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. 2016 Aug 8;18(6):783–797. doi: 10.1111/mpp.12438

YAP1 homologue‐mediated redox sensing is crucial for a successful infection by Monilinia fructicola

Pei‐Ling Yu 1,2,3, Chih‐Li Wang 1, Pei‐Yin Chen 1, Miin‐Huey Lee 1,2,3,✉
PMCID: PMC6638302  PMID: 27239957

Summary

Monilinia fructicola (G. Winter) Honey is a devastating pathogen on Rosaceae which causes blossom blight and fruit rot. Only a few studies related to the plant–pathogen interaction have been published and there is limited knowledge on the relationship between oxidative stress and successful infection in M. fructicola. In this study, we cloned and characterized a redox‐responsive transcription factor MFAP1, a YAP1 homologue. MfAP1‐silenced strains were generated by polyethylene glycol‐mediated protoplast transformation or Agrobacterium T‐DNA‐mediated transformation. Pathogenicity assay demonstrated that MfAP1‐silenced strains caused smaller lesions on rose and peach petals. Transformants carrying extra copies of MfAP1, driven by the native promoter, were generated for MfAP1 overexpression. Interestingly, MfAP1‐overexpressing strains also caused smaller lesions on rose petals. Strains carrying two copies of MfAP1 accumulated reactive oxygen species (ROS) at higher levels and exhibited delayed accumulation of MfAP1 transcripts compared with the wild‐type during pathogenesis. By the analysis of ROS production and the expression patterns of redox‐ and virulence‐related genes in the wild‐type strain and an MfAP1‐overexpressing strain, we found that the M. fructicola wild‐type strain responded to oxidative stress at the infection site, activated the expression of MfAP1 and up‐regulated the genes required for ROS detoxification and fungal virulence. In contrast, MfAP1 expression in the MfAP1‐overexpressing strain was suppressed after the induction of a strong oxidative burst at the infection site, altering the expression of ROS detoxification and virulence‐related genes. Our results highlight the importance of MfAP1 and ROS accumulation in the successful infection of M. fructicola.

Keywords: cutinase, glutathione, oxidative stress, Prunus sp

Introduction

Oxygen (O2) is an essential element to living organisms. However, when electrons are taken from or added to O2, toxic intermediate products (reactive oxygen species, ROS) can be generated (Voeikov, 2006). ROS can react with DNA, proteins and lipids, which can result in damage to nucleotides and proteins or lipid peroxidation, and lead to aging and cell death (Beckman and Ames, 1998).

ROS play important roles in plant–pathogen interactions. On perception of a pathogen attack, plants often accumulate large amounts of ROS, known as the oxidative burst, resulting in programmed cell death (PCD) and cellular defence responses in incompatible interactions (Heller and Tudzynski, 2011; O'Brien et al., 2012). The infection of biotrophic pathogens is extremely restricted by ROS‐mediated PCD in host cells, whereas necrotrophic pathogens, such as Botryotinia fuckeliana (anamorph Botrytis cinerea) and Sclerotinia sclerotiorum, kill host plants before colonization (Asai and Yoshioka, 2009; Govrin and Levine, 2000; Walz et al., 2008), and the accumulation of higher levels of ROS is thought to be important for their infection. Necrotrophic pathogens can produce ROS themselves and stimulate the host to do so (Kim et al., 2008; Shetty et al., 2008). Nevertheless, fungal cells need to activate their ROS detoxification machinery under such oxidative stress regardless of where ROS are produced.

To cope with oxidative stress, cells use effective mechanisms that involve both enzymatic and non‐enzymatic reactions. Glutathione (GSH; l‐γ‐glutamyl‐l‐cysteinyl‐glycine) is one of the major redox buffers in eukaryotic cells (Apel and Hirt, 2004). Under oxidative stress, reduced GSH (GSH) is transformed to oxidized GSH (GSSG) via a GSH peroxidase (GPx; EC 1.11.1.9). To maintain cellular redox homeostasis, cells can convert GSSH back to reduced GSH via a glutathione reductase (GR; EC 1.8.1.7) using NADPH provided by glucose‐6‐phosphate dehydrogenase (G6PD; EC 1.1.1.49) as an electron donor. The cellular redox status is indicated by the ratio of GSH to GSSG, and cells can undergo oxidative stress as this ratio decreases (Han et al., 2006).

Several redox‐sensitive transcription factors required for cellular responses to oxidative/redox conditions have been well studied in mammalian cells (Brigelius‐Flohe and Flohe, 2011; Karin et al., 1997). Among them, activating protein 1 (AP1) has been identified in the budding yeast Saccharomyces cerevisiae and has been named yeast AP1 (YAP1) (Kuge et al., 1997). The expression of YAP1 and its downstream genes, such as GSH reductase, GSH peroxidase, superoxide dismutase and catalase, is highly up‐regulated in cells under oxidative stress (Lee et al., 1999). Recently, YAP1 homologues have been identified in several phytopathogenic fungi, including Cochliobolus heterostrophus (ChAP1), Ustilago maydis (Yap1), B. fuckeliana (BAP1), Alternaria alternata (citrus brown disease, AaAP1; rough lemon pathotype, RLAP1), Magnaporthe oryzae (MoAP1), Fusarium graminearum (FgAP1) and Ashbya gossypii (AgYAP1) (Guo et al., 2011; Lev et al., 2005; Lin et al., 2009; Molina and Kahmann, 2007; Montibus et al., 2013; Temme and Tudzynski, 2009; Walther and Wendland, 2012; Yang et al., 2009). All YAP1 homologues function in the redox stress response in these phytopathogenic fungi. YAP1 homologues found in A. alternata (rough lemon pathotype) and M. oryzae play a role in vegetative growth. YAP1 homologues found in U. maydis, A. alternata (citrus brown disease) and M. oryzae contribute to fungal virulence/pathogenicity. However, all previous studies related to YAP1 function on fungal pathogenicity are based on the gene knockout approach. The changes in cell physiology regulated by ROS are actually dynamic (Gessler et al., 2007; Paul et al., 2014). Therefore, the gene knockout approach may not be able to detect certain uncharacterized functions of ROS‐sensitive genes during fungal pathogenesis.

Monilinia fructicola (G. Winter) Honey, which causes blossom blight and fruit rot on Rosaceae, is also considered to be a necrotroph (Andrew et al., 2012). However, it is unclear whether and how ROS accumulation is involved in virulence in M. fructicola during pathogenesis. Blossom blight and fruit rot caused by M. fructicola are devastating diseases in stone fruits. This fungal pathogen penetrates the cuticle layer directly via appressorial formation (Lee and Bostock, 2006b). Our previous studies have shown that the cutinase‐coding gene MfCUT1 is a virulence factor in M. fructicola (Lee et al., 2010). MfCUT1 expression was up‐regulated by hydrogen peroxide (H2O2) and down‐regulated by antioxidants in axenic culture (Chiu et al., 2013; Lee et al., 2010). The analysis of DNA sequences upstream from the ATG translational initiation codon of MfCUT1 identified several AP1 binding sites (Lee et al., 2010).

