Botrytis cinerea Pers.:Fr. [Botryotinia fuckeliana (de Bary)Whetzel] B05.10 was collected from Vitis vinifera (Buettner et al., 1994) and was used in this study as basis strain and control in all experiments. Further strains are listed in Supplementary Table S1.

For cultivation synthetic complete medium (CM) (Pontecorvo et al., 1953) was used. For transformation, the strains were grown on PDAB medium (Potato dextrose agar [Sigma–Aldrich Chemie, Steinheim, Germany] supplemented with 100 g/l homogenized leaves of French beans (Phaseolus vulgaris). Cultures were grown for 6–8 days at 20°C under light conditions (12 h light/12 h darkness, full spectrum light) to obtain conidia. Sclerotia were induced by incubating the strains for 3 weeks at 20°C in darkness. For DNA preparation, the strains were grown for 3–4 days at 18°C on CM agar overlayed with Cellophane. For stress experiments, the CM was supplemented with agents inducing osmotic, oxidative, cell wall, membrane or ionic stress. Minimal medium was prepared after Czapek Dox (20 g/l sucrose, 3 g/l NaNO₃, 1 g/l K₂HPO₄, 0.5 gl KCl, 0.01 g/l FeSO₄ × 7 H₂O, 0.5 g/l MgSO₄ × 7 H₂O, pH 5.2).

For the generation of deletion and complementation constructs, the yeast homologous recombination system was used (Colot et al., 2006; Schumacher, 2012). For generating the deletion cassette of bcpdi1, the 5′/3′ flank were amplified with the primer 1/2 and 3/4 (Supplementary Table S2) as well as the resistance cassette with the primer pair 16/17. Recombinational cloning was done with S. cerevisiae FY834 and the vector backbone of pRS426 (EcoRI/XhoI). For complementation, bcpdi1 was expressed under its native promotor (primer 10/15) as well as under the constitutive oliC promotor of Aspergillus nidulans (primer 9/10). The constructs for introducing mutated phosphorylation sites were amplified using the primer 9/12 and 10/11 (Δbcpdi1-C-M1), the primer 9/14 and 10/13 (Δbcpdi1-C-M2), 9/12, 11/14 and 10/13 (Δbcpdi1-C-M1/2), 10/29 and 9/28 (Δbcpdi1-C-DA). For the generation of PDI in loco complementation constructs that were fused to the genetically encoded biosensor roGFP2, six different fragments were generated by the primer pairs 1/11, 12/32, 33/34, 35/36, 37/38, 39/4 (PDI_phospho_null) or 1/28, 29/30, 31/32, 33/34, 35/36, 37/4 (PDI_phospho_mimic).

After transforming S. cerevisiae FY834, positive transformants were selected on SD medium lacking uracil since the strain is auxotrophic for this amino acid. Total DNA was isolated and transformed in Escherichia coli. Re-isolation was done by the Nucleo spin^® plasmid easypure kit (Macherey-Nagel, Düren, Germany) and correct assembly was tested by sequencing.

Botrytis cinerea was transformed with 40–60 μg of plasmid DNA as described previously (Gronover et al., 2001). Selection was done via hygromycin (70 μg/ml of hygromycin B (Invitrogen, San Diego, CA, United States) or nourseothricin (50 μg/ml of nourseothricin (Werner-Bioagents, Jena, Germany). Positive transformants were purified by single spore isolation: Conidia were spread on the appropriate selective medium; afterward, germinated conidia were picked and cultivated on new selective plates. Genomic DNA isolation was performed according to Cenis (Cenis, 1992). Correct and ectopic integrations were checked by diagnostic PCR and Southern blot (primer 18–27).

For characterization of the different mutant strains various growth, differentiation and pathogenicity assays were performed. For testing defects in pathogenicity, French bean plants (Phaseolus vulgaris L.) were infected according to Klimpel et al. (2002) with agar plugs or conidia from freshly sporulated PDAB plates. Germination on glass surfaces was tested as described by Doehlemann et al. (2006). Penetration ability was checked on onion epidermal layers after inoculation with washed conidia. Before microscopy, the hyphae growing on top of the layers were stained with lactophenol blue. For observation of ICs, the epidermal layers were inoculated with agar plugs of mycelium and incubated overnight (Siegmund et al., 2015). For visualization of CATs media and strains were prepared following Siegmund et al. (2015).

