar phenotypes. White bar, 1 mm.

FIG 4

qRT-PCR analysis of B. cinerea MAT genes, relative to the internal standard BctubA. (A) Expression in three stages of apothecium development in a wild-type cross: primordia (left), stipes (middle), and disks (right). (B) Expression levels in mutant stipes (ΔMAT1-1-5, left; ΔMAT1-2-10, middle) compared to wild-type stipes in an equivalent stage (Apo34, right). Expression data from Apo34 wild-type stipes are the same between panels A and B.

FIG 5

Comparison of transcriptomes of five stages of sexual development with three asexual tissues (Germ, germinating conidia [53]; PGA, mycelium on polygalacturonate-containing medium [54]; Glu, mycelium on glucose-containing medium [54]). For each gene in these comparisons, the mean CPM was calculated over available replicates. (A) Pairwise correlations between CPM values of B. cinerea total transcriptome samples. Dot size and color represent the Pearson correlation coefficient. (B) Relative expression (Z-scores) of subset of 3,084 genes that were differentially expressed in at least one of the sexual development stages (Scl, Apo12, Apo34, Apo56, and Asc) compared to asexual tissues.

FIG 6

CROC analysis for identification of clusters of coregulated genes. Core chromosomes of B. cinerea strain B05.10 are displayed vertically with Chr1 at the top and Chr16 at the bottom. Size markers are provided at the bottom. Clusters of upregulated genes are displayed in green, while clusters of downregulated genes are displayed in red. For each chromosome, two rows are provided: the top row displays clusters of genes coregulated during the transition from sclerotia to primordia, while the bottom row displays clusters of genes coregulated during the transition from apothecial disks to ascospores. Minichromosomes 17 and 18 (17) did not contain coregulated clusters and were omitted for simplicity.

FIG 7

Transcript abundance of enzyme-encoding genes involved in histone modification and DNA methylation, at five stages in apothecium development. Columns from left to right indicate the respective annotated enzymatic activity, assigned gene symbol, the modified residue on the histone, the Z-score-transformed expression profiles over the five determined life stages, and the mean CPM values over the five stages to relate the relative changes to absolute, respectively. Functional annotations are taken from reference .