In this study, we characterize an AP1‐like transcription factor in M. fructicola, designated as MFAP1, and investigate its role in fungal virulence and the expression of ROS detoxification‐related genes in planta. The generation of gene‐disrupted homokaryotic mutants is difficult in M. fructicola (Lee and Bostock, 2006a). The generation of gene‐silenced strains can be an alternative approach. Fungal strains carrying an additional copy of a gene could also provide extra insights into gene function during fungal pathogenesis. By studying MfAP1‐silenced and MfAP1‐overexpressing strains, we highlight the importance of MfAP1 and ROS accumulation in M. fructicola in the establishment of a successful infection.

Results

Cloning and characterization of MfAP1 and MfG6PD1 from M. fructicola

The open reading frame (ORF) of the M. fructicola YAP1 homologue (designated MfAP1) and its 5′ and 3′ flanking regions were obtained by degenerate‐ and inverse‐polymerase chain reaction (PCR). Comparison of the assembled genomic DNA sequence (∼3 kb) with cDNA revealed that the MfAP1 gene has a 2014‐bp ORF interrupted by three small introns of 46, 93 and 60 bp.

The MfAP1 ORF encodes a polypeptide containing 604 amino acids. The predicted MFAP1 protein contains three conserved regions commonly found in YAP1 and YAP1 homologues: a bZIP DNA‐binding dimerization domain and a nuclear localization domain at the N‐terminus (Fig. S1a, see Supporting Information), as well as a cysteine‐rich domain at the C‐terminus (c‐CRD) (Fig. S1b). Three bipartite nuclear localization signals (NLSs) are found between amino acids 140 and 154 (KRREGDDKSSKKPGR), 150 and 167 (KKPGRKPLTSEPTSKRKA), and 164 and 181 (KRKAQNRAAQRAFRERK) (Lin et al., 2009). A nuclear export sequence (NES; RSTMLSCNTIWDRL), a potential binding site for the Crm1p‐like exporter (Yan et al., 1998), is present in the c‐CRD (amino acids 531–544). The phylogenetic relationship of MFAP1 to other AP1‐like transcription factors revealed two major groups in the dendrogram, clearly separating YAP1 homologues of filamentous fungi from those of yeasts in the clade of Ascomycota (Fig. S2, see Supporting Information). MFAP1 is closely related to the YAP1 homologues identified in B. fuckeliana (CAX15423.1) and S. sclerotiorum (XP_00158939.1), with 84% and 75% identity, respectively.

To understand the cellular redox status, the expression of genes encoding three key enzymes in GSH cycling, including GR (MfGR1), GPx (MfGPx1) and the reducing power NADPH provider G6PD (MfG6PD1), were analysed. We have previously cloned the genes MfGR1 and MfGPx1 from M. fructicola (Chiu et al., 2013). Here, partial sequences corresponding to MfG6PD1 were obtained by PCR with degenerate primers. A 753‐bp MfG6PD1 DNA fragment was amplified. Sequence analysis and similarity searches against fungal sequences deposited in the National Center for Biotechnology Information (NCBI) verified its identity. The deduced partial amino acid sequence of MfG6PD1 contains the expected G6PD motif and is highly similar to the G6PD family of proteins in B. fuckeliana (XP_001553624.1) and S. sclerotiorum (XP_001589839.1) (Fig. S3, see Supporting Information).

Expression of MfAP1 on treatment with redox compounds and during vegetative growth and appressorial formation

Monilinia fructicola was grown in modified Czapek–Dox medium for 3 days, shifted to medium amended with the oxidant H2O2 or the antioxidant caffeic acid, and incubated for an additional 24 h. Quantitative reverse transcriptase‐PCR (qRT‐PCR) analysis revealed that the accumulation of MfAP1 transcripts increased in the presence of H2O2 and decreased when the fungus was grown in medium containing caffeic acid (Fig. 1a). The expression pattern of MfAP1 during fungal growth and appressorium formation was investigated by growing M. fructicola in a 1 mm sucrose solution. MfAP1 expression was down‐regulated in M. fructicola after incubation for 2–9 h and up‐regulated at 24 h when the fungal growth had reached a plateau (Figs 1b, S4, see Supporting Information). The level of MfAP1 expression decreased considerably when M. fructicola was incubated on a plastic surface for 6 h, at which point appressorium formation was observed (Fig. 1c).

Figure 1.

Figure 1

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Relative expression of MfAP1 in Monilinia fructicola under redox stress (a), during vegetative growth (b) and during appressorium formation (c). The fold change in MfAP1 relative expression was compared with that of control treatment (a) or 0 h post‐inoculation (hpi) (b, c). CA, caffeic acid.

Expression of MfAP1 in planta

Monilinia fructicola was inoculated onto rose or peach petals and the accumulation of MfAP1 transcripts was examined by qRT‐PCR. Compared with the expression level at 0 h post‐inoculation (hpi), MfAP1 was expressed at the highest level in M. fructicola assayed at 10 hpi, concomitant with the appearance of necrotic lesions (Fig. 2). The MfAP1 expression levels were down‐regulated at 6 hpi when fungal penetration occurred. As the necrotic lesions expanded at 24–48 hpi, MfAP1 expression levels decreased considerably. In some batches of the experiment, the MfAP1 expression peak appeared at 6 hpi.

Figure 2.

Figure 2

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Relative expression of MfAP1 during pathogenesis of Monilinia fructicola on rose (a) and peach (b) petals. Brown rot lesions at each time point of inoculation are also shown. The brown rot lesion areas of peach petals were semi‐translucent and the background colour appeared after photographing. The fold change in MfAP1 relative expression at each time point was compared with that at 0 h post‐inoculation (hpi).

Generation and characterization of MfAP1‐silenced strains

To investigate the function of MfAP1 in the development and pathogenicity of M. fructicola, MfAP1‐silenced strains were created via polyethylene glycol (PEG)‐mediated protoplast transformation or Agrobacterium T‐DNA‐mediated transformation (ATMT). Transformants carrying an MfAP1 silencing construct, confirmed by PCR, were studied further. The insertion number of the MfAP1 silencing construct was assayed by quantitative PCR or Southern blot, and the MfAP1 expression level was detected by qRT‐PCR. Transformants generated by PEG‐mediated transformation appeared to be heterokaryons, indicating that not all of the nuclei carry the silencing construct (Fig. 3a), but the MfAP1 expression level in these transformants was significantly lower than that in the wild‐type strain (Fig. 3b). Using ATMT, three (B15, E29 and I2) of 10 transformants showed single silencing construct insertion and had a similar band density between the inserted silencing construct and the endogenous MfAP1 fragment (Fig. 3c), indicating that the three transformants carry the same copy number of the MfAP1 silencing construct and the endogenous MfAP1. qRT‐PCR assay revealed that MfAP1 transcripts could be reduced by up to 80% in transformant E6 which carried more than one copy of the silencing construct (Fig. 3d). There were no significant differences in spore germination, appressorial formation and fungal growth between the MfAP1‐silenced strains and the wild‐type strain in vitro (Fig. S5, see Supporting Information, and data not shown).

Figure 3.