For Northern blot analyses, strains were grown on CM overlayed with Cellophane for at least 3 days. The mycelium was harvested, lyophilized and the RNA was isolated via the Trizol procedure (Invitrogen, Groningen, The Netherlands). Samples of 20 μg were loaded to an agarose gel and transferred to Hybond-N+ membranes. Northern blot hybridizations were done according to the method of Church and Gilbert (1984). The different probes used for expression analysis were generated by PCR with the primer pairs 40/41 (Hac1) and 42/43 (BiP/Kar2).

Light microscopy imaging was performed using the Axio Imager 2 and the Axiovert (Zeiss, Jena, Germany). Visualization of ICs was done with the 20× objective lens, while germinated conidia were analyzed with 40× or 63× magnification. ER staining was accomplished using the ER-Tracker^TM Blue-White DPX (Life Technologies, Germany) in McIlvaine standard buffer (McIlvaine, 1921). The samples were observed via the filter set 49 DAPI shift free (excitation G 365, beam splitter FT 395, emission BP 445/50). GFP fluorescence was detected with filter set 38 (excitation BP 470/40, beam splitter FT 495, emission BP 525/50). Images were captured with a Zeiss AxioCamMRm camera and further processed using the Axiovision Rel 4.8 software package.

roGFP2 measurements were done using an inverted microscope (Leica DMIRE2) equipped with a Leica TCS SP2 scan head (Leica Microsystems, Wetzlar, Germany). Conidia were prepared as described previously (Heller et al., 2012). Results were obtained by using the excitation wavelengths 395 (first track) and 488 (second track) as well as a 505–530 bandpass filter for collecting images. Z-stacks were displayed as average projections via the CLSM software. Further evaluation was done with the Image J program (v.1.44f¹) as it was shown before (Marschall et al., 2016b).

Protein–Protein interactions were investigated by a split-ubiquitin based yeast-two-hybrid system (Dual System Biotech). The proteins of interest were fused to the C- or N-terminal half of ubiquitin. Upon interaction, ubiquitin congregates and the transcription factor controlling reporter genes (histidine and adenine – see also manual Dual Systems Biotech, Schlieren, Switzerland) can be released. Proteins without transmembrane regions are additionally fused to a transmembrane domain of the oligosaccharyltransferase subunit 4 (Ost4) of S. cerevisiae for anchoring the proteins at the membrane. Interaction vectors (prey/bait) were generated according to the manufacturer’s protocol. Both vectors (pPR3N = prey; pDHB1 = bait) were restricted with SfiI and ligated with likewise restricted PCR products (for details see Supplementary Table S2). The ligated products were transformed in E. coli and selected with the appropriate selection marker.

For interaction assays, prey and bait were transformed in S. cerevisiae NMY51 according to the manufacturer’s protocol (DUALSystems Biotech) with a modified version of the lithium-acetate method (Gietz and Woods, 2002). Yeast cells were selected on SD-agar lacking the appropriate amino acids (^-leu^-trp as control of vector transformation; ^-leu^-trp^-his^-ade as test for positive interaction). For drop tests, established colonies were grown overnight in SD medium lacking leucine and tryptophan. OD₆₀₀ was adjusted to 1 and cells were pelleted. The medium was removed and starvation was induced by the addition of sorbitol (1 M): incubation then took place for at least 5 h at 30°C and 200 rpm on a shaker. Subsequently samples were diluted up to 1:1000 and 10 μl of each dilution were dropped on selective medium (SD^-leu^-trp^-his^-ade + 5 mM 3-AT + 80 mg/l X-Gal) and control plates (SD^-leu^-trp). Plates were incubated for 3–4 days at 30°C.

For co-IP experiments, proteins of interest were transformed into the wild type strain. The expression is controlled by the constitutive oliC promotor (A. nidulans) and reporter genes were fused to the N- or C terminus of the respective protein. Fusion proteins were generated by the yeast recombination system. Whereas bcnoxA was cloned into the vector pNAH_OCT (restricted with NcoI) containing mcherry as reporter gene, bcpdi1 was cloned into the vector pNDN_OGG (restricted with NcoI) with gfp as fluorescent marker.

