The CHK1 MAPK pathway is crucial in appressorium formation and is highly conserved among plant pathogenic fungi. Here, we investigated a putative upstream regulator of this pathway, BmOPY2, in the maize pathogen Bipolaris maydis. Yeast two-hybrid analysis confirmed the interaction between BmOPY2 and BmSTE50, suggesting that BmOPY2 functions as an upstream regulator of the CHK1 MAPK pathway. To investigate the role of BmOPY2 in appressorium formation, we generated BmOPY2-disrupted (∆BmOPY2) mutants. These mutants formed appressoria normally on maize leaves, but did not form them on plastic Petri dishes. This suggests that BmOPY2 regulates appressorium formation via hydrophobic surface recognition but not via recognition of host-derived chemicals. Plant waxes or cutin monomers are recognized by other fungal pathogens, but these substances failed to restore appressorium formation in ∆BmOPY2 mutants. In comparison with the wild type, the ∆BmOPY2 mutants showed increased appressorium formation on intercellular spaces of maize leaves, suggesting that pectin―a component of these spaces―may promote this process. The addition of pectin restored appressorium formation by ∆BmOPY2 mutants on plastic surfaces. These findings reveal a novel dual regulation of appressorium formation in B. maydis, involving both BmOPY2-mediated hydrophobic surface recognition and a distinct pectin-dependent pathway.

Many plant pathogenic fungi develop specialized structures called appressoria, which are essential for penetrating host tissues during infection. The formation of appressoria is triggered by specific extracellular cues, enabling pathogens to recognize host plants. In the rice blast fungus Pyricularia oryzae, the PMK1 MAP kinase and its upstream regulators, MST7 MAPKK and MST11 MAPKKK, were first identified as essential for appressorium development (Park et al., 2006; Xu & Hamer, 1996). The adaptor protein MST50 directly binds to MST11 and MST7 and facilitates activation of the PMK1 pathway and initiation of appressorium formation (Park et al., 2006). Signaling mucin MoMSB2 functions upstream of MST50 and recognizes physical surface properties, particularly hydrophobicity. The ΔMoMSB2 mutant has severely impaired appressorium formation on hydrophobic surfaces, but plant wax components can substantially restore appressorium formation (Liu et al., 2011). These data strongly suggest the existence of two distinct signaling pathways upstream of MST50 that regulate appressorium formation: one that involves MoMSB2, which recognizes hydrophobic surface properties, and the other one that responds to plant-derived chemical signals, specifically plant waxes (Liu et al., 2011).

Orthologs of PMK1, including CMK1 in Colletotrichum orbiculare (syn. C. lagenarium) (Takano et al., 2000), CHK1 in Bipolaris maydis (syn. Cochliobolus heterostrophus) (Lev et al., 1999), and BMP1 in Botrytis cinerea (Zheng et al., 2000), are essential in appressorium formation. These model plant pathogens represent diverse ascomycete taxa: Sordariomycetes (P. oryzae and C. orbiculare), Dothideomycetes (B. maydis), and Leotiomycetes (B. cinerea). This suggests that this MAP kinase pathway is universally required for appressorium formation across ascomycetes. However, except for the rice blast fungus, the upstream regulators of this conserved MAP kinase pathway remain largely unknown, and it is still unclear what extracellular cues trigger appressorium formation.

Bipolaris maydis is the causal agent of Southern corn leaf blight disease in maize. It requires the CHK1 MAP kinase cascade for infection. We have demonstrated that CHK1 and the upstream components (BmSTE7, BmSTE11, and the adaptor protein BmSTE50, which are orthologs of the PMK1 pathway components) are essential for appressorium formation (Izumitsu et al., 2009; Kitade et al., 2015; Sumita et al., 2020).

In this study, we investigated BmOPY2, an ortholog of the budding yeast OPY2 and a putative upstream regulator of BmSTE50. In Saccharomyces cerevisiae, OPY2 is a membrane-anchored protein that functions as a scaffold or anchor in the HOG and filamentous growth MAPK pathways, interacting with Ste50 to mediate signal transduction (Yamamoto et al., 2010). Although the mutants lacking BmOPY2 did not form appressoria on artificial hydrophobic surfaces, they formed them profusely on host maize leaves. We discovered that pectin, a common plant cell wall component, efficiently restored appressorium formation on hydrophobic surfaces in these mutants. These results suggest the existence of two distinct signaling pathways upstream of BmSTE50 in B. maydis: one is mediated by BmOPY2, which recognizes hydrophobic surface, and the other one functions independently to recognize host-derived chemical signals, specifically pectin.