Figure 3

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Copy number of MfAP1 silencing construct (a, c) and MfAP1 expression level (b, d) in Monilinia fructicola MfAP1‐silenced transformants generated by polyethylene glycol (PEG)‐mediated protoplast transformation (a, b) and Agrobacterium T‐DNA‐mediated transformation (c, d). (a) The copy number of the MfAP1 silencing construct was measured by quantitative polymerase chain reaction (PCR) using primers located within the MfAP1 partial open reading frame (ORF) constructed for double‐strand RNA formation. The relative copy number of the MfAP1 partial ORF in MfAP1‐silenced transformants was calculated by comparison with that in the wild‐type (WT) strain. (b, d) The expression of MfAP1 occurred in MfAP1‐silenced transformants on V8 agar medium. The fold change in MfAP1 relative expression in MfAP1‐silenced transformants was calculated by comparison with that in the WT strain. (c) Southern blot analysis of MfAP1 transformants with a 0.5‐kb MfAP1 ORF fragment probe. The probe was labelled with digoxigenin‐11‐dUTP during PCR using the primer set Yap1_cDNA_F and Yap1_cDNA_R (Table S1, see Supporting Information).

MfAP1‐silenced strains cause smaller brown rot lesions than the wild‐type strain

Pathogenicity assay was conducted on detached peach and rose petals. The brown rot lesions caused by MfAP1‐silenced strains were significantly smaller than those caused by the wild‐type strain (Table 1). qRT‐PCR analysis revealed that MfAP1 transcripts accumulated at significantly lower levels in petals infected with MfAP1‐silenced strains than in petals infected with the wild‐type strain (Table 1). The results suggest that MfAP1‐silenced strains are less virulent than the wild‐type strain on rose and peach petals. When examined with a microscope, no significant difference was observed with regard to conidial germination, appressorium formation and germ tube elongation of the wild‐type strain and the silenced strains on the infected petals at 6 hpi (data not shown).

Table 1.

Lesion size of brown rot caused by Monilinia fructicola wild‐type (WT) strain and MfAP1‐silenced transformants (A6, C2, B15, E6, E29, I2 and J12) on rose and peach petals.

Petal Experiment n * Mean of lesion size (mm2) P † Decrease in lesion size (%) Fold difference of MfAP1 expression ‡
WT Transformant
Rose 1 30 72.4 39.0 (A6) <0.0001 46.2 0.07
22 70.6 25.4 (C2) <0.0001 64.1 0.14
2 23 51.7 12.8 (A6) <0.0001 75.2 0.10
15 58.1 16.7 (C2) 0.003 71.3 0.54
3 6 108.8 35.6 (B15) 0.043 67.2 0.114
5 149.0 72.4 (E6) 0.003 51.4 0.003
4 5 56.5 20.6 (B15) 0.018 63.5 0.130
6 50.4 18.4 (E6) 0.003 64.1 –
5 64.1 24.1 (E29) 0.021 62.4 –
5 61.5 38.9 (I2) <0.001 36.7 –
8 49.4 25.9 (J12) 0.029 47.6 –
Peach 1 28 55.4 39.7 (A6) <0.05 28.4 –
32 26.1 8.7 (C2) <0.0001 66.5 –
2 25 24.3 2.0 (A6) <0.0001 91.8 –
32 44.0 14.8 (C2) <0.0001 66.4 –
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*n, number of plant samples tested.

†Statistical analysis was performed by a paired t‐test.

‡The fold change in MfAP1 relative expression in MfAP1‐silenced strains (A6, C2, B15, E6, E29, I2 and J12) was calculated by comparison with that in the wild‐type strain at 48–72 h post‐inoculation.

The infection of MfAP1‐overexpressing strains also resulted in smaller lesions

To confirm the function of MfAP1 in M. fructicola pathogenicity, MfAP1 driven by the native promoter was transferred into the wild‐type strain. Southern blotting revealed that three transformants (A4‐1, A4‐4 and E4‐1) carried two copies of MfAP1 and two transformants (A1‐2 and A1‐4) carried three copies of MfAP1 (Fig. S6, see Supporting Information). Fungal pathogenicity assayed on detached rose petals revealed that transformants carrying extra copies of MfAP1 induced smaller brown rot lesions than the wild‐type strain (Table 2). qRT‐PCR analysis confirmed that the in planta expression of MfAP1 was significantly higher in the transformants than in the wild‐type strain (Table 2). There was no significant difference in spore germination, appressorial formation and fungal growth between the MfAP1‐overexpressing strains and the wild‐type strain in vitro and in planta (Figs S5, S7, see Supporting Information).

Table 2.

Lesion size and MfAP1 expression in rose petals infected with Monilinia fructicola wild‐type (WT) strain and transformants (A1‐4, A4‐4 and E4‐1) carrying extra copies of the MfAP1 gene.

Experiment n * Mean of lesion size (mm2) P † Decrease in lesion size (%) Fold difference of MfAP1 expression ‡
WT Transformant
1 12 25.0 11.0 (A1‐4) 0.001 55.9 2.2
17 38.0 32.1 (A4‐4) 0.019 15.8 1.6
17 46.1 30.9 (E4‐1) 0.001 32.9 12.9
2 11 78.2 17.2 (A1‐4) 0.001 78.0 3.0
13 128.2 95.9 (A4‐4) 0.007 25.2 2.5
13 118.6 103.8 (E4‐1) 0.099 12.5 9.8
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*n, number of plant samples tested.

†Statistical analysis was performed by a paired t‐test.

‡The fold change in MfAP1 relative expression in MfAP1‐overexpressing strains (A1‐4, A4‐4 and E4‐1) was calculated by comparison with that in the wild‐type strain at 48 h post‐inoculation.

MfAP1‐overexpressing strains induced a higher level of ROS accumulation at the inoculation site

The accumulation of ROS was detected and quantified with a dichlorofluorescin diacetate (DCFH‐DA) probe, which fluoresces after reacting with ROS. Assays of fluorescence intensity at the infection site revealed that inoculation of MfAP1‐silenced strains (B15 and I2) onto rose petals resulted in less ROS accumulation than in the wild‐type strain at the early stages (Fig. S8, see Supporting Information). However, higher levels of ROS accumulation were caused by MfAP1‐overexpressing strains A4‐4 and E4‐1 compared with the wild‐type at the infection site (Fig. 4a,b). Microscopic examinations revealed that conidia of the wild‐type strain germinated, produced appressoria on host tissues and penetrated epidermal cells at 4 hpi, even though plant organelles within the cell remained intact (Fig. 4c). The infected cells became purple and were completely bleached at 6 hpi, which indicated cell death (Fig. 4c). Brown rot lesions appeared on rose petals at 10 hpi and quickly grew into large lesions at 24 hpi (Fig. 2a).

Figure 4.

Figure 4

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Accumulation of reactive oxygen species (ROS) on rose petals inoculated with Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain A4‐4 or E4‐1 (a, b). ROS were detected at various inoculation time points (a) or at 6 h post‐inoculation (hpi) (b). (c) Light microscopy of the wild‐type strain in rose petals observed at 4 and 6 hpi. ap, appressorium; dc, dead cell; ih, infection hypha; rc, rose cell; s, spore. Bar, 10 μm.

