JPWO2009054330A1 - Method for mating incompetent filamentous fungi - Google Patents

Abstract

The present invention relates to a method of mating a deficient mating fungus, wherein the deficient mating fungus as a parent strain and the mating type gene region of the parent strain are derived from a deficient mating strain of the same type that is different from the parent strain. This method relates to a method comprising mating with a strain having a different chromosome type but having a substantially homologous chromosomal pattern, which has been created by exchanging in another mating type gene region.

Description

  The present invention relates to a novel method for mating deficient mating fungi.

  Fungi are eukaryotes having a cell wall mainly composed of chitin. In general, it includes generations called “mold”, “mushroom” and “yeast”. Among these, those mainly consisting of hyphae and spores (mainly having the ability to germinate in the shape of a sphere and form the next generation) and those that are not yeast-like are generally collectively referred to as filamentous fungi. Filamentous fungi include ascomycetes, basidiomycetes, and zygotes on the classification system.

  The life cycle of filamentous fungi is composed of asexual and sexual generations. Mycelia, mycelia, conidia, thick membrane spores, bud cells (yeast-like cells) are asexual generations of nuclear phase n, and their progeny are genetically identical by the formation of mycelia, formation of mycelia, conidia, etc. (Clone) is produced (asexual growth). In many filamentous fungi, asexual growth is repeated, and most of the life cycle is occupied by asexual generations.

  On the other hand, when the environmental conditions are met, the filamentous fungus may switch its life cycle to the sexual generation. In ascomycetes, in basidiomycetes, basidiomycetous organs are formed on the fruiting bodies of ascitic berries (such as ascolic shells) and basidiomycetes (so-called mushrooms). Do and form progeny. In sexual reproduction, two cells in nuclear phase n fuse and become n + n (heteronuclear coexistence), then fuse (2n), paired with homologous chromosomes, and then nuclei by meiosis Create a successor of phase n. Gene recombination may occur through homologous chromosome pairing to chromosomal shuffling or crossover during meiosis, resulting in a genetically different progeny from the parent. As a result, the progeny may have different properties from the parent. In the case of ascomycetes, ascospores and in basidiomycetes, basidiospores (basidiospores) correspond to this progeny. This series of phenomena is called mating. In addition, a series of organs including the formed fruiting body and the next generation and the part between the life forming it are called a complete generation.

  Although filamentous fungi are basically hermaphroditic, there are those that do not require a partner during sexual reproduction. A property that does not require an opponent is called a homothalic, and a property that requires an opponent is called a heterothalic. It can be understood that homothalic is self-compatible, and heterothallic is self-incompatible (Non-patent Document 1). The basic sexual reproduction pattern of ascomycetes is heterothalic, and homothallic is thought to have originated from heterothallic (described later).

  In heterothallic filamentous fungi, there are two mating strains (MAT1-1 and MAT1-2) in one species. The mating type is limited to the two. When the MAT1-1 and MAT1-2 strains meet under appropriate environmental conditions, cell fusion occurs (in this case, since they are hermaphroditic, one cell is fused and the other is female. Functioning), a series of sexual reproduction (described above) proceeds to form progeny. No mating occurs between MAT1-1 strains or between MAT1-2 strains. The mating type of the strain is determined by a set of alleles (MAT1-1-1 and MAT1-2-1) existing in the mating type gene region (MAT1). Holding either one. Both gene products have a DNA binding motif and are suggested to be early regulators of mating (Non-patent Documents 1 and 2).

  Therefore, when cross-type strains of heterothallic filamentous fungi are crossed at appropriate environmental conditions (crossing out), a complete generation is formed. In this case, the appropriate environmental conditions vary from species to species. For example, in Neurospora crassa, it is only necessary to cultivate on a normal medium, and in Cochliobolus heterostrophus, sterilized corn leaves are placed on a minimal medium, and both strains are confronted on the edge, and around 20 ° C, dark, overhumid In Gibberella fujikuroi, the mycelium of one strain is grown on the V8 agar medium, and the conidia suspension of the other strain is applied to the Gibberella fujikuroi, and after leaving it for a while, the excess aqueous phase is removed. Incubate at about 25 ° C.

  For homothallic filamentous fungi that do not require outcrossing with strains of different mating types to form a complete generation, are two mating type genes present in the mating type gene region independently or fused? -1 and MAT1-2-1 genes have been found to exist in distant regions of the genome, suggesting that homotalic bacteria evolved from heterothallic bacteria (Non-Patent Document 3).

