Cch1 and Mid1 are functionally required for vegetative growth under low-calcium conditions in the phytopathogenic ascomycete Botrytis cinerea

Abstract

In the filamentous phytopathogen Botrytis cinerea, the Ca(2+)/calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca(2+)-chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The impact of external Ca(2+) was investigated by supplementing with CaCl(2) and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1-green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea.

Fig 1

Gene replacement in the B. cinerea Δmid1, Δcch1, and Δcch1 Δmid1 strains. (A) Deletion strategies for the Δcch1 (top) and Δmid1 (bottom) strains. All primers used for cloning of the replacement vectors and for diagnostic PCR analyses to prove homologous integration are indicated by numbers from 1 to 20, and their use is described further in Materials and Methods. Introns are depicted as open vertical bars in arrows representing the genes. Restriction sites for cloning and for Southern blot analyses are indicated, as are expected fragment sizes and probes for the hybridization. (Top) Physical maps of wild-type (WT; strain B05.10) cch1 and the Δcch1 locus. The wild-type B. cinerea strain B05.10 was transformed with the cch1 replacement fragment (derived from cloning of both flanking regions into vector pNR1 containing the nat^R resistance cassette), resulting in the Δcch1 strain via homologous recombination and insertion of nat^R. (Bottom) Physical maps of the WT mid1 locus and the mutated Δmid1 locus. The mid1 gene was replaced by the hph^R cassette (derived from vector pOliHP) in the opposite direction via homologous recombination. (B) Southern blot analyses of the Δmid1, Δcch1, and Δcch1 Δmid1 mutants. Two to four different mutants were tested for any additional ectopic integration of the respective resistance cassettes. The wild type, the Δmid1 T1 and T7 strains, all four Δcch1 mutants, and the Δcch1 Δmid1 T3 and T7 strains each displayed just one hybridizing fragment with the expected size (for comparison, see panel A). Asterisks indicate strains which were used for further phenotypic analyses.

Fig 2

Domain structures of the putative calcium channel proteins Cch1 and Mid1 in B. cinerea. (A) DAS transmembrane prediction analysis profile of Cch1. Four domains (I, II, III, IV) are present, each with six transmembrane regions (S1 to S6). As known from other fungi, the pore loops (P) are arranged between segments S5 and S6 in each domain. (B) Domain structure of Mid1. Mid1 has four hydrophobic regions (H1 to H4) and a putative N-terminal signal peptide ending within H1. Sixteen predicted N-glycosylation sites (arrowheads) and an EF-hand-like structure (a putative calcium-binding domain) are also indicated. Fourteen cysteine residues (black dots) are present; they are concentrated in the C-terminal cysteine-rich regions C1 and C2.

Fig 3

Phenotypic growth analyses of the B. cinerea Δmid1 and Δcch1 gene deletion strains and the Δcch1 Δmid1 double deletion mutant in comparison to the wild-type strain B05.10 and the Δmid1/mid1-gfp complementing strain. (A) Strains were grown on complete medium (CM). (Left) Strains were cultured under day/night conditions (12 h/12 h) for 1 week. All strains produced conidia; mutant strains initially grew circularly, and subsequently they formed sectors. (Right) Growth in permanent darkness for 2 weeks. All strains show the formation of sclerotia. (B) Graphic presentation of colony diameters (in centimeters) on minimal medium (CD), minimal medium supplemented with 50 mM CaCl₂, and CM. Mean values for three biological replicates with at least four plates per strain and condition are shown. Error bars, standard deviations. Strains were grown for 3 days on the specified medium (3 dpi). The WT displays a 4-fold colony diameter relative to those of the deletion mutants on CD medium and a 1.5-fold colony diameter on CM. Delayed growth was rescued by the addition of 50 mM CaCl₂ to CD medium. Complementation of the Δmid1 strain with the mid1-gfp fragment resulted in wild-type-like colony diameters. (C) Biomass quantification. The same media were used as for panel B, but liquid cultures were inoculated with conidial suspensions of the indicated strains. Different primary transformants were used for verification of the phenotypes. As in panel B, the growth of the mutants, which had been reduced, was restored by the addition of CaCl₂.

