2021 年 62 巻 5 号 p. 289-296
In this study, we identified Sec5 in Bipolaris maydis, a homologue of Sec5 in Saccharomyces cerevisiae and a possible exocyst component of the fungus. To examine how Sec5 affects the life cycle of B. maydis, we generated null mutant strains of the gene (Δsec5). The Δsec5 strains showed a strong reduction in hyphal growth and a slight reduction in pathogenicity. In sexual reproduction, they possessed the ability to develop pseudothecia. However, all ascospores were aborted in any of the asci obtained from crosses between Δsec5 and the wild-type. Our cytological study revealed that the abortion was caused by impairments of the post-meiotic stages in ascospore development, where ascospore delimitation and young spore elongation occur.
The exocyst, which is highly conserved in eukaryotic systems, is thought to regulate the transport of the secretory vesicle and to tether the secretory vesicle to the plasma membrane before vesicle fusion (Chen, Ebbole, & Wang, 2015). The exocyst was first identified in Saccharomyces cerevisiae and consists of eight components: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (TerBush, Maurice, Roth, & Novick, 1996). These exocyst components, which encoded a single gene in each fungus, were confirmed by co-purification and co-immunoprecipitation experiments to also form an octameric complex in the filamentous fungi Magnaporthe oryzae and Neurospora crassa (Riquelme et al., 2014; Gupta et al., 2015). Subsequently, these exocyst components were characterized in several fungi, indicating the possibility that exocyst components are involved in morphogenesis and pathogenicity in fungi (e.g., Finger & Novick, 1997; Guan et al., 2019). However, most of exocyst components are essential for viability, with some exceptions, and their importance is dependent on the fungal species. This suggests that the exocyst components should be investigated in many fungal species to gain further insight into the roles of exocysts in their life cycles.
Among the exocyst components, Sec5 plays unique and important roles in the construction of the exocyst complex in S. cerevisiae. The structure of the yeast exocyst complex was made up of two subcomplexes: subcomplex I, consisting of Sec3, Sec5, Sec6, and Sec8; and subcomplex II, consisting of Sec10, Sec15, Exo70, and Exo84. Sec5 mediates the interaction of these subcomplexes (Mei et al., 2018; Mei & Guo, 2018). In N. crassa, only the Sec5 gene is dispensable with regard to viability, but required for proper vegetative growth (Riquelme et al., 2014). In M. oryzae, the disruption of Sec5 significantly reduces pathogenicity (Giraldo et al., 2013). However, little has been reported on the functional role of Sec5 in development and morphogenesis in filamentous fungi, especially in sexual reproduction.
The filamentous ascomycete Bipolaris maydis (syn.: Cochliobolus heterostrophus) is a necrotrophic pathogen that causes southern corn leaf blight (Rossman, Manamgoda, & Hyde, 2013). The fungus uses fusiform asexual spores, called conidia, as a primary inoculum. In addition, B. maydis is a heterothallic fungus with bipolar mating types (MAT1-1 and MAT1-2) and easily mates between strains with different mating types in laboratory conditions. In the mating process, the fungus develops ascocarps, called pseudothecia. As a result of mating, filiform ascospores within pseudothecia can be observed readily in the laboratory. Therefore, B. maydis has often been used as a model organism to investigate fungal asexual and sexual development (Turgeon, 1998; Raju, 2008; Sumita, Izumitsu, & Tanaka, 2017; Kitade, Sumita, Izumitsu, & Tanaka, 2019).
In the present study, an orthologue of S. cerevisiae Sec5 was identified in B. maydis. Then, we performed gene disruption and obtained null mutant strains of the gene. Here, we characterize the roles of Sec5 in the life cycle of B. maydis by comparing it with the wild-type and mutants.
All strains used in this study are shown in Table 1. To express gene symbols in this paper, a proposed genetic nomenclature by Yoder, Valent, and Chumley (1986) was generally followed, and a two-lettered acronym of a fungal scientific name (species prefix) was placed in front of a gene symbol, if necessary. Bipolaris maydis HITO7711 (SEC5, ALB3, MAT1-2) was used as a wild-type strain throughout this study. The compatible albino strain, M3alb3 (SEC5, alb3, MAT1-1; Tanaka, Kubo, & Tsuda, 1991), was used in crossing experiments. Pseudothecia of the alb3 strain were light tawny colored; thus, they were easily distinguishable from black pseudothecia developed by strains that carry a wild-type allele (ALB3) at the Alb3 locus, e.g., HITO7711, etc. Disruptants of the Sec5 gene derived from the wild-type strain HITO7711 were generated by homologous recombination. All strains were maintained on a complete medium agar (CMA; Tanaka et al., 1991) or V8 agar (V8A; Ribeiro, 1978) at 25 °C.