  The above indicates that the MAT1-1-1 and / or MAT1-2-1 genes are required in any case for the fungi to cross and form a complete generation. .

  There are many species of filamentous fungi that have not previously been observed to cross and form full generations. These filamentous fungi are called imperfect bacteria because they do not form a complete generation. In the past, these fungi have been grouped as Deuteromycotina (Incomplete fungi) or Fungi imperfecti (Incomplete fungi), but in recent years, with the progress of molecular phylogenetic analysis, strains of closely related mating species The relationship has been clarified, and a fungus group such as past Deuteromycotina or Fungi imperfecti is understood as meaningless (Non-patent Document 4). Here, a filamentous fungus in which no mating has been observed is referred to as a “cross-competent filamentous fungus”. Since incompetent filamentous fungi cannot be mated, genetic analysis of gene function (mating test) and breeding by mating were impossible. If mating becomes possible, the analysis of gene function by mating test will be greatly advanced, and breeding of bacteria with better traits will be possible, such as Neisseria gonorrhoeae, which will be scientifically and industrially significant.

  There are many types of incompetent filamentous fungi. As an important phytopathogenic fungus, many mating imperfect species such as Fusarium oxysporum, Verticillium dahliae, Alternaria alternata, Penicillium italicum have been reported (see Non-Patent Document 5, item of incomplete fungi). In addition, yellow mold fungus Aspergillus oryzae, which is said to be the national fungus of Japan, is also an incompletely mated species. In addition, many of the antibiotic-producing Penicillium spp. And black molds (Cladosporium spp. And Alternaria spp.) That are problematic in the home are incompletely mated.

  In the past, the only way to cross a deficient filamentous fungus was to find a strain that retains the mating ability from the origin of the species (which is assumed to be rich in diversity), and cross it with this. It was. As an example, the rice blast fungus (Magnaporthe oryzae, incomplete generation Pyricularia oryzae) has long been known as a mating deficient fungus, but the rice blast fungus from China's Kunming has a mating ability at the laboratory level, It has been reported that rice blast fungus with mating ability has been collected from northern Vietnam and northwestern India. Furthermore, there is also an example in which mating of basidiomycetes, which has been difficult to mate conventionally, is enabled by selection of mating strains by isozyme analysis (Patent Document 1).

  Crossing of deficient filamentous fungi by such a method is possible only when a strain having the same type of mating ability as that of the deficient hybrid is found by chance, and the mating deficient strain can be crossed. I can't say I did it. That is, the rice blast fungus strain actually occurring in the field in Japan, Korea, etc. remains incompetent.

  In heterothallic filamentous fungi known to have mating ability (eg Neurospora crassa), the mating type (MAT1-1 or MAT1-2) of each strain is determined by mating tests with tester strains. It is easy to test. Based on the results, strains of different mating types can be crossed under appropriate conditions and mated to form a complete generation.

  However, in the case of incompetent filamentous fungi, it was previously impossible to even test the mating type of the strain in the first place. In other words, it was not known whether there was a mating type in the incompetent filamentous fungal strain.

  Non-patent document 6 and a related patent “a primer for amplifying a specific site on a mating type gene; patent 3718755” (patent document 2) are based on the PCR method. The test was made possible. (Of course, this method is also applicable to filamentous fungi having mating ability.)

  Using this technique, we investigated whether there are strains with different mating types in the species of Fusarium oxysporum, Alternaria alternata, and Magnaporthe oryzae from Japan, and there are strains of both mating types. (Non-Patent Documents 6, 7, 8, and 9). Furthermore, based on the result of the test, a crossing test between strains of different mating types was performed, but none of them resulted in mating (Non-Patent Documents 7 and 9).

  The following items (1), (2), and (3) were presumed as causes of the mating failure of the mating deficient bacteria. (1) The mating type gene region that is the initial regulator of mating or the gene of the mating type gene region (mating type gene) does not exist or is dysfunctional. (2) Signal transduction that functions downstream (hereinafter meaning) System failure, (3) There is only one mating type strain (absence of mating partner). The example of the conventional research which verified these is outlined.

  A mating deficient bacterium has a mating type gene region equivalent to a related bacterium with mating ability, and the mating type gene encoded in that region is almost homologous to a related bacterium with mating ability This is shown using Bipolaris sacchari, Fusarium oxysporum, Alternaria alternata, Japanese Magnaporthe grisea, etc. (Non-patent Documents 7, 9, and 10). Moreover, it is shown that these mating type genes are expressed (Non-Patent Documents 9, 10, and 11).