We used B. maydis HITO7711 (MAT1-2) as the wild-type strain. Other B. maydis strains are listed in Table 1. All cultures of B. maydis were maintained on complete medium agar (CMA: Tanaka et al., 1991) or V8 juice agar medium (V8A; Ribeiro, 1978) at 25 °C.

A BmOPY2 gene disruption cassette was constructed using the PCR fusion method (Izumitsu et al., 2009). Genomic DNA from HITO7711 was used as a template. The 5′ flanking region was amplified with primers BmOPY2-fusion-f1 and BmOPY2-fusion-r1. The 3′ flanking region was amplified with primers BmOPY2-fusion-f2 and BmOPY2-fusion-r2. The hygromycin resistance (HPH) marker was amplified from the pCB1004 plasmid with primers pCB1004-HPH-f1 and pCB1004-HPH-r1. To construct a gene disruption cassette containing the three fused fragments, we mixed these three amplified fragments and used the mixture as a template for a subsequent PCR with primers BmOPY2-fusion-f1 and BmOPY2-fusion-r2. The amplified gene disruption cassette was concentrated by ethanol precipitation.

To construct the complementation plasmid pBmOPY2C, the full-length BmOPY2 gene was PCR-amplified from genomic DNA of HITO7711 with primers BmOPY2-comp-f1 and BmOPY2-fusion-r2, and ligated into the EcoRV site of the pZNAT1 plasmid (Sumita et al., 2020). The protoplast-PEG method was used to transform each of the constructs, as previously reported (Izumitsu et al., 2007). All primers used in this study are summarized in Supplementary Table S1.

Conidia (10⁵ conidia/mL) were (i) cultured on φ55×17 mm plastic Petri dishes (AS ONE Corporation, Osaka, Japan; product no. 1-8549-02) and observed under an inverted microscope or (ii) inoculated onto maize leaves (Zea mays ‘Takanestar’) for 18 h, and the leaves were stained with lactophenol cotton blue solution, then decolorized (Sumita et al., 2017) and observed under a light microscope. (iii) Beeswax and candelilla wax (both from Tree of Life Co., Ltd., Gifu, Japan) were directly applied onto plastic Petri dishes. (iv) Citrus- and apple-derived pectin powders (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were each dissolved in either tap or distilled water using a microwave; the corresponding water without pectin served as a control. (v) Leaf segments were immersed into methanol or hexane in glass Petri dishes for 1 h, removed, and dried on filter paper. The extracts were left to evaporate for several hours.

Maize plants were grown in pots under room conditions at 25 °C in 14 h light/10 h dark for 3 wk. Leaves were harvested and placed on a paper towel soaked with water in a polystyrene box. The leaves were inoculated with conidial suspensions (10⁵ conidia/mL) prepared from 2-wk-old colonies of B. maydis strains grown on V8A.

The Matchmaker Two-Hybrid System (Clontech, Carlsbad, CA, USA) was used according to the manufacturer’s instructions. Mycelia of B. maydis (strain HITO7711) were cultured in complete medium (CM) broth for 2 d at 25 °C, harvested by filtration, and immediately frozen in liquid nitrogen. The frozen mycelia were ground to a fine powder using a pre-chilled mortar and pestle. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and treated with DNase I (Thermo Fisher Scientific) to remove genomic DNA contamination. First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) with oligo(dT) primers. Full-length BmSTE50 was used as bait, and the cytoplasmic domain of BmOPY2 (amino acids 168-485, containing the CR-A domain) served as prey. The respective plasmid vectors were constructed using the In-Fusion HD Cloning Kit (Clontech). Coding sequences of BmSTE50 and BmOPY2 were amplified by PCR from cDNA using primer pairs BmSte50-bait-f1/BmSte50-bait-r1 and BmOpy2-prey-f1/BmOpy2-prey-r1, respectively. The PCR amplicons containing BmSTE50 and BmOPY2 coding sequences were cloned into the pGBKT7 DNA-BD vector (Clontech) and pGADT7 AD vector (Clontech), respectively, both pre-digested with BamHI and EcoRI. The resulting plasmids (pBaitBmSte50 and pPreyBmOpy2) were transformed into S. cerevisiae strains Y2HGold and Y187, respectively, using the lithium acetate transformation method (Akada et al., 2000). Transformants were selected on synthetic defined (SD) medium lacking tryptophan (SD/-Trp) for pBaitBmSte50 or lacking leucine (SD/-Leu) for pPreyBmOpy2. The transformed strains were mated, and the resulting diploids were selected on SD/-Trp/-Leu plates and grown on SD/-Trp/-Leu/-Ade/-His plates and SD/-Trp/-Leu plates supplemented with Aureobasidin A (+AbA). Vectors for positive and negative controls included in the Matchmaker system were used: the interaction between murine p53 and SV40 large T-antigen (pGBKT7-53 and pGADT7-T) served as a positive control, while human lamin C and SV40 large T-antigen (pGBKT7-Lam and pGADT7-T) were used as a negative control.