The amino acid sequence of Sec5 in S. cerevisiae (Accession No. KZV12402) was obtained from the DDBJ/EMBL/GenBank. To identify its orthologue in B. maydis, we used it as a query in PSI-BLAST searches of the NCBI protein database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sec5 orthologues in other fungal species were also acquired from the NCBI protein database. Amino acid sequence alignments were carried out using CLUSTAL X (Larkin et al., 2007).
All primers used in this study are shown in Table 2. Molecular experiments were performed according to Sambrook, Fritsch, and Maniatis (1989). The entire coding regions and introns of the wild-type allele (SEC5) were deleted using the targeted replacement method in homologous recombination. A fungal genomic DNA was obtained using the method of Izumitsu et al. (2012). A DNA fragment for gene disruption was constructed using the PCR fusion method (Szewczyk et al., 2006) with a HygR cassette (Aspergillus nidulans TrpC promoter (PTrpC) fused to a hygromycin B phosphotransferase gene (HPH)) obtained from the plasmid pCB1004 (Carroll, Sweigard, & Valent, 1994). In the first-round PCR, 5′- and 3′-noncoding regions of the SEC5 gene were amplified from a genomic DNA of the wild-type HITO7711 using an ExTaq polymerase (Takara Bio, Otsu, Japan) and primer set (BmSec5-f1/BmSec5-r1 for the 5′-noncoding region; BmSec5-f2/BmSec5-r2 for the 3′-noncoding region; Table 2). The HygR cassette was also amplified from the plasmid pCB1004 using ExTaq polymerase and the primers pCB1004-f1 and pCB1004-r1 (Table 2). These DNA fragments were used as substrates for the second-round PCR in order to fuse the three fragments into one deletion cassette using primers BmSec5-f1 and BmSec5-r2 (Table 2). The resulting major PCR product was purified by ethanol precipitation and resuspended at half of the original volume in a TE buffer for fungal transformation. Transformation experiments were performed using the method described by Izumitsu et al. (2009). Integration of the HygR cassette into the targeted region was confirmed by PCR using primers BmSec5-f3 and BmSec5-r3, which annealed to a flanking region outside the deletion cassette (Table 2; Fig. 1A).
The underlines show a reverse complement sequence of the HygR cassette.
For functional complementation of the null mutant, we amplified the SEC5 gene including 5′- and 3′-noncoding regions from genomic DNA of the wild-type HITO7711 with a primer set BmSec5-f1/BmSec5-r4 using PrimeStar GXL DNA polymerase (Takara Bio; Table 2). The amplified fragment was ligated into the EcoRV site of pZNAT1 (Izumitsu et al., 2009) carrying a nourseothricin-resistance gene (NAT) under the control of the TrpC promoter. The plasmid in which SEC5 was oriented in a tail-to-tail configuration with NAT was selected by PCR with BmSec5-f4 and NAT-chk-f1. Reconstitution of the SEC5 gene in the Δsec5 strain was made by a double crossing over event of homologous recombination within 5′-noncoding regions of the Sec5 genes and the TrpC promoter regions of resistance gene cassettes. The constructed vector was linearized by SpeI digestion, and the vector’s transformation into a Δsec5 strain was performed using a method described previously (Izumitsu et al., 2009). Strains showing resistance to nourseothricin (300 µg/mL) were isolated individually, and the reintroduction of the gene was confirmed by PCR using two primer sets: BmSec5-f3/BmSec5-r5 and BmSec5-f5/BmSec5-r6 (Table 2).
A penetration assay was performed on onion epidermises. These epidermises were killed by heat in an oven (65 °C, 1 h). Conidial suspensions (100 µL, 1 × 104 conidia/mL) of each strain were prepared and inoculated on these epidermises. These samples were incubated for 24 h at 25 °C in the dark. After staining with cotton blue (0.05% (w/v) in lactophenol), the penetration sites were observed microscopically.