  In addition, the mating type gene of a mating deficient bacterium has the function that the mating type gene of the mating deficient bacterium has been removed from the mating type gene region of a closely related bacterium having heterothallic mating ability. It was reported that mating ability was restored when sex mutants were introduced as recipients. There are only two reports (Non-Patent Documents 7 and 11) on the deficiency of Bipolaris sacchari and Alternaria alternata. The genomic region of the recipient into which the region has been introduced is not a mating gene region (MAT1).

  As described above, the mating type gene product has a DNA binding site and binds to genomic DNA, thereby regulating the expression of pheromones and pheromone receptor genes involved in mating. Pheromones bind to pheromone receptors of different strains, and the information is transmitted to the nucleus through regulators such as G-protein, MAP kinase, cAMP, and STE12 protein, which are signal transduction systems. It is said that a series of mating behaviors such as fusion, formation of fruiting bodies, and formation of progeny occur.

  So far, some dysmorphic fungi have been analyzed for genes such as pheromones, pheromone receptors, G-proteins, and MAP kinases, and they function in the same way as those with mating ability. It is shown.

  The above may deny the above (1) and (2), which are considered to be the cause of mating deficiency, and may indicate the possibility that the mating deficient bacteria basically have mating ability.

  Non-patent document 8 describes that a mating-deficient tomato wilt fungus (F. oxysporum f. Sp. Lycopersici) strain forms three strains in the F. oxysporum phylogenetic tree. It was reported that the fusion groups were different and that the wilt fungal strains contained in each strain were not only the same mycelium fusion group, but also limited to one mating type.

Non-Patent Document 12 tried to produce a replacement strain of the mating type gene region (MAT1) of Fusarium oxysporum as part of the pursuit of the cause of mating deficiency.
Japanese Patent Laid-Open No. 07-227164 Japanese Patent No. 3718755 “Primers for amplifying specific sites on mating type genes” Debuchy R, Turgeon BG (2006) Mating-type structure, evolution, and function in euascomycetes.In The Mycota I (Kues & Fisher eds.), Springer-Verlag, Berlin.pp. 293-323 Shiu PK, Glass NL (2000) Cell and nuclear recognition mechanisms mediated by mating type in filamentous ascomycetes. Curr Opin Microbiol 3: 183-188 Yun SH, Berbee ML, Yoder OC, Turgeon BG (1999) Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors.Proc Natl Acad Sci USA 96: 5592-5597 Kazuo Shishido et al., Basic Science and Biotechnology of Mushrooms and Molds, IPC, 2002 Edited by Katsumi Yoneyama et al., Plant Pathogenic Atlas, Soft Science, 2006 Arie, T., Christiansen, SK, Yoder, OC and Turgeon, BG (1997) .Effecient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG box. Fungal Genet Biol 21: 118-130 Arie, T., Kaneko, I., Yoshida, T., Noguchi, M., Nomura, Y. and Yamaguchi, I. (2000). Mating type genes from asexual phytopathogenic ascomycetes, Fusarium oxysporum and Alternaria alternata. Mol Plant- Microbe Interact 13 (12): 1330-1339 M. Kawabe, Y. Kobayashi, G. Okada, I. Yamaguchi, T. Teraoka and T. Arie (2005). Three evolutionary lineages of tomato wilt pathogen, Fusarium oxysporum f. Sp. Lycopersici, based on sequences of IGS, MAT1 , and pg1, are each composed of isolates of a single mating type and a single or closely related vegetative compatibility group.J Gen Plant Pathol: 71 (4): 263-272 Masaki Kanamori, Hana Kato, Nobuko Yasuda, Shinzo Koizumi, Tobin L. Peever, Takashi Kamakura, Tohru Teraoka, Tsutomu Arie (2007) Novel mating type-dependent transcripts at the mating type locus in Magnaporthe oryzae. Gene 403: 6-17 Sharon A., Yamaguchi K., Christiansen S., Horwitz BA, Yoder OC, Turgeon BG, 1996. An asexual fungus has the potential for sexual development. Mol Gen Genet 250: 11-122 Yun, Sung-Hwan, Arie, Tsutomu, Kaneko, Isao, Yoder, OC, Turgeon, B. Gillian, 2000. Molecular Organization of Mating Type Loci in Heterothallic, Homothallic, and Asexual Gibberella / Fusarium Species. Fungal Genet Biol 31: 7 -20 Shusuke Imai, Tohru Teraoka, Tsuyoshi Arie 2007. “Creating a replacement strain of the mating type gene region (MAT1) of Fusarium oxysporum” Proceedings of the 2007 Annual Meeting of the Kanto Division of the Japanese Society of Plant Pathology 8

  As described above, in the case of Fusarium oxysporum, which is a mating deficiency, the partner was absent in the close population that can be mated, suggesting that this may be the cause of the mating deficiency. That is, this suggests the possibility of the above (3) in the background art section presumed as the cause.