MfAP1 expression was suppressed in MfAP1‐overexpressing strains on exposure to oxidative stress

To understand why the MfAP1‐overexpressing strains showed reduced virulence, MfAP1‐overexpressing strain A4‐4 was used for further study. The T‐DNA insertion site in the A4‐4 strain was identified in a non‐coding region by inverse‐PCR amplification and sequence analysis. The MfAP1‐overexpressing and wild‐type strains were inoculated onto individual petals to characterize further, in time course experiments, the expression patterns of MfAP1 and other genes. The fold change in MfAP1 expression at various time points was calculated by comparison with the level at 0 hpi. MfAP1 expression in the wild‐type strain was down‐regulated at 4 hpi and then up‐regulated at 10 hpi (Fig. 5a). The expression levels of MfAP1 reached a maximum at 10–24 hpi in the wild‐type strain. In contrast, MfAP1 expression in the A4‐4 strain was down‐regulated at 4‐10 hpi and increased to a maximum at 24–48 hpi (Fig. 5b). To compare the MfAP1 expression level in the A4‐4 strain with that in the wild‐type strain at each time point, the fold change in MfAP1 expression in the A4‐4 strain relative to that in the wild‐type strain was calculated. The accumulation of MfAP1 transcripts was suppressed at 10 hpi in the A4‐4 strain compared with the wild‐type strain (Fig. 5c). These data suggest that the down‐regulation of MfAP1 in the MfAP1‐overexpressing strain at 10 hpi might be caused by the overexpressing strain encountering the oxidative burst generated at the early infection stage. In vitro assay by the culture of fungal strains in broth medium containing 1 mm H2O2 also showed a similar phenomenon. With the presence of H2O2, MfAP1 expression increased in the wild‐type strain, but decreased in A4‐4 (Fig. S9a, see Supporting Information). When compared with the wild‐type strain, A4‐4 accumulated a higher level of MfAP1 transcripts in the control treatment and expressed a lower level of MfAP1 in the presence of H2O2 (Fig. S9b).

Figure 5.

Figure 5

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MfAP1 expression of Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4) on rose petals. The expression of MfAP1 occurred in the wild‐type (a) and A4‐4 (b) strains on rose petals. The fold change in MfAP1 relative expression at all assayed time points was calculated by comparison with that at 0 h post‐inoculation (hpi). The fold change values are indicated (a, b). The fold change in MfAP1 relative expression of the A4‐4 strain was calculated by comparison with that of the wild‐type strain at each time point (c).

MfCUT1 expression was down‐regulated in the MfAP1‐overexpressing strain at the lesion expansion stage

MfCUT1, encoding a cutinase, is a virulence factor of M. fructicola (Lee et al., 2010). When compared with the expression level at 0 hpi, MfCUT1 expression was up‐regulated at two stages during the pathogenesis of the wild‐type strain: the penetration stage (4–6 hpi) and the lesion expansion stage (24–48 hpi) (Fig. 6a). However, up‐regulation of MfCUT1 did not exist at the lesion expansion stage in the A4‐4 strain (Fig. 6b). Furthermore, a comparison of the expression level of the A4‐4 strain with that of the wild‐type strain at each hpi showed that A4‐4 appeared to accumulate fewer MfCUT1 transcripts than the wild‐type strain at the late infection stage (24–48 hpi) (Fig. S10, see Supporting Information). Although MfCUT1 was overexpressed at the early infection stage in A4‐4 (Fig. S10), the up‐regulation of MfCUT1 at 4–6 hpi in A4‐4 was not as noticeable as that in the wild‐type strain (Fig. 6).

Figure 6.

Figure 6

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Relative expression of MfCUT1 in Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4) during pathogenesis on rose petals. The fold change in MfCUT1 relative expression at all assayed time points was calculated by comparison with that at 0 h post‐inoculation (hpi).

The expression patterns of glutathione cycling genes were changed in A4‐4 during pathogenesis

ROS detoxification genes can be induced via the regulation of YAP1 homologues in fungal cells under oxidative stress (Lee et al., 1999; Lev et al., 2005; Temme and Tudzynski, 2009). To study the expression patterns of MfGR1, MfGPx1 and MfG6PD1 in the wild‐type and A4‐4 strains during pathogenesis on rose petals, the fold change in the target gene expression at various time points was calculated by comparison with the level at 0 hpi (Fig. 7). MfGR1 expression was down‐regulated at 6–10 hpi, and the expression level dramatically increased from 10 to 24 hpi in the wild‐type strain (Fig. 7a). In the A4‐4 strain, the expression of MfGR1 was down‐regulated from 0 to 10 hpi and then up‐regulated at 24 hpi (Fig. 7b). However, the up‐regulation of MfGR1 from 10 to 24 hpi in the A4‐4 strain (14‐fold increase) was not as strong as that shown in the wild‐type strain (300‐fold increase). The expression pattern of MfGPx1 in the wild‐type strain was similar to that in the A4‐4 strain during pathogenesis on rose petals (Fig. 7c,d). MfGPx1 was slightly up‐regulated at 6 hpi and down‐regulated at 10 hpi. The down‐regulation of MfGR1 and up‐regulation of MfGPx1 from 4 to 6 hpi indicate that the fungal cells are under oxidative stress. The time course expression pattern of MfG6PD1 in the wild‐type strain was also similar to that in the A4‐4 strain (Fig. 7e,f). An expression peak of MfG6PD1 was shown at 24 hpi, in which MfGR1 expression also displayed a significant peak (Fig. 7).

Figure 7.

Figure 7

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Relative expression of MfGR1 (a, b), MfGPx1 (c, d) and MfG6PD1 (e, f) in Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4) during pathogenesis on rose petals. The fold change in the relative expression of the target gene at all assayed time points was calculated by comparison with that at 0 h post‐inoculation (hpi).

To determine whether the three genes were regulated by MfAP1, the fold change in the expression of the three genes in the A4‐4 strain relative to that in the wild‐type strain at each time point was calculated. The A4‐4 strain expressed higher levels of MfGR1, MfGPx1 and MfG6PD1 from 0 to 6 hpi (Fig. S11, see Supporting Information). However, the overexpression of the three genes in the A4‐4 strain was not consistent between 24 and 48 hpi.

Discussion

Monilinia fructicola is one of the most important pathogens in Rosaceae. However, studies on plant–pathogen interactions in M. fructicola are quite rare. Here, we identified an AP1‐like transcription factor from M. fructicola, which is referred to as MFAP1. MfAP1 functional analysis with gene‐silenced strains suggests that MfAP1 is required for full virulence of M. fructicola on peach and rose petals. In addition, by the analysis of ROS production and the expression patterns of MfAP1, redox‐ and virulence‐related genes in an MfAP1‐overexpressing strain and the wild‐type strain, we found that an appropriate MfAP1 expression level is vital for M. fructicola to coordinate the expression of genes required for ROS detoxification and fungal virulence (Fig. 8).

Figure 8.