After transformation, the strain expressing both constructs was purified by single spore isolation and grown on CM medium with Cellophane overlay for 3–4 days. The mycelium was harvested, lyophilized and proteins were isolated by grinding and applying disruption buffer [20 mM Tris/HCL pH 8, 150 mM NaCl, 0.05% Triton X-100 supplemented with 10 μl Phosphatase inhibitor 1/2 and 10 μl of Protease inhibitor (Sigma–Aldrich, Steinheim, Germany)]. For the NoxA_mcherry control, the extract was stored until Western blotting. The protein extract of the PDI_GFP control as well as of the strain expressing both constructs was loaded to μMACS GFP beads/columns and purified according to the instruction manual of the μMACS GFP tagged protein isolation kit (Milteny Biotech Bergisch Gladbach, Germany). After Western blotting, detection was accomplished using the anti-gfp antibody (Milteny Biotech, Bergisch Gladbach, Germany). For mcherry detection, the anti-mcherry antibody (Thermo Fisher Scientific, Schwerte, Germany) was used as primary antibody and for the secondary antibody the donkey-anti-rabbit antibody (Thermo Fisher Scientific, Schwerte, Germany) was used. Visualization was conducted using the enhanced chemi-luminescence (ECL) detection (Bio-Rad, Munich, Germany).

Nucleotide and protein sequences of B. cinerea strain B05.10 were obtained by the database Ensembl². For the analysis of the sequences, different programs were used: Signal peptides were predicted by SOSUIsignal (Gomi et al., 2000), subcellular localization patterns of proteins were predicted by ProtComp v.9.0³, transmembrane regions were predicted by using the programs TMHMM⁴ and SACS MEMSAT2 (Jones et al., 1994). Phosphorylation sites were predicted by KinasePhos (Huang et al., 2005).

Since our studies had revealed that the NoxA complex has a distinct function inside the ER, putative targets/partners were investigated. An essential ER protein, which in mammalian systems is associated with subunits of the Nox complex (Janiszewski et al., 2005; Laurindo et al., 2008; Fernandes et al., 2009; de A Paes et al., 2011) is the PDI. Furthermore, the putative homolog to the mammalian protein was identified as a putative interaction partner of BcNoxA in a cDNA library screen (Siegmund and Tudzynski, unpublished). Blastp analysis and multiple sequence alignments using ClustalΩ (Sievers et al., 2011) displayed a query coverage of 85% as well as a sequence identity of 37% by comparing the mammalian PDI with the fungal protein (Bcin06g05730). According to the Ensemble Botrytis genome database⁵ the ORF consists of 1596 bp encoding a protein of 531 aa. Bioinformatic analyses revealed that the protein (BcPdi1) probably contains two transmembrane domains with a small cytosolic loop (Supplementary Figure S1A), with both ends located inside the ER. Its two catalytic domains (CXXC-motif) are predicted to be located in the cytosolic loop (1 in Supplementary Figure S1A) and inside the ER, respectively (4 in Supplementary Figure S1A). Moreover, two cytosolic phosphorylation sites are predicted (2/3 in Supplementary Figure S1A). Interestingly, the first putative phosphorylation site is highly conserved among different fungal species (Supplementary Figure S1A) while the second one is poorly conserved.

For functional characterization, deletion mutants of bcpdi1 were generated (Supplementary Figure S1B). Transformants were checked via diagnostic PCR (Supplementary Figure S1B) and Southern blot analysis (Supplementary Figure S1C – for details see Supplementary Tables S1, S2). Since all mutants displayed an identical phenotype, the results of the mutant Δbcpdi1_T2 are presented. In order to elucidate the role of the predicted cytosolic phosphorylation sites of the protein, mutants with mutated phosphorylation sites were generated. The key amino acids were replaced by an alanine to mimic the constitutively dephosphorylated state (phospho null) (BcPdi1_M1 = T65A; BcPdi1_M2 = Y208A). Moreover, the putative first phosphorylation site was transferred into its constitutively phosphorylated form (phospho mimic) by replacing threonine by glutamic acid (T65E).