  Therefore, the development of a new method for mating deficient mating fungi is desired.

  An object of the present invention is to provide a novel method for mating deficient mating fungi.

  In order to solve the above-mentioned problems and achieve the object of the present invention, the present inventors have determined that the mating-deficient filamentous fungus that is a mating strain and the mating type gene region of the parent strain are different from the parent strain but of the same species. A mating deficient filamentous form by a method comprising mating with a substitutive strain of different mating type while having a substantially homologous chromosomal pattern by exchanging with another mating type gene region derived from the mating deficient strain We found a way to make it possible to cross the fungus.

  Here, although not necessarily limited, it is preferable that the filamentous fungus is a phytopathogenic filamentous fungus or a koji mold. Moreover, although not necessarily limited, it is preferable that a phytopathogenic filamentous fungus is Fusarium oxysporum. Moreover, although not necessarily limited, it is preferable that replacement | exchange is performed by homologous recombination.

  The method of mating deficient filamentous fungi of the present invention has the advantage of enabling the analysis of gene functions of industrially important or useful filamentous fungi and the generation of bacteria with good traits, that is, breeding.

This figure shows the chromosomal pattern of Fusarium oxysporum by CHEF separation. In the figure, A represents the NBRC6531 strain, B represents the 880621a-1 strain, C represents the MAFF103038 strain, D represents the MAFF103036 strain, E represents the tomino 1-c strain, and F represents the F-1-1 strain. . The agarose gel electrophoresis conditions are as follows. Gel: 0.8% Certified Megabase Agarose (Bio-rad); Running buffer: 0.5 × TBE; Temperature: 10 ° C .; Voltage: 2.0 V / cm; Running time: 120 hours; Initial switching time: 10 minutes: Final switching time :Half an hour. This figure shows the chromosomal pattern of Gibberella fujikuroi by CHEF separation. The agarose gel electrophoresis conditions are as follows. Gel: 0.8% Certified Megabase Agarose (Bio-rad); Running buffer: 0.5 × TBE; Temperature: 10 ° C .; Voltage: 2.0 V / cm; Running time: 120 hours; Initial switching time: 10 minutes: Final switching time :Half an hour. This figure shows the positions of the primers used in TAIL-PCR and the structure of the MAT1 region of 880621a-1 and tomino1-c. A white box indicates a region where the base sequence is 95% or more homologous. This figure shows the reaction conditions and procedures of TAIL (thermal asymmetric interlaced) -PCR and LA (long accurate) -PCR. All TAIL-PCR reagents are from New England Biolabs, while LA-PCR reagents are all from Takara. This figure shows the construction of the MAT1 region replacement vector (FIG. 5A) and the mating type test result of the transformant (FIG. 5B). This figure shows the chromosomal pattern of the transformant. The agarose gel electrophoresis conditions are as follows. Gel: 0.8% Certified Megabase Agarose (Bio-rad); Running buffer: 0.5 × TBE; Temperature: 10 ° C .; Voltage: 2.0 V / cm; Running time: 120 hours; Initial switching time: 10 minutes: Final switching time :Half an hour. This figure shows the ascending shell (FIGS. 7A and 7B), ascending (FIG. 7C) and ascospore (FIG. 7D) formed on the tomato stem.

  Hereinafter, the best mode for carrying out the invention according to the method for mating filamentous fungi will be described.

  The present invention relates to a method of mating a deficient mating fungus, wherein the deficient mating fungus as a parent strain and the mating type gene region of the parent strain are derived from a mating deficient strain of the same type that is different from the parent strain. A method is provided that comprises crossing with a replacement strain that is generated by replacement with another hybrid-type gene region, and that has a substantially homologous chromosomal pattern but a different cross-type.

  As used herein, a “cross-hybrid filamentous fungus” refers to a filamentous fungus in which no mating has been observed. Here, the filamentous fungi refers to fungi composed of hyphae and spores, and includes ascomycetes, basidiomycetes, zygotes, and the like. Since mating deficient filamentous fungi cannot be mated, genetic analysis of gene function (mating test) and breeding by mating are impossible, so if mating is possible by the method of the present invention, The progress of the analysis will be remarkable, and the breeding of bacteria with better traits will be possible with gonorrhea and the like, which will be scientifically and industrially significant.