For pathogenicity assays, maize plants (Zea mays cv. Takanester; Sato, Sawano, Shigemori, Mejima, & Miki, 2008) were grown in a greenhouse. Leaves were harvested from 3- to 4-wk-old plants and placed in a transparent polystyrene box lined with paper moistened with sterile water. Ten microliters of the conidial suspensions (5 × 104 conidia/mL) of each strain was inoculated onto the leaves. The conidial suspensions were prepared from a 2-wk-old culture on V8A. The inoculated leaves were incubated for 3 d under dark conditions at room temperature. Pathogenicity assays were repeated three times using three leaves per assay.
Crossing was performed on a sterilized corn leaf on Sachs’s agar medium according to the method described by Tanaka et al. (1991). The wild-type HITO7711 and Δsec5 strains were crossed with their compatible albino strain, M3alb3. The inoculated cultures were incubated at 25 °C for 4 wk, and the observation of pseudothecia on the corn leaf was made with a stereomicroscope. To inspect the ascus and ascospore development, mature pseudothecia were harvested and crushed in sterilized water on glass slides. Subsequently, these asci and ascospores were observed microscopically.
For ascus cytology, nuclei in an ascus were stained with fluorochrome bisbenzimide H33258 trihydrochloride (Hoechst; Nacalai Tesque, Kyoto, Japan). Developing pseudothecia on the corn leaf from 10 to 30 d after inoculation were harvested and dissected with tweezers on a glass slide. They were fixed with 5% (v/v) glutaraldehyde at 4 °C for 1 d and washed with phosphate buffer. These samples were then stained with Hoechst solution (10 µg/mL) for 20 min in the dark and washed with phosphate buffer. After the addition of 5% (v/v) glycerol, a cover slip was applied, and the stained material was carefully spread by pressure exerted with a toothpick. The samples were inspected using DIC-epifluorescent microscopy (Leica DML with A cube (BP 340–380 nm excitation filter, 400 nm dichromatic mirror, LP 425 nm suppression filter); Leica Microsystems, Wetzler, FRG). Over 200 pseudothecia were dissected, and thousands of developing asci at various stages of nuclear division and ascospore developments were examined.
In order to identify a Sec5 orthologue in B. maydis, an S. cerevisiae Sec5 protein sequence was subjected to PSI-BLAST searches with default parameters. The resulting hit was only one protein (ID: 1193032); thus, that protein was designated as Sec5 in this study. A gene encoding the Sec5 protein in B. maydis is composed of four exons interrupted by three introns; it also encodes a protein of 1065 amino acids. It showed 20% identity with S. cerevisiae Sec5. On the other hand, the sequence of BmSec5 of B. maydis showed higher identity with Sec5 orthologues in other filamentous fungi: 45% identity with AnSec5 in A. niger (Accession No. XP_001392674), 47% identity with NcSec5 in N. crassa (Accession No. XP_962284), 47% identity with MoSec5 in M. oryzae (Accession No. XP_003715348), and 47% identity with VdSec5 in Verticillium dahliae (Accession No. RBQ89220). Previous studies have reported that orthologues of Sec5 in V. dahliae and N. crassa contain a Vps51/Vps67 domain (Yang et al., 2013; Riquelme et al., 2014). The region predicted to be the Vps51/Vps67 domain was highly conserved among some filamentous fungi, including B. maydis (Supplementary Fig. S1). These results suggested that BmSec5 was an orthologue of the S. cerevisiae Sec5.
To investigate the role of Sec5 in B. maydis, we generated null mutants of the gene. We obtained 19 transformants that showed resistance to hygromycin B. Subsequently, all hygromycin B-resistant transformants were screened using the PCR method described above, and four strains were characterized as Sec5 null-mutant (Δsec5) strains. Two null mutants were arbitrarily chosen for subsequent experiments (Fig. 1B). To confirm the effects of Sec5 gene disruption, we also attempted to generate a reconstituted strain by reintroducing the wild-type allele (SEC5) into the Δsec5 strain. The reconstituted strain was generated by a double-crossover event in which the wild-type gene (SEC5) was inserted into a null allele (Δsec5). The transformant—named CSE5—was confirmed by PCR and characterized as reconstituted strain (data not shown).