To check whether BcPdi1 is involved in stress response pathways, the radial growth was determined on CM containing various stress agents.

The radial growth of the Δbcpdi1 mutant on CM was reduced by 20% in comparison to the wild type. Whereas the complemented strain, Δbcpdi1-C-M2 and Δbcpdi1-C-DA grew as well as the wild type, the colony diameters of Δbcpdi1-C-M1 (87.5%) and Δbcpdi1-C-M1/2 (83.5%) were slightly reduced.

The deletion of bcpdi1 led to a higher sensitivity of the mutants to oxidative and osmotic stress. The addition of 10 mM H₂O₂ and on 1 M NaCl was sufficient to reduce the rate of radial growth of Δbcpdi1 significantly, but the addition of menadione had no effect. While the complemented strain displayed wild type-like growth on all selective media, the mutation of the first predicted phosphorylation site correlated with the deletion mutant’s phenotype. The mutation of the second putative phosphorylation site had no effect (Figure 1). The ‘phospho mimic’ mutants mostly resembled the growth behavior of the wild type. However, on NaCl-supplemented medium the mutant grew much better than all other strains. In contrast to oxidative and osmotic stress, the addition of Calcofluor White (2 mg/ml) as a cell wall stressor, or SDS (0.02%) as a membrane stressor did not differentially influence the radial growth of the mutants compared to the wild type.

Botrytis cinerea is able to infect its host from spore- or mycelium-mediated penetration processes. Whereas appressoria are formed after the short germination of a fungal spore, so-called ICs are mycelium-derived penetration structures that facilitate the breakthrough of the plant cell wall (Armentrout and Downer, 1987).

The deletion of bcpdi1 attenuated the spore-mediated infection of bean leaves, but the way the mutant formed appressoria appeared as in the wild type (Figures 2A,C). For this study, spores were inoculated on onion epidermal layers and fungal material was stained with lactophenol blue after 16 h, allowing discrimination between the stained surface hyphae and those left unstained after penetration of the host. The mutants were able to penetrate the plant’s surface but the subsequent colonization was severely inhibited (Figure 2A). A similar phenotype was recorded for the strains Δbcpdi1-C-M1 as well as Δbcpdi1-C-M1/2. In marked contrast, the complemented strain (Δbcpdi1-C) and Δbcpdi1-C-M2 appeared to infect in the manner of the wild type.

All strains that displayed a retarded colonization of plant tissue produced significantly fewer spores than the wild type and the strains that restored the wild type phenotype (Figure 2B). However, since all plants were infected with the same concentration of conidiospores, there is no causal correlation between reduced production of conidia and impaired virulence.

When studying the mycelium-mediated infection process of B. cinerea similar results were obtained as previously for the spore-mediated infection. In contrast to the wild type and the strains Δbcpdi1-C and Δbcpdi1-C-M2, the deletion mutant was impaired in its ability to achieve mycelium-mediated penetration as well as in the colonization stage (Figure 3A). Primary lesions were not visible until the 3rd day of infection. Similar results were obtained for Δbcpdi1-C-M1 and Δbcpdi1-C-M1/2.

The defects in infecting plant tissue may be due to the impaired production of ICs. All strains that were unaffected in the mycelium-mediated infection process were still able to produce ICs. In contrast, the mutant strains Δbcpdi1, Δbcpdi1-C-M1, and Δbcpdi1-C-M1/2 were unable to produce wild type-like ICs, but instead produced twisted hyphae which were not targeted toward one infection site as is normal for the wild type (Figure 3B).

Since it has been shown previously that members of the NoxA complex as well as putative interaction partners are impaired in the formation of CATs (Roca et al., 2012; Siegmund et al., 2015; Marschall and Tudzynski, 2016b), the different PDI strains were investigated with respect to this differentiation process. These specialized hyphae are formed to exchange cellular material between two hyphae of the same organism. Whereas the wild type, as well as the strain Δbcpdi1-C, were still capable of forming CATs, the deletion mutant and the strains Δbcpdi1-C-M1, Δbcpdi1-C-M1/2, and Δbcpdi1-C-DA were impaired in the formation of hyphal fusions. The mutant Δbcpdi1-C-M2 was still able to form the hyphal structures, but produced markedly less CATs than the wild type (Figure 2D).