  The incompetent filamentous fungi in the present invention are not limited to the following, but for example, important phytopathogenic filamentous fungi such as Fusarium oxysporum, Verticillium dahliae, Alternaria alternata, Penicillium italicum, etc. (Katsumi Yoneyama, 2006 (Non-patent Document 5) ), See “Incomplete fungi”), gonococci such as Aspergillus oryzae and Aspergillus spp., Or other ascomycetes such as Acremonium spp., Alternaria spp., Ambrosiella spp., Arthrobotrys spp. , Aureobasidium spp., Beauveria spp., Blastomyces spp., Botriosporium spp., Botrytis spp., Chalara spp., Cercospora spp., Cephalosporium spp., Chrysomnia spp. spp., Cryptococcus spp., Curvularia spp., Cylindrocarpon spp., Cylindrocladium spp., Drechslera spp., Epicocumcum spp., Fusarium spp., Geotrichum spp., Gliocladium spp., Graphium spp., Heliumyp , Heli scus spp., Helmithosporium spp., Hyalodendron spp., Hystoplasma spp., Isaria spp., Lemonniera spp., Metarhizium spp., Microsporium spp., Monilia spp., Monocillium spp., Nigrospora spp., Nodulipor spid. Paicilomyces spp. Trichiphyton spp., Trichoderma spp., Trichothecium spp., Tripospermum spp., Varicosporium spp., Verticillium spp., Ascochyta spp., Endothiella spp., Leucocytospora spp., Melasmia spp., Pestalotia spp., Pestalotia spp. ., Phomopsis spp., Septoria spp., Rhizoctonia spp., Sclerotium spp.

  As used herein, a “mating gene” is a gene that determines the mating type of a strain. Generally, in heterothallic filamentous fungi, there are two mating strains (for example, MAT1-1 and MAT1-2) in one species, and cell fusion occurs when these strains meet in appropriate environmental conditions. A series of reproductive processes proceeds to form progeny. The name of the mating type may differ depending on the bacterial species, but it is standard that the mating type gene region is organized as MAT1-1 and MAT1-2 (Turgeon, BG and Yoder, OC (2000 ). Proposed nom enclature for mating type genes of filam entous ascom ycetes. Fungal genet. Biol. 31: 1-5.), This specification adopts such a standard designation as a mating type (and therefore other All of the notation is also included).

  In the present invention, a parental strain of a mating deficient filamentous fungus, and a replacement in which the chromosome pattern is substantially homologous by exchanging in another mating type gene region that is different from the parent strain but derived from the same type of mating deficient strain Cross with stocks. Here, the mating type gene of the parent strain and the mating type gene of the replacement strain are two genes that determine the mating type that can cause cell fusion between strains. As exemplified above, MAT1-1 and MAT1-2 are a pair of mating type determining genes.

  The sequence information of the region containing mating type genes such as MAT1-1 and MAT1-2 is described in sequences registered in gene banks such as GenBank (US NCBI) or publications for various mating deficient filamentous fungi. Can be used. For example, the sequence information of the genome region including the MAT1 region of Fusarium oxysporum and its flanking regions is registered as GenBank AB011379, AB011378, and the like. Moreover, the sequence information regarding the genome sequence including the mating type gene region of Aspergillus oryzae is registered in Galagan et al., (2005) Nature Vol 438: 22-29.