To understand the role of Sec5 in colonial development and conidiation, all strains were grown on CMA or V8A at 25 °C. Δsec5 strains developed a melanized colony, as did the wild-type. However, Δsec5 strains showed a drastic reduction in radial growth as compared with the wild-type and reconstituted strains (Fig. 2; Table 3). On the other hand, the conidial morphology of Δsec5 strains was similar to that of the wild-type strain. The conidial density (number of conidia per cm2) of Δsec5 strains was slightly increased as compared to those of the wild-type and reconstituted strains (Table 3); however, there is no statistic significance among categories of the wild-type, Δsec5, and reconstituted strains (statistic results from Tukey’s multiple comparison test are not shown).
Values represent the mean±standard errors (n = 5). Different letters indicate a significant difference between strains (P < 0.05) according to the Tukey’s multiple comparison test of mean separation.
We investigated whether Sec5 is important for penetration and pathogenicity. Conidia of the wild-type and reconstituted strains developed numerous appressoria on the heat-killed onion epidermis. Infection hyphae from these appressoria were observed in onion epidermises (Fig. 3A). Δsec5 strains also showed normal appressoria and infection hypha (Fig. 3A). The infection rate was similar to those of the wild-type and reconstituted strains (Fig. 3B).
For a pathogenicity test, we inoculated conidial suspensions of the wild-type, Δsec5, and reconstituted strains on host corn leaves. The wild-type and reconstituted strains produced well-developed lesions. On the other hand, the lesion size formed by Δsec5 strains was slightly reduced, as compared to those of wild-type and reconstituted strains (Fig. 4A, B). These observations suggested that Sec5 is required for full virulence after infection.
To assess the role of the Sec5 in sexual reproduction, we crossed the Δsec5 strains (MAT1-2) with the compatible albino strain, M3alb3. In crossing the wild-type strain with the albino strain, the former formed black pseudothecia, and the latter developed light tawny-colored ones along the interface of the two strains (Fig. 5A). In the crosses of Δsec5 strains with the albino strain, black and light tawny pseudothecia were formed along the interface of the strains, as in the cross of the wild-type and the albino strains. These results suggested that Sec5 is not required for pseudothecial development. In order to assess the involvement of Sec5 in ascus and ascospore development, pseudothecia obtained by crossing of the Δsec5 strains with the albino strain were inspected microscopically. In results of crosses of the wild-type strain with the albino strain, seven or eight mature ascospores per ascus were typically formed in both black and light tawny pseudothecia (Fig. 5B). In asci from a cross between the reconstituted and the albino strains, ascospores developed. However, in the crosses of Δsec5 strains with the albino strain, any asci in either black or light tawny pseudothecia had no ascospores (>1000 asci; Fig. 5B). These results implied that Sec5 is not essential for female organ development but is essential for female and/or male functions in the sexual reproduction process of this fungus. Further attempts to clarify the defective step affected by Δsec5 in sexual reproduction were carried out by cytological study. Black pseudothecia from 10 to 30 d old after inoculation were dissected, and each ascus was stained with Hoechst dye. In pseudothecia from crosses between wild-type and albino strains, we could observe all stages of asci with a single nucleus, as well as 2-nucleus, 4-nucleus, 8-nucleus, 16-nucleus, and multinucleate ascospores (Fig. 6A–F), as Raju (2008) reported previously. In crosses of Δsec5 strains with the albino strain, meiotic stages of asci were found (Fig. 6G–I). Asci with 8 and 16 nuclei were also observed, which implied that the post-meiotic first and second mitoses progressed (Fig. 6J–N). However, few asci with 8 nuclei were delimited (Fig. 6K). In most of the asci with 16 nuclei, the nuclei were not enclosed in ascospores (Fig. 6L), and they migrated to lower part of the ascus later (Fig. 6M). Delimited binucleate ascospores might be unable to elongate, meeting the same fate as unenclosed nuclei (Fig. 6N). No further stages for multinucleating ascospores were found in the crosses of Δsec5 strains. Thus, they seemed to be delimitation failures or impaired in the elongation of delimited young ascospores, and further processes including the third post-meiotic mitosis were aborted. Our observations implied that a defection of Sec5 did not affect the mating process prior to the meiosis of the fungus but was critical for ascospore development posterior to meiosis.