Statistical analyses were conducted using R software (version 4.4.2). A one-way analysis of variance (ANOVA) was performed to determine significant differences among groups. Post-hoc multiple comparisons were carried out using Tukey’s Honest Significant Difference (HSD) test. A p-value of less than 0.05 was considered statistically significant.

In BLAST analysis, we used the S. cerevisiae OPY2 sequence as a query and identified its ortholog in B. maydis, which we designated BmOPY2. It exists as a single copy in the B. maydis genome; the encoded protein of 485 amino acids is similar to that encoded by S. cerevisiae OPY2 and contains one transmembrane domain and one OPY2-specific conserved domain (IPR018571: Membrane anchor Opy2, N-terminal) at its N terminus (Supplementary Fig. S1). It also contains a CR-A domain; this domain constitutively binds to STE50 in S. cerevisiae (Yamamoto et al., 2010).

To elucidate the function of BmOPY2, we generated null mutants through homologous recombination using HPH marker. Gene disruption was verified by PCR analysis using primers that annealed to regions flanking the disruption cassette and within the HPH marker (Supplementary Fig. S2). Three independent null mutants (∆BmOPY2) were established and designated DOPY2-1, DOPY2-2, and DOPY2-3. A complemented strain (COPY2-1) was generated by ectopically introducing a BmOPY2-containing plasmid, pBmOPY2C, into DOPY2-1.

The colony morphology of the ∆BmOPY2 strains was indistinguishable from that of the wild type (Fig. 1). No significant differences were detected in radial growth or conidiation among the wild-type strain, null mutants, and the complemented strain (Table 2). These results demonstrate that BmOPY2 is dispensable for vegetative growth and asexual development.

Fig. 1 - Colonies of the wild-type, ∆BmOPY2, and the complemented strain. Strains were cultured on CMA medium for 4 d.

We next investigated the role of BmOPY2 in appressorium formation and pathogenicity. The ∆BmOPY2 mutants did not form appressoria on plastic Petri dishes, whereas both the wild-type and complemented strains formed appressoria (Figs. 2A, 3). The complemented strain showed slightly reduced appressorium formation compared to the wild-type, possibly due to ectopic integration of the BmOPY2 gene rather than insertion at the native locus. These findings suggest that BmOPY2 is crucial for appressorium formation on artificial surfaces. In pathogenicity assays on maize leaves (the host plant), the ∆BmOPY2 mutants produced distinct disease lesions, albeit slightly smaller than those caused by the wild-type and the complemented strain (Fig. 4A). Examination of appressorium formation on host leaves revealed that, in contrast to their behavior on plastic surfaces, the ∆BmOPY2 mutants formed appressoria at rates comparable to that of the wild-type strain (Figs. 2B, 3). These results indicate that while BmOPY2 is essential for appressorium formation on simple hydrophobic surfaces such as plastic, the host leaf surface likely provides additional chemical cues (e.g., plant-derived compounds or cuticular waxes) beyond hydrophobicity that can trigger appressorium formation.

Fig. 2 - Micrographs of appressorium formation in the wild-type, ∆BmOPY2, and the complemented strain. A: Strains cultured on plastic Petri dishes for 4.5 h. B: Strains cultured on maize leaves for 6 h. Arrows indicate appressoria.

Fig. 3 - Quantification of appressorium formation in the wild-type, ∆BmOPY2, and the complemented strains. White bars represent appressorial formation on plastic petri dishes; black bars represent appressorial formation on maize leaves. Error bars represent standard error of the mean (SEM). Different lowercase letters indicate statistically significant differences among groups (Tukey’s HSD test, p < 0.05).