Figure 8

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Schematic overview of the infection process, reactive oxygen species (ROS) accumulation, gene expression and pathogenicity of Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4) on rose petals.

MFAP1, similar to YAP1 in S. cerevisiae and other YAP1 homologues in filamentous fungi, contains a DNA‐binding bZIP domain, an oxidative stress‐sensitive cysteine‐rich region, a nuclear localization domain and a Crm1 exporter protein‐binding motif (Lev et al., 2005; Molina and Kahmann, 2007; Temme and Tudzynski, 2009; Toone et al., 2001). The YAP1 and YAP1 homologues are a vital element for the survival of different yeasts and fungal species under oxidative stress (Guo et al., 2011; Kuge et al., 1997; Lev et al., 2005; Lin et al., 2009; Molina and Kahmann, 2007; Montibus et al., 2013; Temme and Tudzynski, 2009; Walther and Wendland, 2012; Yang et al., 2009). YAP1 locates in the cytosol in unstressed conditions and is activated under oxidative stress by rapid translocation to the nucleus (Toone et al., 2001). The induction of MfAP1 expression by H2O2 indicates that it plays a crucial role in cellular resistance to oxidative stress in M. fructicola, which is consistent with previous findings in other fungi (Kuge and Jones, 1994; Lin et al., 2009; Shanmugam et al., 2010). In contrast, MfAP1 expression is inhibited by caffeic acid, an antioxidant compound that has been shown previously to decrease cellular redox and virulence of M. fructicola in peach (Chiu et al., 2013; Lee and Bostock, 2007). MfAP1 expression occurs in response to the disturbance of cellular redox homeostasis. In human cells, studies have demonstrated that phenolic antioxidants can trigger the up‐regulation of AP1 expression and enhance its DNA‐binding activity (Choi and Moore, 1993). The activation of the YAP1 homologue by maize phenolics has also been observed in the maize fungal pathogen Cochliobolus heterostrophus (Shanmugam et al., 2010). However, plant phenolic compounds can act as antioxidants or pro‐oxidants depending on the dose applied (Maurya and Devasagayam, 2010). In the medium shift system, caffeic acid reduces the level of cellular H2O2, indicating that it is an antioxidant (Chiu et al., 2013). Therefore, under the same medium shift condition, it is possible that caffeic acid treatment would reduce the nuclear retention of MFAP1 compared with the control treatment. An AP1‐binding site was found at position −313 to −319 in the minus strand of MfAP1, indicating that MFAP1 might be able to trigger gene self‐expression (Seldeen et al., 2009).

A change in redox status can affect fungal growth and development (Gessler et al., 2007; Heller and Tudzynski, 2011; Tudzynski et al., 2012). In Neurospora crassa, the generation of ROS results in a hyperoxidant status, and a disturbance of the redox balance impacts spore formation and germination (Lledias et al., 1999; Toledo et al., 1995). Monilinia fructicola expresses MfAP1 during vegetative growth at levels that are lower than those in hydrated conidia and in the stationary stage of growth, indicating that the last two stages of fungal cells might be under oxidative stress. An AP1‐like gene, MoAP1, in the rice blast pathogen M. oryzae has also been shown to be expressed at higher levels in conidia than during vegetative growth (Guo et al., 2011).

MfAP1 expression was investigated during the invasive growth of M. fructicola inside rose and peach petals. In the wild‐type strain, MfAP1 expression levels were significantly lower during the early infection stage (4–6 hpi) and increased during lesion development (10–24 hpi). Quantitative analysis revealed that higher levels of ROS, indicative of the oxidative burst, were detected within 10 hpi. The increased expression of MfAP1 observed in the wild‐type strain at 10–24 hpi might be a response to the increase in ROS around the infection site.

The glutathione cycling system is a pivotal mechanism in the response to extracellular oxidative stress and for the maintenance of redox homeostasis in cells. Under oxidative stress, cellular GSH is converted to GSSG by GPx, concomitantly leading to the detoxification of H2O2. GSSG can be reduced to GSH by GR at the expense of NADPH generated by G6PD. Infection of the wild‐type strain in rose petals induced the accumulation of ROS, reduced the expression of MfGR1 and increased the expression of MfGPx1 at 6–10 hpi, indicating that fungal cells were under oxidative stress. Consequently, MfGR1 and MfG6PD1 were up‐regulated, probably to maintain cellular redox homeostasis, and the induction might be a result of the accumulation of MfAP1 transcripts at 10 hpi. The glutathione cycling system have been reported to be regulated by YAP1 (Temme and Tudzynski, 2009).

Our previous study has shown that the cutinase‐coding gene MfCUT1 is a potent virulence determinant of M. fructicola (Lee et al., 2010). In addition, several potential AP1‐binding sites were found in the promoter region of MfCUT1, suggesting that the expression of MfCUT1 might be regulated by MFAP1. Their expression patterns in this study also support this assertion. MfCUT1 expression was up‐regulated at the penetration stage (4–6 hpi) and the lesion expansion stage (24–48 hpi), which followed the two up‐regulation events in MfAP1 at 0 and 10 hpi, respectively, in the wild‐type strain. MfCUT1 might be involved in the penetration of the host cuticle at the early infection stage and in the degradation of a large amount of cuticle during lesion expansion. The regulation of fungal virulence genes by the YAP1 homologue has also been reported in M. oryzae (Guo et al., 2011). As illustrated in Fig. 8, M. fructicola is capable of sensing and responding to changes in redox status to coordinate the expression of virulence and antioxidant genes that can deal with the oxidative burst at the infection site, and contribute to the establishment of a successful infection.

The MfAP1‐overexpressing strain A4‐4 showed a different behaviour from the wild‐type strain on induction of the oxidative burst during pathogenesis and different expression patterns of the various genes investigated here. The A4‐4 strain induced ROS accumulation at levels greater than those of the wild‐type strain in planta. In the overexpressing strain A4‐4, some fungal factors might be expressed at different levels. If these factors affect ROS production by the plant, this could explain the finding that the MfAP1‐overexpressing strain caused higher levels of ROS accumulation than the wild‐type strain at the infection site. For example, MfPG1, encoding an endopolygalacturonase in M. fructicola, could affect ROS production at the early infection stage (Chou et al., 2015). It is also possible that ROS were produced by the pathogen induced by an in planta factor. The high level of ROS induced in planta was followed by a down‐regulation of MfAP1 in the MfAP1‐overexpressing strain A4‐4. Furthermore, the treatment of the A4‐4 strain with H2O2 resulted in a lower accumulation of MfAP1 transcripts. These results indicate that excess ROS could inhibit MfAP1 expression in A4‐4. MfAP1 down‐regulation in the MfAP1‐overexpressing strain under oxidative stress might occur via a feedback control. YAP1 activation is dynamic and highly autoregulated (Toledano et al., 2004). Cellular H2O2 can maintain YAP1 activation until its concentration is corrected by peroxidase. Although the autoregulatory nature occurs at the protein level, gene transcription might also be regulated. Under oxidative stress, the overexpressed MFAP1 protein of the A4‐4 strain may remain activated for a shorter period than the protein of the wild‐type strain. Therefore, the up‐regulation of MfAP1 appeared to be delayed in A4‐4 during pathogenesis. To test this hypothesis, the overexpression construct could be replaced with a MFAP1‐GFP fusion driven by the native promoter to generate a green fluorescent protein (GFP)‐fused MfAP1‐overexpressing strain. GFP localization and fluorescence intensity in the cytosol and in the nucleus should be monitored at the infection site during pathogenesis. ROS accumulation and gene expression levels should also be detected to verify the role of ROS signalling in MfAP1 expression and the antioxidant response in M. fructicola infection. In other pathosystems in which fungal gene knockout can be achieved, the use of AP1 knockout mutants carrying single or double copies of the AP1‐GFP fusion construct for the experiments mentioned above will provide clear evidence.