Apart from hyphal fusions, BcPdi1 also affects the production of perennial survival structures called sclerotia. These structures, which are produced under constant darkness, were formed by the wild type and the complemented strain Δbcpdi1-C (Figure 3C). All other strains were defective in the production of sclerotia. Surprisingly, the mutant strain Δbcpdi1-C-M2 also failed to complement the defect in sclerotial development, although this mutant behave as the wild type in all other respects.

To summarize, BcPdi1 affects important vegetative and pathogenic processes, such as sclerotial formation, hyphal fusions and penetration structures. Essential for these developmental processes is the cytosolic region of BcPdi1 containing the two putative phosphorylation sites as well as one catalytically active site. Whereas the second predicted phosphorylation site seems to have an exclusive effect on the production of sclerotia, the first site seems to be essential for all essential protein functions.

To study the localization pattern of BcPdi1, a GFP fusion construct was generated under the control of the native promoter (1 kb upstream of the ORF) and the constitutive oliC promoter. Both were transformed into the deletion mutant’s background. The wild type phenotype was fully restored by both complementation constructs (Data not shown). Additionally, in both cases the target protein localized to identical structures. The strain BcPdi1-C was used since the fluorescence of the protein under the constitutive oliC promoter was much brighter for localization studies and later experiments. Apart from re-integrating bcpdi1 into its wild type version, GFP-fusion constructs with the mutated alleles (Δbcpdi1-C-M1, Δbcpdi1-C-M2, Δbcpdi1-C-M1/2, Δbcpdi1-C-DA-M1) were generated.

BcPdi1 localized to filamentous structures (Figure 4) belonging to the ER (verified by co-staining with ER tracker) and was additionally found in the nuclear envelope (verified by co-staining with the DNA dye Hoechst 33342 – Data not shown). Although the mutation of the second putative phosphorylation site (M2) into its phospho null form had no influence on the localization, the pattern changed upon mutation of M1. BcPdi1_M1 was detected in a faint network of filamentous structures belonging to the ER, but was hardly visible in the nuclear envelope. Moreover, the proteins seemed to adhere as aggregates that were detected throughout the hyphae. Similar results were obtained for the BcPdi1_GFP fusion construct containing both mutated phosphorylation sites.

The localization pattern of the phospho mimic mutants differed from all other observed PDI-constructs. The fusion constructs localized to filamentous structures, which show high similarity to mitochondria. Co-localization with the mitoIDred mito tracker as well as with the ER tracker revealed that those structures are overlapping with ER and mitochondrial structures (Figure 4).

In summary, we have established that BcPdi1 is indeed an ER protein that is located within the nuclear envelope and filamentous structures. Mutations of the first predicted cytosolic phosphorylation site of the protein directly influenced the distribution of the protein within hyphae, a most unexpected result.

To elucidate the role of BcPdi1 both inside and outside the ER, studies with the genetically encoded biosensor roGFP2 were performed. The modified version of the reporter gene was suitable for the visualization of the glutathione pool, which mirrors the current redox state of the monitored compartment (Schwarzlaender et al., 2008; Heller et al., 2012). These studies aimed to establish, whether BcPdi1 affects the cytosolic redox state and, therefore, is involved in the maintenance of the intracellular redox balance. Under non-inducing conditions, the phospho null version (Δbcpdi1-C-M1_roGFP2) appeared like the wild type and the deletion mutant (data not shown) whereas the phospho mimic version (Δbcpdi1-C-DA_roGFP2) exhibited an elevated _395/488ratio (Figure 5A top).

Since previous studies with mutants of both Nox complexes revealed that CaCl₂ is sufficient to induce an oxidative burst inside the cytosol, the mutants (Δbcpdi1-C-M1_roGFP2; Δbcpdi1-C-DA_roGFP2) were also tested under those conditions (50 mM CaCl₂). Surprisingly, both patterns of the mutants did not resemble the wild type control. In the wild type, the addition of CaCl₂ induced a massive oxidative burst, changing the _395/488ratio from 0.3 to 1.4. In comparison, the _395/488ratio of Δbcpdi1-C-M1_roGFP2 remained unaffected as the deletion mutant Δbcpdi1 (data not included in the figure, identical curve), and the _395/488ratio of the phospho mimic version (Δbcpdi1-C-DA_roGFP2) was slightly shifted to a more oxidized state than the wild type (Figure 5A below). The induction via CaCl₂ led only to minor changes of the _395/488ratio.