  The present invention utilizes the replacement (for example, recombination or substitution) of the mating type gene region between the same type of filamentous fungi, particularly between strains. In the examples described later, among Fusarium oxysporum, between the 880621a-1 strain and the tomino1-c strain, the MAT1-1 region (about 4.5 kb) of the 880621a-1 strain and its adjacent regions (each about 2.5 to 95% or more (preferably 97%), depending on the genomic region containing 3.0 kb) and each of the MAT1-2 region of the tomino1-c strain and each of the adjacent regions of the MAT1-1 region (about 4.5 kb) of the 880621a-1 strain As described above, more preferably 98% or more of the genomic region including both adjacent regions (about 2.5 to 3.0 kb) having homology is recombined by the homologous recombination method (see FIGS. 3 and 5). In order to carry out homologous recombination, PCR (Polymerase Chain Reaction) using primers for the genomic region to be inserted (total length: about 7-10 kb) consisting of the mating type gene region and its adjacent regions in the target filamentous fungus After the amplification product is incorporated into a vector such as a plasmid, it is cleaved with a restriction enzyme to make a single strand, and this is used to recombine with the homologous region of the genome of the parent strain. At this time, a protoplast (or spheroplast) is produced from the parent strain, and a plasmid-derived DNA for homologous recombination is introduced therein. Such a method can be implemented by a general method. Protoplasts (or spheroplasts) are obtained by treating a cultured filamentous fungus with a cell wall lytic enzyme (for example, cellulase, protease, pectinase, chitinase, lysozyme, etc.) to partially or totally lyse the cell wall of the fungus. Obtainable. A polyethylene glycol (PEG) method, an electroporation method, or the like can be used for introducing foreign DNA (here, a plasmid) into protoplasts (or spheroplasts). The molecular weight of PEG that can be used in the PEG method is generally in the range of about 4,000 to about 10,000. Electroporation is a technique in which fine holes are made by applying an electric pulse to a cell suspension to introduce foreign DNA into cells. Any plasmid can be used as long as it is for transformation of filamentous fungi, and includes, for example, pCSN43 (Staben, C. et al., (1989) Fungal Genet. Newsl. 36: 79-81). Plasmids include promoters, enhancers, selectable markers (eg, drug resistance markers (eg, G418, oligomycin, brassicidin S, etc.), auxotrophic markers (eg, pryG, argB, tryC, etc.), thymidine kinase (tk) gene, etc.) Multiple cloning sites can be included.

  In the present invention, in order to achieve successful mating, the chromosome pattern of the parent strain and the chromosome pattern of the mating partner replacement strain must be substantially homologous. Here, the chromosome pattern can be confirmed by CHEF electrophoresis as described in Examples described later (see FIG. 6). Here, “substantial” means that they are not completely matched but visually matched. The chromosome pattern means EFHEH of the number and size of chromosomes of the strain that can be identified by CHEF electrophoresis. It is synonymous with karyotype.

  Crossing between the transformed strain (replacement strain) produced as described above and the parent strain is carried out by culturing both strains together in a medium supplemented with nutrients necessary for a solid medium such as an agar medium. Culturing is conducted by bringing the target to be infected into contact, and a strain having a trait different from the parent strain is selected by crossing. Differences in traits can be determined, for example, by the formation of a cyst-like structure, the formation of internal cysts and cysts, and the release of cysts from the cyst.

  Strains with homologous chromosomal patterns and different mating genes are not necessarily limited to mating gene replacement strains of the same strain. Even if the chromosome pattern is homologous (including cases where they are not the same and similar), it is only necessary that the homologous chromosomes can be paired.

  The present invention is not limited to the best mode for carrying out the above-described invention, and various other configurations can be adopted without departing from the gist of the present invention.

  Next, specific examples of the present invention will be described. However, it goes without saying that the present invention is not limited to these examples.

In order to investigate the genetic background in Fusarium oxysporum, a mating-deficient bacterium, CHEF (Clamped Homogeneous Electric Fields) electrophoresis was performed, and chromosomal patterns were investigated by karyotype analysis. The strains used were 4 strains of F. oxysporum f. Sp. Lycopersici MAT1-1 (NBRC6531, 880621a-1, MAFF103038, MAFF103036) and 2 strains of MAT1-2 (tomino1-c, F -1-1). Prepare protoplasts for each strain (Protoplast preparation method will be described later), and reconstitute with STE (1 M Sorbitol, 25 mM Tris-HCl, 50 mM EDTA, pH 7.5) to a concentration of 1.0 x 10 ⁸ protoplasts / ml or more. Suspended. 1.4% InCert agarose (low melting point agarose, FMC) was melted in an equal amount of STE, mixed with the protoplast solution, and the protoplast was embedded. The resulting plug was sliced to a thickness of about 3 mm and incubated in SE buffer (1 M Sorbitol, 50 mM EDTA, pH 8.0) at 55 ° C. for 36 hours. The plug was removed and incubated in NDS (100 mM Tris, 500 mM EDTA, 1% N-laurylsarcosyl) containing 2 mg / ml Proteinase K for an additional 36 hours at 55 ° C. to disrupt the cells and expose the chromosomes . After immersing the plug in 50 mM EDTA and shaking for 10 minutes, the EDTA was discarded. This operation was repeated three times, and the completed plug was used for CHEF Mapper (Bio-rad). The electrophoresis conditions are shown in FIG. It can be seen that each strain has a different chromosome pattern (FIG. 1). In particular, there is no similar chromosomal pattern between MAT1-1 and MAT1-2 strains that can be crossed.

