Figure 1.
Sexual crosses of the WT and Δbcpls1 mutant.
A: Sexual crosses between B. cinerea SAS405 microconidia (standard strain of the opposite mating type to B05.10) and sclerotia of the B05.10 WT, two independent Δbcpls1 transformants and the Δbcpls1:GFP-Pls1 complementation strain. Sclerotia of the Δbcpls1 deletion strain show no morphological differences during their development but are sterile. B: Sexual crosses between B. cinerea SAS405 sclerotia and microconidia of the B05.10 WT, the Δbcpls1 mutants and the Δbcpls1:GFP-Pls1 complementation strains. Microconidia of the Δbcpls1 deletion strain are fertile.
Figure 2.
Germination assay and vegetative hyphal fusions of Δbcpls1 and the WT. A:
Hyphal germling fusions of the WT and the Δbcpls1 mutant. 3×107 conidia were plated on Vogel’s minimal medium and incubated for 14 h in darkness. CAT formation was analyzed using light microscopy. The diagram represents the mean values of three replicates evaluating 300 spores each. The fusion frequency (CAT fusion %) was slightly enhanced in the Δbcpls1 mutant; statistical analysis (T-test) revealed no significant differences. B: Comparison of germination efficiencies of B. cinerea B05.10 WT and Δbcpls1 conidia after 3, 6 and 24 hours. Germination was monitored in liquid Gamborg-B5 medium amended with 10 mM glucose. The diagram represents one experiment with triplicate counting. Germination rates of Δbcpls1 conidia were increased at 3 hpi. Asterisks above the bars denote significant differences in the measurements of the indicated strains to the WT. ** = P<0.01.
Figure 3.
Virulence of the Botrtytis cinerea strain Δbcpls1 in comparison to the wild-type B05.10 (WT) and the complemented strain Δbcpls1:GFP-Pls1.
A: Time course of infection of French bean (Phaseolus vulgaris). Primary leaves of 10 day old plants were inoculated with 7 µl droplets of conidial suspensions (2×105 spores/ml in Gamborgs B5+2% glucose). Progression of infections was documented 3, 5 and 7 days after the infection (dpi). Results of the mutant were not consistent as in some cases infection could be detected between 3 and 5 dpi (see middle row and B). However, at 7 dpi all the leaves infected with the mutant strain showed strong infection symptoms (see right row). B: Statistical evaluation of infection efficiencies at different time points. At 3 dpi in only about 50% of the Δbcpls1-infected leaves symptoms could be detected while infection efficiencies of the WT and the complemented strain amounted to 100%. At 6 dpi also in the Δbcpls1 strain infection symptoms were detected in 100% of infected leaves ((separate set of experiments from A: repeated 3 times with a sample size of n = 16, 16, 12 per strain; standard deviations are indicated by the error bars). C: Statistical evaluation of infection spreading. Only the lesion diameters of successful infection events were measured at 3 dpi. Lesion diameters of Δbcpls1 infections were only slightly reduced when compared to WT and to the complemented strain at this early time point (The indicated values are means of 16 different infection events; standard deviations are indicated by the error bars; a biological replicate yielded similar results). Asterisks above the bars denote significant differences in the measurements of the indicated strains to the WT. ** = P<0.01; *** = P<0.001.
Figure 4.
Penetration assay on onion epidermis.
The hydrophobic side of onion epidermis was inoculated with 10 µl droplets of conidial suspensions (5×104 spores/ml). After 24 h incubation at 18°C lactophenole blue staining was used to visualize the fungus on top of the onion epidermis. Fungal cells within the onion cells are not stained. Conidia of the WT and ΔbcnoxA form a short germ tube with a terminal thickening, which are able to directly penetrate the plant surface. Conidia of the deletion strains ΔbcnoxB, ΔbcnoxR, ΔbcnoxAB and Δbcpls1 germinate on the plant epidermis and differentiate appressoria-like structures, but fail to penetrate the plant via these structures. Black arrows indicate appressoria-like structures. Scale bars = 10 µm.
Figure 5.
In planta penetration assay on bean leaves.
Scanning electron microscopy (SEM) images of germinating conidia of the B. cinerea WT, Δbcpls1, ΔbcnoxA, ΔbcnoxB, ΔbcnoxAB and ΔbcnoxR were taken on the surface of bean leaves. Detached bean leaves were inoculated with conidial suspensions (2×106 spores/ml) and 18 h after the infection samples were prepared for SEM by glutharaldehyde fixation, osmification, ethanol series, critical point drying and gold sputtering. Arrowheads indicate appressoria-like structures. Scale bars = 10 µm.
Figure 6.
Cellular localization of BcNoxA, BcNoxB and BcNoxR.