The MfAP1‐overexpressing strain A4‐4 showed an up‐regulation of the expression of MfGR1, MfGPx1, MfG6PD1 and MfCUT1 at the early infection stage. However, up‐regulation compared with the wild‐type strain was not observed at 24 hpi, which might be a result of the significant down‐regulation of MfAP1 in the A4‐4 strain at 10 hpi, the assayed time point immediately before 24 hpi. As discussed above, the MfAP1‐overexpressing strain caused excess ROS accumulation, followed by the down‐regulation of MfAP1 in planta. Low levels of MfAP1 could, in turn, down‐regulate the genes required for fungal virulence and ROS detoxification. The identification of the downstream genes regulated by MfAP1 will provide an insight into their possible regulatory roles in ROS detoxification during M. fructicola pathogenesis. In conclusion, our results highlight the importance of the M. fructicola response to the oxidative burst during the early infection stages. Furthermore, we have demonstrated that MfAP1 plays an important role in pathogenicity.

Experimental Procedures

Fungal strains and growth conditions

The wild‐type strain MUK‐1 of M. fructicola (Bostock et al., 1999) was used as recipient for T‐DNA‐mediated transformation. Fungal strains were grown on V8‐juice agar medium for sporulation (Lee et al., 2010). For medium shift assays, spore suspensions were added to modified Czapek–Dox broth (Lee et al., 2010) containing 0.1% glucose, and incubated for 3 days. The medium was removed and the mycelia were washed twice with sterile water. Modified Czapek–Dox broth (20 mL) containing caffeic acid or H2O2 was applied to the washed mycelia and incubated for an additional 24 h, as indicated. Mycelia were collected for RNA isolation and gene expression.

Cloning of MfAP1 and MfG6PD1

The primers used in this study are listed in Table S1 (see Supporting Information). A YAP1 homologue of M. fructicola was amplified by PCR with the primer pair yAP‐5n and yAP‐6n using a KOD‐Plus polymerase (Toyobo, Kita‐ku, Osaka, Japan). The product obtained was cloned into pGEM‐T easy vector (Promega, Madison, WI, USA) for sequencing. The 5′ and 3′ flanking sequences were obtained by inverse‐PCR with two primer pairs, 3′_nest_primer_F/3′_nest_primer_R and 5′_nest_primer_F/5′_nest_primer_R, from BamHI‐ and EcoRI‐digested and self‐ligated genomic DNA. The DNA sequences obtained were assembled and blasted against the NCBI database using the blastn program. The identification of the exons was determined by comparison of MfAP1 genomic DNA and cDNA sequences. A 1.8‐kb MfAP1 cDNA fragment was amplified using the primer pair MfAP1_cDNA_F and MfAP1_cDNA_R. Phylogenetic analysis of MFAP1 and other AP1‐like proteins was performed with PHYLIP (Workbench, San Diego Supercomputer Center (SDSC), UC San Diego, San Diego, CA, USA) using full‐length amino acid sequences. A phylogenetic tree was generated with the TreeView program available at http://taxonomy.zoology.gla.ac.uk/rod/treeview.html. Functional domains of MFAP1 were predicted using PROSITE (http://prosite.expasy.org), InterPro (http://www.ebi.ac.uk/interpro/) or Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan) programs.

The genes encoding G6PD (MfG6PD1) were obtained from M. fructicola genomic DNA by PCR with degenerate primers G6PD‐1 and G6PD‐R (Table S1). The PCR products were cloned and sequenced. The similarity of the amplified fragments was determined by searching against the databases at NCBI using the blastx program. The deduced amino acids of each gene were aligned with published sequences using ClustalW. Functional domains were predicted using the PROSITE program.

Vector construction and fungal transformation

Gene silencing vector pSilent‐Dual1, which contains a multiple cloning site within two opposite promoters (gpd and trpC), kindly provided by Dr Nakayashiki (Kobe University, Kobe, Japan; Nguyen et al., 2008), was used to generate the MfAP1 silencing vector pSDMfAP1. Briefly, a 584‐bp MfAP1 ORF fragment was amplified using the primer pair Yap1_cDNA_F and Yap1_cDNA_R (Table S1). The PCR fragment was ligated to EcoRV‐digested pSilent‐Dual1 to form the MfAP1 silencing construct (Pgpd‐MfAP1 partial ORF‐PtrpC), resulting in pSDMfAP1 (Fig. S12a, see Supporting Information). pSDMfAP1 was transferrred into M. fructicola via PEG‐mediated protoplast transformation. To transfer the MfAP1 silencing construct into M. fructicola by ATMT, pBHt2SDAP1 (Fig. S12b) was constructed by ligating the MfAP1 silencing construct into SmaI‐digested binary vector pBHt2 (Mullins et al., 2001). For the construction of MfAP1 overexpression vector, a 3‐kb fragment, including a 0.7‐kb native promoter, a MfAP1 ORF and a 0.3‐kb terminator, was amplified using a high‐fidelity KOD polymerase with the primer pair EcoRI_MfAP1_P_F and EcoRI_MfAP1_T_R (Table S1). The amplified fragment (∼3 kb) was cloned into EcoRI‐digested pBHt2 carrying a hygromycin resistance gene for fungal transformant selection to generate pMfAP1.

To perform PEG‐mediated protoplast transformation, M. fructicola protoplasts were prepared (Lee et al., 2010). The freshly prepared protoplasts (200 μL, 1 × 108/mL) were mixed gently with 5 μg of ApaI‐linearized pSDMfAP1 and 10 μL of 50 mm spermidine, and incubated on ice for 20 min. A 1.25‐mL PEG solution (40% PEG, 50 mm Tris, pH 8.0, and 50 mm CaCl2) was then added to the protoplast–DNA mixture and incubated at room temperature for 25 min. The PEG–protoplast–DNA mixture (100 μL) was spread onto a 9‐cm Petri dish containing 20 mL of SH agar medium (0.6 M sucrose, 5 mM HEPES, pH 5.3, 1 mM NH4H2PO4 and 1.2% agar) (Lee et al., 2010), and incubated at 23 oC in the dark for 3–5 h for protoplast regeneration. To screen the transformants, 10 mL of SH agar medium with G418 sulfate was placed on the protoplast layer to bring the final concentration of G418 sulfate to 75 μg/mL. Hyphal tips from individual colonies were transferred to plates containing potato dextrose agar (PDA; Difco Co.) and 75 μg/mL G418 sulfate (PDAG75). Three continuous single hyphal tip transfers and three runs of single spore isolations on PDAG75 plates were conducted to purify the transformants. pBHt2SDAP1 and pMfAP1 were introduced into the M. fructicola MUK‐1 strain through ATMT using Agrobacterium tumefaciens strain EHA105. The transformation was conducted according to the protocol described by Lee and Bostock (2006a) with slight modifications. Briefly, the spore suspension was mixed with bacterial cells, and the mixture was spread onto a Whatman nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) placed on top of an inducing medium agar plate. After incubation for 3 days, the membranes were transferred onto PDA containing 100 μg/mL hygromycin, 50 μg/mL cephalexin and 200 μg/mL cefotaxime. Membranes were removed after a 7‐day incubation period and fungal hyphae that had grown into the agar medium were picked and transferred to PDA containing 100 μg/mL hygromycin. All transformants were purified by streaking three times on the medium for single spore isolation.