  Gibberella fujikuroi, which is closely related to F. oxysporum, has a group called mating population, and mating occurs only between strains of different mating types in the same mating group. When the chromosomes of G. fujikuroi strains were separated by CHEF in the same manner as described above, within one mating group, similar patterns were shown even among the outcrossing strains (Fig. 2).

  However, even if F. oxysporum strains have the potential to cross, each strain has a different chromosomal pattern, so even if a cross-bred strain starts mating, the process of meiosis from chromosomal pairing It was suggested that this could be a failure, and that this was the cause of F. oxysporum mating failure. It is speculated that this is because F. oxysporum has repeated asexual growth without mating so far.

  The above shows that in F. oxysporum, each strain has a different chromosomal pattern, indicating the absence of mating partners, and strongly supporting (3), which was speculated above as the cause of mating failure. To do.

  Therefore, in F. oxysporum, a strain having a homologous chromosome pattern but having a different mating type was artificially produced, and a mating test was attempted using it. As shown in FIG. 3, since both sides of the MAT1 region are homologous to the MAT1-1 strain and the MAT1-2 strain, it was intended to completely replace the MAT1 region based on the twice homologous recombination method. As a result, a new type of inserted strain different from the conventionally produced MAT1 region inserted strain described above is generated. In addition, the replacement of the gene region by twice homologous recombination itself is a universal technique.

  In order to replace a gene region by twice homologous recombination, base sequence information of about 0.5 to 5 kb adjacent regions on both sides of the gene region is required. Then, the base sequence of the peripheral region of the MAT1 region of F. oxysporum and the base sequence of the adjacent region (Arie et al. 2000; GenBank AB011379, AB011378) that had already been determined was determined. For this purpose, TAIL-PCR (Non-patent Document 7 (Arie et al. 2000)) was performed using F. oxysporum 880621a-1 strain (mating MAT1-1) genomic DNA as a template.

  Perform TAIL-PCR several times under the conditions shown in Fig. 4 using specific primers designed based on the nucleotide sequence registered in GenBank AB011379 and seven non-specific primers (Table 1). The nucleotide sequence of the adjacent region of 2.8 kb and about 2.6 kb downstream was determined (FIG. 3). These base sequences showed high homology of 95% or more with the same region of F. oxysporum tomino 1-c strain (mating MAT1-2) (FIG. 3).

  LA-PCR was performed using primers X and Y (Table 1) and F. oxysporum 880621a-1 genomic DNA as a template under the conditions shown in FIG. 4, and the resulting MAT1 region (MAT1-1- DNA fragment containing 1 gene) is purified using TOPO XL Gel purification kit (Invitrogen) and introduced into the pCR-XL-TOPO (Invitrogen) vector to obtain the MAT1 region replacement vector pMAT1-1 (Fig. 5A) .

The mycelium of tomino 1-c grown on PSA (potato sucrose agar) medium was cut out together with the medium, put into 50 ml of PSB (potato sucrose broth) liquid medium, and cultured with shaking at 120 rpm for 5 days. The mycelium was removed by filtration through double sterilized gauze, centrifuged at 3000 rpm for 10 minutes, the spores were submerged at the bottom of the tube, and the supernatant was discarded. Sterile RO water was added to thoroughly wash the precipitated spores, and the mixture was centrifuged again at 3000 rpm for 10 minutes. The supernatant was discarded, and an appropriate amount of sterile RO water was added to form a spore suspension. 1.0 × 10 ⁷ spores were added to 100 ml of PSB and allowed to stand at room temperature. After about 36 hours, germinated spores were collected using a Buchner funnel and filter paper, suspended in 50 ml of sterile RO water, centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. After the same work sterile RO water was again added, and the recovered germinated spores were resuspended in 1.2 M MgSO ₄ in 10 ml, the enzyme solution of equal volume to it (2% Lysing Enzymes, 2% Driselase (cellulase・ Protease-pectinase complex enzyme (manufactured by Kyowa Hakko Kogyo Co., Ltd.), 0.0005% chitinase was added, and the mixture was incubated at 70 rpm for 3 hours. , 10 mM CaCl ₂ · 2 H ₂ O), and centrifuged at 3000 rpm for 20 minutes, and the precipitated protoplasts were collected and resuspended in 120 μl of STC to prepare a protoplast solution. Adjusted to 1.0 x 10 ⁸ protoplasts / ml or more.