Fig. 4 - Pathogenicity of the wild-type, ∆BmOPY2, and complemented strains in inoculation tests. A: Non-wounded maize leaves. B: Wounded maize leaves.

To distinguish between penetration defects and post-penetration functions, we assessed whether mechanical damage would affect the infection process. In wound inoculation tests, the ∆BmOPY2 mutants consistently produced smaller lesions than both the wild-type and complemented strains (Fig. 4B). These results suggest that BmOPY2 functions not only in appressorium formation on hydrophobic surfaces but also in post-penetration development during host infection.

To identify the substances in maize leaves that induced appressorium formation in the ΔBmOPY2 mutants, we first investigated those reported in other plant pathogenic fungi. Plant wax components such as beeswax and 1-triacontanol induce appressoria in P. oryzae (Gilbert et al., 1996; Liu et al., 2011), and cutin monomers serve as host-derived appressorium-inducing factors in both P. oryzae and C. orbiculare (Gilbert et al., 1996; Kamakura et al., 2002; Kodama et al., 2017). However, none were able to restore appressorium formation by the ΔBmOPY2 mutants on plastic Petri dishes (Fig. 5; Supplementary Fig. S3). These results suggested that B. maydis likely responds to distinct host-derived chemical signals for appressorium formation.

Fig. 5 - Effects of known plant-derived appressorium-inducing substances (plant waxes or cutin monomers) on appressorium formation in a ∆BmOPY2 strain on plastic plates. None of these substances restored appressorium formation by ∆BmOPY2. Error bars represent standard error of the mean (SEM).

To investigate whether appressorium-inducing factors are conserved across plant species, we examined appressorium formation on leaves from diverse plants, including monocots (wheat) and dicots (cucumber, eggplant, and taro). A ΔBmOPY2 mutant formed appressoria on leaves of all plants tested (Supplementary Fig. S4). These results suggest that the chemical factors inducing appressorium formation are present across diverse plant species.

We next attempted to extract appressorium-inducing compounds using solvents of different polarities: water (polar), methanol (moderately polar), and hexane (non-polar). None of these extracts restored appressorium formation by the ΔBmOPY2 mutant on plastic Petri dishes (Supplementary Fig. S5). Interestingly, the mutant readily formed appressoria on the air-dried residual leaf material following extraction (right panels). These findings suggest that the appressorium-inducing compounds are not readily extractable with hexane, methanol or water. Since plant wax components are typically well-extracted by methanol, these results further supported our hypothesis that the appressorium-inducing factors are distinct from plant waxes.

Although both wild-type and complemented strains showed a preference for appressorium formation over the middle lamella (the intercellular spaces functioning as an adhesive interface), this tendency was notably enhanced in the ΔBmOPY2 mutants (Fig. 6). This observation suggested that components within these spaces induce appressorium formation. Pectin is the primary constituent of the middle lamella in plants (Baba & Verger, 2024). In Poaceae species, including maize, the pectin content in leaf cell walls outside the middle lamella (2-10% of cell wall dry weight) is much lower than in dicotyledonous plants (30-35%) (Mohnen, 2008). Therefore, we hypothesized that pectin serves as the appressoria-inducing compound in B. maydis.

Fig. 6 - Appressorium formation by ∆BmOPY2 on the intercellular spaces of maize leaves. A: Quantification of the appressoria formed on the intercellular spaces relative to the total appressoria formed. B: Micrographs and schematic illustration of appressoria formed on the intercellular spaces and those formed elsewhere. Error bars represent standard error of the mean (SEM). Different lowercase letters indicate statistically significant differences among groups (Tukey’s HSD test, p < 0.05).

In a preliminary experiment, we dissolved citrus- and apple-derived pectins in tap water at a concentration of 0.1% using microwave heating. Both pectins strongly restored appressorium formation in the ΔBmOPY2 mutant on plastic Petri dishes (Fig. 7). This observation strongly supported our hypothesis that pectin serves as a host-derived appressorium-inducing compound. Intriguingly, when pectin from either source was dissolved in distilled water, the restoration effect was either diminished or completely absent (data not shown). This suggested that one or more minerals (ions) present in tap water are essential for appressorium formation.