To elucidate, why the basic _395/488ratio of Δbcpdi1-C-DA_roGFP2 increased in comparison to the wild type, we did expression studies with known marker genes of the UPR. Since previous studies in plant cells revealed that changes in the redox state are due to the activity of the UPR machinery (Merksamer et al., 2008) we analyzed the bcpdi1 mutants expressing roGFP2 with respect to UPR activity. We used the putative B. cinerea homologs of the genes BiP/Kar2 (Bcin13g00960) and Hac1 (Bcin08g06730). Whereas BiP/Kar2 is a chaperone responsible for the correct folding of proteins (Jung et al., 2013), the transcriptional activator Hac1 binds to UPR-responsive elements to increase the amount of ER-resident proteins required for the folding machinery as well as components of the secretory pathway (Mori et al., 1992, 1998). Both were analyzed in the wild type in comparison to Δbcpdi1-C-M1_roGFP2 and Δbcpdi1-C-DA_roGFP2. The expression of the BiP/Kar2 homolog appeared to be wild type like in both mutants. The Hac1 homolog was upregulated in Δbcpdi1-C-DA_roGFP2 (Figure 5B). In summary, the results suggest that BcPdi1 is involved in the maintenance of the cytosolic redox state and may play a role in the UPR.

To examine whether the overlapping functions between the Nox complex and PDI are based on physical/direct interaction of these proteins we used a yeast-two hybrid approach; since membrane associated proteins are involved, we chose the split-ubiquitin system which is optimized for monitoring interactions between transmembrane proteins. The protein BcPdi1 was shown to interact with the catalytic subunit of the NoxA complex, and to a minor extent with BcNoxB (Figure 6A and Supplementary Figure S2). The positive interaction of BcNoxA and BcPdi1 was confirmed in co-IP experiments (Figure 6B). Strains expressing BcNoxA.mcherry (lane 1) or BcPdi1_GFP (lane 2) were used as controls (BcPdi1_GFP = 84 kDa; BcNoxA_mcherry = 102 kDa) (see also Materials and Methods). Additional interactions were shown for BcPdi1 with thioredoxin 2 (BcTrx2), an oxidoreductin (BcEro1) and the catalytic subunit of the calcineurin complex (BcCnA).

To elucidate the impact of the putative phosphorylation sites on the interaction with the different proteins, and also for the yeast-two hybrid based method, the putative cytosolic phosphorylation sites were mutated (BcPdi1_M1 = T65A; BcPdi1_M2 = Y208A). Again, the first putative phosphorylation site was transferred into its putatively dominant active form (phospho mimic) (T65E).

Interaction tests were done comparatively with the mutated versions of BcPdi1. While the mutation of the second predicted phosphorylation site (M2) had no effect on any of the interactions, mutation of M1 (T65A) and the phospho mimic led to the loss of the interaction with BcCnA. There was no effect on the binding to NoxA/B, Trx2, and Ero1. (Figure 6 and Supplementary Figure S2).

To summarize these results it is clear that BcPdi1 interacts with members of various signaling cascades such as Nox-, Ca²⁺-, and redox related pathways. The catalytic subunit of calcineurin (BcCnA) seems to bind to the cytosolic region of the protein and in particular to a segment exhibiting a single putative phosphorylation site.

In conclusion, we present evidence that BcPdi1 is involved in all major developmental processes, such as the formation of conidia, sclerotia and CATs. Moreover, the ER protein affects the virulence of the necrotrophic plant pathogen and mediates stress resistance to oxidative and osmotic stress. The phenotype of Δbcpdi1 is similar to those of ΔbcnoxA and ΔbcnoxD mutants suggesting a link between Nox signaling and BcPdi1 in these differentiation processes. A cytosolic phosphorylation site, which is highly conserved among different species, clearly is essential for the function of BcPdi1. In its phospho null form, the respective mutants behave like Δbcpdi1. In contrast, the phospho mimic mostly restores the wild type phenotype.