DNA extraction and Southern blot analysis

Monilinia fructicola strains were cultured on V8 agar medium for 7 days, and DNA was extracted as described previously (Lee et al., 2010). Integration of the hygromycin resistance cassette in fungal transformants was verified by PCR with the primer pair hygT and hygR, and confirmed further by Southern blotting. Genomic DNA was digested with EcoRV (transferred with pBHt2SDAP1) or BamHI (transferred with pMfAP1), electrophoresed in an agarose gel, blotted onto a nylon membrane and hybridized with a digoxigenin‐labelled probe. The probe was labelled with digoxigenin‐11‐dUTP during PCR using the primer set Yap1_cDNA_F and Yap1_cDNA_R (Table S1). Hybridization and detection using the DIG High Prime DNA Labelling and Detection Starter Kit II (Roche Applied Science, Mannheim, Germany) were conducted according to the instruction manual.

T‐DNA insertion site identification

The T‐DNA insertion site of transformants was identified with inverse‐ and nested‐PCR. The T‐DNA flanking sequences were amplified by primers LB1 and MfAP1_cDNA_F from XhoI‐digested and self‐ligated genomic DNA. The PCR products were then diluted and amplified using the nested primers LB2 and N_MfAP1_RT_F (Table S1). The resulting DNA fragments were cloned and sequenced. The similarity of the amplified fragments was determined by searching against the databases at NCBI using the blastn and blastx programs.

RNA extraction and qRT‐PCR

Total RNA was extracted from mycelia as described previously (Lee et al., 2010). Total RNA from inoculated plant tissues was isolated using a Plant Total RNA Miniprep Purification Kit (Genemark, Taichung, Taiwan), treated with RQ1 RNase‐free DNase (Promega), and cDNA was synthesized with Oligo‐dT (5′‐(T)25VN‐3′) using the MMLV High Performance Reverse Transcriptase (Epicentre, Madison, WI, USA). The primer pairs (Table S1) for qRT‐PCR were designed using the online resource (http://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10001&tab=Assay±Design±Center&identifier=Universal±Probe±Library&langId=‐1). qRT‐PCR was performed using 5X HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne, Tartu, Estonia) with a Rotor‐Gene Q PCR machine (QIAGEN, Valencia, CA, USA). The gene encoding β‐tubulin was employed as an internal control to determine the relative expression of target genes using the comparative CT method (Schmittgen and Livak, 2008). The primers designed for qRT‐PCR are specific for the fungal genes.

Spore germination, appressorium formation and fungal growth assay

A spore suspension (5 × 105 spores/mL) was prepared in a 1 mm sucrose solution, and 80 μL of spore–sucrose mixture was loaded into each well of a 96‐well plate. Spore germination and appressorial formation were examined under a light microscope at 6 h post‐incubation. Fungal growth was detected by transferring a mycelial disc to the centre of a 9‐cm PDA plate. The diameter of each colony was measured and analysed. To study MfAP1 expression during fungal growth, 50‐mL centrifuge tubes, each containing 20 mL of 1 mm sucrose solution, were prepared and inoculated with spore suspension to a final concentration of 5 × 105 spores/mL. At each time point, fungal mycelia were collected by centrifugation for RNA extraction. For appressorium formation, 5 µL of spore suspension containing 1 mm sucrose were inoculated on a Petri dish and incubated for 6 h. The droplets were collected for RNA isolation. Fungal biomass was detected with a LabSystems 352 Multiskan MS Microplate Reader (Thermo Scientific, Waltham, MA, USA) at a wavelength of 550 nm. Experiments were repeated at least twice with three replicates each time.

Pathogenicity assay

Flower petals collected from Rosa cv. Flaming Peace or Prunus persica cv. Tian Tao were inoculated with 5 μL of spore suspension (105 spores/mL) prepared from the wild‐type strain or transformants by paired inoculation (Lee et al., 2010). Brown rot lesion size was measured at 40 or 48 h post‐inoculation (Lee et al., 2010). Experiments were repeated at least three times.

ROS detection

Spore suspensions prepared from the wild‐type and transformants were incubated in a 96‐well plate containing sucrose solution to make a final concentration of sucrose of 1 mm and spores of 5000 spores/well. For in planta assays, spore suspensions were inoculated onto rose petals, one strain on one side of a petal, 30 μL (3000 spores/drop) for each inoculation drop, and two drops for one strain on one side. For ROS detection, 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) solution was applied to make a final concentration of 60 μm at each time point, and the petals or 96‐well plates were incubated at 37°C for an additional 30 min. A total of 50 μL from two inoculation drops was recovered and placed into a 96‐well plate for fluorescence detection. The fluorescence of DCF was measured with a multimode reader (Infinite M200; Tecan Group, Mannedorf, Switzerland) with excitation and emission at 485 and 528 nm, respectively. Experiments were repeated at least twice with three replicates in each experiment.

MfAP1 expression of fungal strains in response to H2O2

The spore suspension (2 × 105 conidia) prepared from the transformants or wild‐type strain was incubated on a 9‐cm Petri dish containing 20 mL of 10‐fold‐diluted potato dextrose broth (PDB; Difco, Spark, MD, USA) and incubated for 16 h. H2O2 was added to each plate to a final concentration of 1 mm. Mycelia were collected for RNA isolation after the plates had been incubated for an additional 48 h. Experiments were repeated at least twice with three replicates in each experiment.

Sequence information

Sequence data can be found in the EMBL/GenBank data libraries: MfAP1, accession no. KT119835; MfG6PD1, accession no. AFK82641.1; T‐DNA insertion in transformant A4‐4, accession no. KT119836; T‐DNA insertion in transformant E4‐1, accession no. KT119837.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Fig. S1 Functional domains in the N‐terminus (a) and C‐terminus (b) of MFAP1. Amino acid sequence of MFAP1 aligned with the YAP1‐like proteins from Botryotinia fuckeliana (BAP1, CAX15423.1), Magnaporthe oryzae (MoAP1, XP_001408783.1), Aspergillus fumigatus (AfAP1, EAL88844), Cochliobolus heterostrophus (ChAP1, ASS64313) and Alternaria alternata (AaAP1, ACM50933.1). The basic leucine zipper domain (bZIP) is boxed with a full line and the cysteine‐rich domain (CRD) is boxed with a broken line. The putative nuclear export sequence (NES) is also indicated.