Fig. 7 - Recovery of appressorium formation by ∆BmOPY2 on plastic Petri dishes by pectin. A: Micrographs. Arrows indicate appressoria. B: Quantification of appressorium formation in the wild-type, ∆BmOPY2, and complemented strains with or without pectin. Tap water was used to dissolve pectin. Error bars represent standard error of the mean (SEM). Different lowercase letters indicate statistically significant differences among groups (Tukey’s HSD test, p < 0.05).

Pectin molecules are known to gel through cross-linking with Ca²⁺: without Ca²⁺, they exhibit very low gelling capacity. Pectin, naturally present in the plant cell wall and middle lamella, is generally in a gelled state (Daher & Braybrook, 2015). We hypothesized that Ca²⁺ ions in tap water are essential for pectin gelling and the induction of appressorium formation. When 0.1% citrus-derived pectin and Ca²⁺ were added to distilled water, the appressorium formation ability of a ΔBmOPY2 mutant was significantly restored (Figs. 8, 9). Neither Ca²⁺ nor pectin alone induced this recovery (Fig. 9). These findings strongly suggest that gelled pectin acts as an appressorium formation-inducing factor derived from the host. These findings revealed that gelled pectin is needed for appressorium formation in B. maydis.

Fig. 8 - Recovery of appressorium formation by ∆BmOPY2 on plastic plates by pectin + CaCO₃ in distilled water. Arrows indicate appressoria.

Fig. 9 - Quantification of appressorium formation by the wild-type, ∆BmOPY2, and complemented strains on plastic plates. Pectin, CaCO₃, or both were added to distilled water. Error bars represent standard error of the mean (SEM). Different lowercase letters indicate statistically significant differences among groups (Tukey’s HSD test, p < 0.05).

The CHK1 MAPK pathway is essential for appressorium formation in B. maydis and is regulated by the adaptor protein BmSTE50 (Izumitsu et al., 2009; Kitade et al., 2015). In S. cerevisiae, OPY2 directly interacts with STE50 (Yamamoto et al., 2010). To investigate whether BmOPY2 interacts with BmSTE50, we performed a yeast two-hybrid assay using full-length BmSTE50 as bait and the cytoplasmic domain of BmOPY2 (amino acids 168-485, including CR-A domain) as prey. In this assay, a yeast diploid strain containing both constructs was able to grow on SD/-Trp/-Leu/-Ade/-His plates and SD/-Trp/-Leu/+Aba plates, as did the positive control (Fig. 10). The results suggest that BmOPY2 functions upstream of BmSTE50, directly interacts with it, and is involved in hydrophobic surface recognition and direct interaction with it.

Fig. 10 - Yeast two-hybrid analysis of the interaction between BmSte50 and BmOPY2. Yeast cells (10-fold serial dilution; 4 mL per dilution) were spotted on plates to examine the expression of the reporter genes Ade2, His3, and AUR1-C. Diploid yeast strains contained both pGBKT7-53 and pGADT7-T (positive control) or both pGBKT7-Lam and pGADT7-T (negative control; all plasmid vectors from Clontech).

We then investigated whether pectin treatment could restore appressorium formation in the ΔBmSTE50 mutant and downstream mutants of the CHK1 MAPK pathway, specifically ΔCHK1 and ΔBmSTE11 mutants. Since these mutants produce few conidia, we examined appressorium formation on hyphal cells. Pectin failed to restore appressorium formation on plastic plates by these mutants (Fig. 11). These results strongly suggest the existence of two distinct recognition pathways upstream of the CHK1 MAPK pathway: one involving OPY2-mediated physical surface (hydrophobic surface) recognition and another involving pectin recognition through unknown factors (Fig. 12).

Fig. 11 - Effect of pectin on appressorium formation by CHK1 MAPK pathway mutants on plastic Petri dishes. Unlike the ∆BmOPY2 mutant, the ∆BmSte50, ∆BmSTE11, and ∆CHK1 mutants did not form appressoria regardless of pectin supplementation. Arrows indicate appressoria. All conditions use pectin dissolved in tap water.

Fig. 12 - Model of signal transduction pathways for host plant recognition and appressorium formation by Bipolaris maydis as inferred from this and previous studies (Izumitsu et al., 2009; Kitade et al., 2015; Sumita et al., 2019).

In this study, we investigated BmOPY2, a putative upstream regulator of the CHK1 MAPK pathway, which is essential for appressorium formation in B. maydis. BmOPY2 contains a CR-A domain, which constitutively binds to STE50 in S. cerevisiae (Yamamoto et al., 2010). Our yeast two-hybrid analysis confirmed the interaction between BmOPY2 and BmSTE50, indicating that BmOPY2 is an upstream regulator of the CHK1 MAPK pathway in B. maydis.