Protein localization was determined by epifluorescence microscopy in germlings of the strains WT:GFP-NoxA, WT:NoxB-GFP and WT:GFP-NoxR expressing gfp-bcnoxA, bcnoxB-gfp and gfp-noxR gene fusions, respectively. 10-µl droplets of a conidial suspension in GB5 medium (105 conida/ml) were placed on a glass slide and allowed to germinate over night before microscopic analyses were performed. A: BcNoxA localized to intracellular membrane structures and at times also to the plasma membrane. Staining with the ER-Tracker™ Blue-White DPX and a respective overlay show that the intracellular structures are partly consistent with the ER (from left to right: GFP-NoxA, ER-tracker, overlay, white light). B: BcNoxB localized to similar intracellular membrane structures and to the plasma membrane as visible for BcNoxA. Staining with the ER-Tracker™ Blue-White DPX and a respective overlay show that the intracellular structures visual are similar to ER structures (from left to right: NoxB-GFP, ER-tracker, overlay, white light). C: BcNoxR accumulated in cellular granules, which were distributed irregularly throughout the hyphae (left) and at the hyphal tip, where it seemed to determine the point of outgrowth (right, see also movie S2). Scale bars = 10 µm.
Figure 7.
Complementation analyses of ΔbcnoxA and bcnoxR with gfp-constructs.
Strains ΔbcnoxA:GFP-NoxA and ΔbcnoxR:GFP-NoxR expressing gfp-noxA and gfp-noxR gene fusions were tested for their phenotypical characteristics. The strains WT, ΔbcnoxA and ΔbcnoxR served as a control. The GFP-fusion constructs did complement the deletion phenotypes of ΔbcnoxA and ΔbcnoxR A: The ability to form sclerotia after 2 weeks incubation in darkness on CM media at 18°C is restored. B: The pathogenicity defect of the mutants is reinstated. Primary leaves of 10 day old French bean plants were inoculated with 7 µl droplets of conidial suspensions (2×104 spores/ml in Gamborgs B5+2% glucose). Progression of infections was documented 3, 4 and 6 days after the infection (dpi).
Figure 8.
Cellular localization of the protein BcPls1.
Protein localization was determined by confocal laser scanning microscopy (CLSM) in germlings of the strain Δbcpls1:gfp-Pls1 expressing a gfp-bcpls1 gene fusion. 10-µl droplets of a conidial suspension in GB5 medium (105 conida/ml) were given on a glass slide and were allowed to germinate over night before microscopic analyses were performed. A: Overview of BcPls1 localization in a Botrytis cinerea germling. BcPls1 mainly localizes to the border of circular structures in germinating hyphae that seem to be coherent (middle row). Higher magnification showed that the protein also localizes to structures at the edge of the hyphal tip (top row). Fluorescence is clearly limited to the last segment of the germling. The septal pore shows no fluorescence (white arrow, bottom row). B: Germlings were treated with the endocytosis marker FM4-64 (red, top right panel) to stain intracellular membranes. BcPls1 localization is shown in green (top left panel). White arrows indicate co-localization of BcPls1 fluorescence and the FM4-64 stained plasma membrane (see overlay, bottom left panel). Yellow arrows indicate localization of BcPls1 in membrane structures that are not stained by FM4-64 (indicative for the nuclear envelope/endoplasmic reticulum). C: Germlings were treated with the dye Hoechst (blue, top right panel) to stain nuclei. BcPls1 localization is shown in green (top left panel). White arrows indicate Hoechst- stained nuclei that are surrounded by the intracellular membrane structures BcPls1 localizes to ((see overlay, bottom left panel). D: Germlings were treated with the dyes FM4-64 (red, second row)) and Hoechst (blue, third row), simultaneously. BcPls1 localization is shown in green (first row). In addition to the membrane structures seen before (nuclear envelope), white arrows indicate localization of BcPls1 to the interior space of round structures, whose membranes are stained by FM4-64 (indicative for vacuoles). Scale bars in all images = 3 µm.
Figure 9.
Hypothetical schematic overview of the localization and putative functions of Nox in B. cinerea.
Nox in B. cinerea might function at two different locations. On the one hand (top) they are located in the ER (green) membrane, where they possibly contribute to the ER redox status. Besides GSH (glutathion), Nox might be a second supplier of electrons to PDI. PDI is oxidized by Ero1 by transfer of electrons to O2, which in turn becomes H2O2 (scheme of Ero1-PDI pathway based on [72]. Upon an activation signal Nox are translocated to the plasma membrane (bottom) via vesicles (yellow dots), where they form the active Nox complex with the subunits NoxR, Rac and other so far unknown components (possibly Bem1 and Cdc24 [17]). This complex then transfers electrons through the membrane, reducing oxygen to superoxide, using NADPH as an elctron donor. The BcNoxA complex is necessary for CAT fusions, colonization of plant tissue and sclerotia formation, BcNoxB, which additionally might be regulated by BcPls1, is involved in penetration via appressoria-like structures and female fertility. Nuclei are depicted in blue.
Table 1.
Strains used in this study.