The composition of the exocyst complex is evolutionally conserved among eukaryotic cells. In fungi, each of the eight exocyst components presents a single gene, and most of these genes are essential for viability, with the exceptions of Sec3 in S. cerevisiae, Sec5 in N. crassa, and Sec5 and Exo70 in M. oryzae (Finger & Novick, 1997; Giraldo et al., 2013; Riquelme et al., 2014). This suggests the possibility that the same exocyst components may not have a common function in different fungi. Thus, these homologues were needed to investigate functions in different fungi. Moreover, to our knowledge, only a few studies have focused on the broad function of exocyst components in the fungal life cycle. In this study, we identified the Sec5 orthologue of B. maydis and investigated its broad functions in the fungal life cycle, using null mutants.
Our results revealed that BmSec5 is required for hyphal growth (Fig. 2; Table 3). Similar results were reported for the Sec5 mutant of N. crassa (Riquelme et al., 2014). These results show that an exocyst complex or Sec5 is important for normal hyphal growth. In addition, the study indicated that BmSec5 is not important for conidiation (Table 3). However, few studies have focused on the function of Sec5 in conidiation of other fungi.
In infectious development of B. maydis, Δsec5 strains formed numerous appressoria on the onion epidermis and infectious hypha in the onion epidermis, which was similar to the wild-type strain (Fig. 3A, B). The results indicated that BmSec5 is not required for infectious development. In M. oryzae, lack of the exocyst component MoSec5 or MoExo70 shows significant pathogenicity to host plants (Giraldo et al., 2013; Gupta et al., 2015). These exocyst components are thought to be required for the secretion of cytoplasmic effectors after the infection of host plants (Giraldo et al., 2013). These results suggest that the exocyst complex or several of its components play important roles in the secretion of virulence factors. In B. maydis, here, Δsec5 strains showed a slight reduction in lesion size. Unlike the case of M. oryzae, BmSec5 is not critical for pathogenicity in the postinfectious stage in B. maydis (Fig. 4A, B). The result implies that the role of Sec5 in pathogenicity can be different between two species.
In this study, we found that BmSec5 is required for ascospore formation, but not for the development of the female organ, the pseudothecium. Ascospore formation processes in Bipolaris (syn.: Cochliobolus) spp. has been studied by Hrushovetz (1956); Guzman, Garber, and Yoder (1982); and Raju (2008). Meiosis occurs in a common cytoplasm of the ascus, which has arisen from an ascogenous hypha. Meiosis is followed by the first post-meiotic mitosis, which generates eight daughter nuclei in the ascus. Typically, delimitation happens after the first post-meiotic mitosis to enclose a single nucleus in each ascospore. These delimited young ascospores elongate to filiform shape and are parallel to one another along the long axis of the ascus. The following second mitosis synchronously or asynchronously occurs in each uninucleate ascospore, producing binucleate ascospores, which occupy about 75–80% of the ascus length. The ascospores elongate further with the third and additional mitoses, and septa appear at random in the spores, resulting in helically coiled mature ascospores. Hrushovetz (1956) has described his observation of such asci that failed to delimit ascospores after the first post-meiotic mitosis, noting the possibility that they never do delimit and form normal spores in B. sorokiniana. In the crosses of Δsec5, we did not find any filiform young ascospores or further stages of ascus resulting from the second post-meiotic mitosis.
The functions of exocysts in mating and ascospore formation have been studied in yeast (Sharifmoghadam, de Leon, Hoya, Curto, & Valdivieso, 2010). In fission yeast S. pombe, exocyst components were reported to be involved in mating and ascospore development. Exocyst component mutants in S. pombe reduce mating efficiency as compared with a wild-type. These mutants also exhibit abnormal ascospores or no ascospore formation (Sharifmoghadam et al., 2010). Exocyst components are known to be important for forespore membrane (FSM) development (Sharifmoghadam et al., 2010; Imada & Nakamura, 2016). An FSM is a double membrane within the cytoplasm of the diploid mother cell, later becoming an ascospore membrane. These results may show that BmSec5 has similar functions in the delimitation of ascospores or in the elongation of delimited young ascospores for a filiform shape to that of exocysts in ascospore formation of the yeast, although there are some differences in mating behavior and ascospore development between the yeast and B. maydis.
The authors declare that they have no conflict of interest. All of the experiments undertaken in this study comply with the current laws of Japan, where the research was performed.
Part of this work was supported financially by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to CT (Nos. 15K07311, 15H05249, and 19K06052).