The ΔBmOPY2 mutants did not form appressoria on plastic Petri dishes. However, they maintained nearly wild-type levels of appressoria on plant surfaces and had only slightly reduced pathogenicity toward maize. This phenotype distinctly differed from those of other CHK1 MAPK pathway deletion mutants, including ΔCHK1, ΔBmSTE7, ΔBmSTE11, and ΔBmSTE50, which form no appressoria on artificial hydrophobic surfaces or host plant surfaces (Izumitsu et al., 2009; Kitade et al., 2015; Lev et al., 1999; Sumita et al., 2019). These findings indicate two independent recognition pathways upstream of the CHK1 MAPK cascade: one for hydrophobic surfaces, where BmOPY2 is essential, and the other one for a host plant-derived signal.

We initially hypothesized that host-derived wax components induced appressoria in B. maydis, as they do in P. oryzae (Liu et al., 2011), a major model organism among plant pathogenic fungi. However, wax components and wax extracts from maize leaves failed to restore appressorium formation by a ΔBmOPY2 mutant on plastic plates. Notably, this mutant formed normal appressoria on dewaxed leaves (Supplementary Fig. S5). These findings indicate that B. maydis recognizes different plant-derived substances than P. oryzae.

Further investigation revealed that the ΔBmOPY2 strain preferentially formed appressoria above the intercellular spaces on maize leaves, leading us to examine pectin, the primary constituent of plant intercellular spaces, as a potential appressorium-inducing substance. Indeed, pectin, presumably in its Ca²⁺-induced gel form, strongly restored appressorium formation on plastic plates, demonstrating that distinct host-derived substances induce appressorium formation in these species: plant waxes in P. oryzae and pectin in B. maydis.

It should be noted, however, that while addition of commercial apple or citrus-derived pectin in its Ca²⁺-induced gel form significantly restored appressorium formation in the ΔBmOPY2 strain, the restoration was not completely equivalent to wild-type or complemented strains (Fig. 8). This partial restoration suggests that maize-derived pectin might potentially achieve complete recovery due to host-specific structural variations. Alternatively, we cannot exclude the possibility that additional minor substances may also contribute to appressorium induction in B. maydis, acting in concert with pectin to achieve the full induction observed in wild-type strains.

Interestingly, despite the ΔBmOPY2 mutant forming appressoria on maize leaves at wild-type levels, it develops slightly smaller lesions in pathogenicity assays (Fig. 4A). This reduced virulence persists even with wound inoculation (Fig. 4B), indicating that BmOPY2 has a minor but distinct role in post-penetration development, separate from its essential function in hydrophobic surface recognition. While the mechanism underlying this additional role remains unknown, our results clearly demonstrate that BmOPY2 contributes to both appressorium formation and in planta fungal development, meriting further investigation.

The infection strategies fundamentally differ between the hemibiotrophic pathogen P. oryzae and the necrotrophic pathogen B. maydis. While P. oryzae must establish immediate biotrophic infection within plant cells through penetrating hyphae that form from the appressorium, necrotrophic pathogens Alternaria and Bipolaris initially spread through plant intercellular spaces before necrotrophically killing host cells (Rajarammohan, 2021; Santén et al., 2005). Our observation that wild-type B. maydis preferentially formed appressoria above intercellular spaces suggests that these differences in infection strategies may be directly linked to the differences in appressorium-inducing substances.

Future studies generating mutants defective in hydrophobic surface recognition across various plant pathogenic ascomycetes would provide valuable insights into species-specific appressorium-inducing substances. Such studies may reveal whether the use of wax components by hemibiotrophic fungi and pectin by intercellular space-colonizing necrotrophic fungi represents a general pattern, or whether diverse chemical substances serve as inducers depending on taxonomic group or host plant species.

The authors declare no conflicts of interest.

This research was supported by JSPS KAKENHI Grant Number 15K07311 (Grant-in-Aid for Scientific Research (C)) from the Japan Society for the Promotion of Science (JSPS). The funding period was from Apr 1, 2015, to Mar 31, 2018. Since Apr 2023, this research has been further supported by JSPS KAKENHI Grant Number 23K26908 (Grant-in-Aid for Scientific Research (B)).

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