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Fig. S2 Phylogenetic relationship of MFAP1 to other AP1 family proteins.

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Fig. S3 Partial sequence alignment of MFG6PD1 with glucose‐6‐phosphate dehydrogenase (G6PD) family proteins from Botryotinia fuckeliana (anamorph Botrytis cinerea; XP_001553624.1), Sclerotinia sclerotiorum (XP_001586308.1), Maganaporthe oryzae (XP_003710026.1), Aspergillus flavus (XP_002373576.1), Neurospora crassa (XP_958320.3) and Saccharomyces cerevisiae (AJT06396.1). G6PD active site is boxed.

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Fig. S4 Growth curve of Monilinia fructicola wild‐type strain in 1 mm sucrose solution. hpi, hours post‐inoculation; OD550, optical density at 550 nm.

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Fig. S5 Growth (a, c, e) and spore germination (b, d) of MfAP1‐silenced transformants (a, b, e) and transformants carrying extra copies of the MfAP1 gene (c, d). WT, wild type.

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Fig. S6 Southern blot analysis of MfAP1 transformants with a 0.5‐kb MfAP1 open reading frame (ORF) fragment probe.

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Fig. S7 Spore germination, appressorial formation and germ tube elongation of Monilinia fructicola wild‐type strain (a) and MfAP1‐overexpressing strain A4‐4 (b) on rose petals at 6 h post‐inoculation (hpi). ap, appressorium; gt, germ tube; s, spore. Bar, 10 μm.

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Fig. S8 Accumulation of reactive oxygen species (ROS) on rose petals inoculated with Monilinia fructicola wild‐type (WT) strain and MfAP1‐silenced strain B15 (a) or I2 (b) at various time points. hpi, hours post‐inoculation.

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Fig. S9 MfAP1 expression of the MfAP1‐overexpressing strain (A4‐4) and wild‐type (WT) strain in axenic culture amended with H2O2. The fold change in MfAP1 expression in response to H2O2 treatment was calculated by comparison with that of the control treatment (H2O) (a). The fold change in MfAP1 expression in the A4‐4 strain was calculated by comparison with that of the wild‐type strain (b).

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Fig. S10 Relative expression of MfCUT1 in Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4). The fold change in MfCUT1 relative expression of the A4‐4 strain was calculated by comparison with that of the wild‐type strain.

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Fig. S11 Relative expression of MfGR1 (a), MfGPx1 (b) and MfG6PD1 (c) in Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4). The fold change in the relative expression of the target gene of the A4‐4 strain was calculated by comparison with that of the wild‐type strain.

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Fig. S12 Map of MfAP1 silencing vectors pSD‐MfAP1 (a) and pBHt2SDAP1 (b). The MfAP1 silencing construct is marked with two arrows.

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Table S1 Oligonucleotide primers used in this study.

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Acknowledgements

The authors thank Drs R. M. Bostock (University of California at Davis, USA) and K.‐R. Chung (National Chung‐Hsing University, Taiwan) for their helpful comments and suggestions. Research was supported by grants (NSC‐102‐2911‐I‐005‐301, NSC‐103‐2911‐I‐005‐301, MOST‐104‐2911‐I‐005‐301 and MOST‐105‐2911‐I‐005‐301) from the Ministry of Science and Technology, Taiwan to M‐HL, and by the Ministry of Education, Taiwan, under the ATU plan.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Fig. S1 Functional domains in the N‐terminus (a) and C‐terminus (b) of MFAP1. Amino acid sequence of MFAP1 aligned with the YAP1‐like proteins from Botryotinia fuckeliana (BAP1, CAX15423.1), Magnaporthe oryzae (MoAP1, XP_001408783.1), Aspergillus fumigatus (AfAP1, EAL88844), Cochliobolus heterostrophus (ChAP1, ASS64313) and Alternaria alternata (AaAP1, ACM50933.1). The basic leucine zipper domain (bZIP) is boxed with a full line and the cysteine‐rich domain (CRD) is boxed with a broken line. The putative nuclear export sequence (NES) is also indicated.

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Fig. S2 Phylogenetic relationship of MFAP1 to other AP1 family proteins.

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Fig. S3 Partial sequence alignment of MFG6PD1 with glucose‐6‐phosphate dehydrogenase (G6PD) family proteins from Botryotinia fuckeliana (anamorph Botrytis cinerea; XP_001553624.1), Sclerotinia sclerotiorum (XP_001586308.1), Maganaporthe oryzae (XP_003710026.1), Aspergillus flavus (XP_002373576.1), Neurospora crassa (XP_958320.3) and Saccharomyces cerevisiae (AJT06396.1). G6PD active site is boxed.

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Fig. S4 Growth curve of Monilinia fructicola wild‐type strain in 1 mm sucrose solution. hpi, hours post‐inoculation; OD550, optical density at 550 nm.

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Fig. S5 Growth (a, c, e) and spore germination (b, d) of MfAP1‐silenced transformants (a, b, e) and transformants carrying extra copies of the MfAP1 gene (c, d). WT, wild type.

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Fig. S6 Southern blot analysis of MfAP1 transformants with a 0.5‐kb MfAP1 open reading frame (ORF) fragment probe.

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Fig. S7 Spore germination, appressorial formation and germ tube elongation of Monilinia fructicola wild‐type strain (a) and MfAP1‐overexpressing strain A4‐4 (b) on rose petals at 6 h post‐inoculation (hpi). ap, appressorium; gt, germ tube; s, spore. Bar, 10 μm.

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Fig. S8 Accumulation of reactive oxygen species (ROS) on rose petals inoculated with Monilinia fructicola wild‐type (WT) strain and MfAP1‐silenced strain B15 (a) or I2 (b) at various time points. hpi, hours post‐inoculation.

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Fig. S9 MfAP1 expression of the MfAP1‐overexpressing strain (A4‐4) and wild‐type (WT) strain in axenic culture amended with H2O2. The fold change in MfAP1 expression in response to H2O2 treatment was calculated by comparison with that of the control treatment (H2O) (a). The fold change in MfAP1 expression in the A4‐4 strain was calculated by comparison with that of the wild‐type strain (b).

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Fig. S10 Relative expression of MfCUT1 in Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4). The fold change in MfCUT1 relative expression of the A4‐4 strain was calculated by comparison with that of the wild‐type strain.

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Fig. S11 Relative expression of MfGR1 (a), MfGPx1 (b) and MfG6PD1 (c) in Monilinia fructicola wild‐type (WT) strain and MfAP1‐overexpressing strain (A4‐4). The fold change in the relative expression of the target gene of the A4‐4 strain was calculated by comparison with that of the wild‐type strain.

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Fig. S12 Map of MfAP1 silencing vectors pSD‐MfAP1 (a) and pBHt2SDAP1 (b). The MfAP1 silencing construct is marked with two arrows.

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Table S1 Oligonucleotide primers used in this study.

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