Transcriptome profiling of Botrytis cinerea conidial germination reveals upregulation of infection-related genes during the prepenetration stage

Abstract

Botrytis cinerea causes gray mold on a great number of host plants. Infection is initiated by airborne conidia that invade the host tissue, often by penetration of intact epidermal cells. To mimic the surface properties of natural plant surfaces, conidia were incubated on apple wax-coated surfaces, resulting in rapid germination and appressorium formation. Global changes in gene expression were analyzed by microarray hybridization between conidia incubated for 0 h (dormant), 1 h (pregermination), 2.5 h (postgermination), 4 h (appressoria), and 15 h (early mycelium). Considerable changes were observed, in particular between 0 h and 1 h. Genes induced during germination were enriched in those genes encoding secreted proteins, including lytic enzymes. Comparison of wild-type and a nonpathogenic MAP kinase mutant (bmp1) revealed marked differences in germination-related gene expression, in particular related to secretory proteins. Using promoter-GFP reporter strains, we detected a strictly germination-specific expression pattern of a putative chitin deacetylase gene (cda1). In contrast, a cutinase gene (cutB) was found to be expressed only in the presence of plant lipids, in a developmentally less stringent pattern. We also identified a coregulated gene cluster possibly involved in secondary metabolite synthesis which was found to be controlled by a transcription factor also encoded in this cluster. Our data demonstrate that early conidial development in B. cinerea is accompanied by rapid shifts in gene expression that prepare the fungus for germ tube outgrowth and host cell invasion.

Fig 1

Pregermination swelling of B. cinerea wild-type conidia in minimal medium with or without 10 mM fructose. Bars 1, 3, 4, and 5, apple wax-coated glass slides; bars 2 and 6, untreated glass slides. For bar 3, heat-killed conidia (15 min at 65°C) were used. For bar 4, a high conidium concentration (10⁷/ml) was used. For the other bars, 10⁵/ml conidia were used. Swelling of each spore until germination was determined by subtracting the calculated volume of each spore immediately after inoculation from the volume at the moment of germination. Under conditions that did not lead to spore germination (bars 3 and 4), swelling of the spores was measured after 5 h of incubation. Values are the means of three independent experiments (n ≥ 30 spores each).

Fig 2

Germination kinetics of B. cinerea conidia. (A) Germination kinetics of B05.10 wild-type and bmp1 mutant conidia on different surfaces in minimal medium containing 10 mM fructose as follows: the wild type on apple wax-coated polystyrene (black), the wild type on glass (light gray), the wild type on polypropylene (dark gray), and the bmp1 mutant on apple wax-coated polystyrene (hatched). Standard deviations of four independent experiments are shown. (B) Time course of germination of wild-type and bmp1 mutant conidia on apple wax-coated polystyrene. Scale bars, 10 μm.

Fig 3

Nuclear division of B. cinerea conidia during germination. (A) Hoechst 33342 staining of germinating B. cinerea conidia and increase in average number of nuclei during early germination. Scale bars, 10 μm. (B) Graph showing the average numbers of nuclei in germinating conidia incubated on apple wax-coated polystyrene in minimal medium containing 10 mM fructose. Error bars show standard errors of three independent experiments (n = 50).

Fig 4

Changes in gene expression between different stages of wild-type conidium development. Numbers of genes showing at least 2-fold upregulation (dark gray) or downregulation (light gray) and at least 4-fold upregulation (dark hatching) or downregulation (light hatching) are shown (P < 0.05; Student's t test).

Fig 5

Identification of gene groups with similar transcriptional profiles across the five time points of germination and germling development.

Fig 6

Distribution of functional categories among genes belonging to different expression groups. For each functional category, the observed percentage of genes within the group is shown. Numbers indicate the total number of genes in each group. Asterisks indicate P values showing significant differences between observed and expected frequencies for a functional category within the total number of genes in the respective group (*, P < 0.01; **, P < 0.001; Fisher test). Red asterisks indicate overrepresentation and blue asterisks underrepresentation. The colored boxes above the bars indicate functional categories that are absent.

Fig 7

Time course of expression of selected genes in the wild type (black bars) and in the bmp1 mutant (gray bars). (A) Germination-induced genes, downregulated during germination in the bmp1 mutant. (B) Germination-induced cutA gene, similar expression in the wild type and the bmp1 mutant. (C) Late germination induced genes, similar expression in the wild type and the bmp1 mutant. (D) Constitutive (CON-H) genes.

Fig 8

Germination-specific expression of cda1 revealed by a B. cinerea cda1-GFP promoter reporter strain. Conidia were germinated on a polypropylene surface in minimal medium with 10 mM fructose. (A) Time course of germination and appearance of GFP fluorescence. The means of three experiments are shown, with standard deviations (n > 150). (B) Time-lapse microscopy of germinating conidia during a 3.5-h observation period. Conidia that started to show fluorescence before germ tube outgrowth are circled in red. A nonfluorescent spore that did not germinate during the observation period is circled in blue.

Fig 9

Analysis of cutB expression using a B. cinerea cutB-GFP promoter reporter strain. (A) Correlation of germination (dark gray) and increased GFP expression (light gray) of conidia incubated in the presence of 10 mM fructose and THPA. Values are the means of three experiments, with standard deviations (n = 90). (B) The top shows induction of GFP fluorescence in the presence of 10 mM fructose on polyethylene foil (PE) or on glass slides coated with 10 μg/cm² of either apple wax (AW), cutin hydrolysate (CH), or THPA (3.5 h). On the THPA-coated surface, two germlings each showing strong (solid arrowheads) and very low (open arrowheads) fluorescence are highlighted. The lower part shows partial suppression of cutB promoter-driven GFP expression by 10 mM fructose (fruc) or glucose (gluc) (4 h). Fluorescence intensity levels are scored as described in Materials and Methods. Values for each condition are the means of two experiments (n = 50). (C) GFP fluorescence of conidia during germination and penetration on heat-killed onion epidermis cells. Solid arrows indicate penetration sites, and the open arrow indicates intracellular infection hyphae. Scale bars, 20 μm.