Fig 4

Depiction of the calcium rescue phenotype of B. cinerea Δmid1, Δcch1, and Δcch1 Δmid1 mutants in comparison to the wild-type strain B05.10. Strains were grown on minimal medium (CD) alone or containing either the indicated amounts of CaCl₂ at increasing concentrations (from 50 to 400 mM) or the Ca²⁺-chelating agent EGTA (10 or 15 mM). (A) Graphic presentation of relative growth 2 days after inoculation. Strain were incubated under light/dark conditions (12 h/12 h). The growth of the wild-type strain B05.10 on a control medium (CD) was set at 100%; that for all other strains and conditions was calculated proportionally. Mean values for three biological replicates with at least four plates per strain and condition were used for the calculation of percentages. Error bars, standard deviations. High standard deviations result from enhanced sector formation on minimal medium. (B) Representative plates after 3 days postinoculation.

Fig 5

Effect of the calcium chelator BAPTA (1 mM) on growth on minimal medium (CD). The indicated strains were inoculated with mycelium plugs and were grown for 5 days under day/night conditions.

Fig 6

Effect of increasing the cytosolic Ca²⁺ concentration by the presence of the calcium ionophore A23187 on the growth of B. cinerea Δmid1, Δcch1, and Δcch1 Δmid1 mutants in comparison to that of the wild-type strain B05.10. The indicated mutant strains and the wild-type strain B05.10 were grown on minimal medium (CD) with or without additional CaCl₂ (50 mM) for 4 days. Circular filter discs supplied with a 10-μl droplet of EtOH (control) or with the calcium ionophore A23187 at 4.725 or 9.5 mM were laid on the agar plates just before inoculation. The extent of growth is outlined by white dots. Schematic views of growth rates in the indicated directions are shown below the plates. All strains exhibited similar zones of inhibition around the ionophore-containing filter discs. Apart from that, the wild type showed circular growth in all directions on CD medium, whereas the mutant strains displayed less growth in the direction of the control than in the direction of the ionophore filter discs. The addition of 50 mM CaCl₂ reversed the effect. The mutants' defect in growth toward the control was rescued, and like the wild type, they displayed approximately circular growth, except for the indicated inhibition zones of all strains around the ionophore-containing discs.

Fig 7

Localization studies of the Mid1 protein in B. cinerea. (A) The Δmid1 strain was transformed with a mid1-gfp construct, where mid1 was fused N-terminally to gfp and was expressed under the control of the constitutively active oliC promoter. In germinated conidia (germination occurred for 20 h in GB5 medium in darkness), fluorescence was observed in network-like filaments and around the nuclei. Costaining of nuclei by Hoechst 33342 is depicted in Fig. S1 in the supplemental material. (B) The Δmid1 strain was transformed with a gfp-mid1 construct, where mid1 was fused C-terminally to gfp and was expressed under the control of the constitutively active oliC promoter. The fluorescence signal observed was similar to that in panel A. (C) Staining of the Δmid1/mid1-gfp strain with ER-Tracker. In the top three images, the intracellular GFP-fluorescent structures are mostly consistent with the signal of the ER marker (white), as shown in the overlay (merge; false color blue for ER-Tracker). At the bottom, a DIC image of the germinated conidia is shown. Scale bars, 10 μm.

Fig 8

Cytosolic calcium measurements using aequorin. Microtiter wells were inoculated with B. cinerea strains expressing aeqS. Aequorin luminescence monitoring was begun, and at 20 s (indicated by a vertical line), 100 μl of GB5 medium plus EtOH (control) or GB5 medium plus 100 μM amiodarone (AMD; final concentration, 50 μM) was injected. Luminescence was monitored for 180 s. The free cytosolic calcium concentration ([Ca²⁺]_cyt) was calculated from relative luminescence units. Means for 3 technical experiments, along with standard errors, are shown for each curve. The total experiments were repeated at least twice.