  pMAT1-1 and pCSN43 having a hygromycin B resistance gene were each extracted with QIAfilter Plasmid Midi kit (QIAGEN), and 20 μg each was single-stranded with restriction enzyme Not I. Both plasmid solutions were heated at 65 ° C. for 20 minutes or longer to inactivate the restriction enzyme, and then the entire amount was added to the protoplast solution described above, and left on ice for 20 minutes. To this solution, 60% PEG (polyethylene glycol) 4000 solution was added stepwise while mixing in the order of 200 μl, 200 μl, and 800 μl, and left on ice for 20 minutes to incorporate both plasmids into the cells. 6 ml of STC was added to dilute the PEG, mixed with RM medium cooled to about 40 ° C., and dispensed into a petri dish. Approximately 24 hours later, 100 μg / ml hygromycin B (non-transformant F. oxysporum-sensitive antibiotic) was overlaid with 1.0% water agar, and the strain that had grown in the upper layer was used as the transformant. Used for. This transformation is performed using two plasmids simultaneously. It is known that two plasmids are simultaneously incorporated into the genome with high efficiency, and this method is called cotransformation. In this example, pCSN43 that makes the filamentous fungus resistant to hygromycin is used as one of the two, so that the transformant can be selected using a medium supplemented with hygromycin B.

  Genomic DNAs of the two transformants obtained were extracted, PCR was performed with combinations of A + B and C + D primers (Table 1, FIG. 5), and the mating type was investigated. As a result, MAT1-2 of MAT1-2 and MAT1-1 of 880621a-1 were replaced by TOM1-1 and MAT1-1 was not replaced with the strain tomino1-c (ΔMAT1-2; MAT1-1) [hereinafter referred to as R1]. It was confirmed that the strain tomino1-c (MAT1-2; MAT1-1) [hereinafter referred to as E1] inserted in the ectopic site was obtained (FIG. 5B).

  The chromosomes of these transformants were separated by CHEF electrophoresis as described above, and the chromosome pattern was investigated. As a result, it was found that the chromosome pattern of the transformants (R1 and E1) was homologous to the parent strain (tomino 1-c) (FIG. 6).

  As a result, in F. oxysporum, a strain (R1) having a chromosomal pattern homologous to tomino 1-c (MAT1-2) but having a different mating type (MAT1-1) could be artificially produced. The feature of this example is not that strains having different mating types were simply obtained, but that strains having different mating types were produced while homogenizing chromosome patterns.

  A crossover test of the transformant (R1, MAT1-1) produced so far and its parent strain tomino 1-c (MAT1-2) was performed. About 3 cm of water agar was added to the bottom of the test tube and autoclaved. Both strains are cultured for about 5 days on a PSA or MMA plate medium, and the tip of the fungus cell with good growth is cut out about 1 cm square together with the medium and placed on water agar in the above-mentioned test tube. It was. Next, the air roots of tomatoes (variety: Ponderosa) grown in sterile soil for more than a month were removed, and the cut pieces of about 5 cm length were washed with shaking in 70% ethanol for 5 minutes. Furthermore, it was sterilized by shaking up and down for 5 minutes in an aqueous sodium hypochlorite solution adjusted to an effective chlorine concentration of 1%. The aqueous sodium hypochlorite solution was discarded and washed with sterile RO water. This washing operation was repeated three times. Place the obtained surface-sterilized tomato stalk vertically so that the lower end of the stalk reaches the water agar in the test tube, plug the test tube into a silico, and set it at 25 ° C in a 12 hour light / dark cycle. In culture.

  After 14 days of culturing, it was confirmed that an ascocar, an organ specific to the sexual generation, was formed in a test tube containing a combination of tomino1-c × R1. Further observation under a microscope revealed ascending sac and 8 ascospores inside the sac. Therefore, it was shown that mating was performed under the above-mentioned conditions (FIG. 7).

  Filamentous fungi require different environmental conditions for each species for mating. In this example, it was also the first report that the above-mentioned was found to be suitable conditions for mating F. oxysporum, which is significant.

  As described above, by producing strains having very close genetic background and different mating types and crossing them under suitable mating conditions, mating of deficient mating fungi became possible. This is also the first proof that F. oxysporum has fully maintained mating functions.

  The E1 strain carrying the double-crossing gene formed the same complete generation as described above when only E1 fungus was planted under the above-mentioned conditions. The E1 strain used here is reasonable because it retains the double-crossing gene in the same manner as the homothallic strain described in the background art.

  In the sequence listing, 880621a-1 MAT1-1 locus is a base sequence including the MAT1-1 region, and the MAT1-1 region is 2897-7501 bp. tomino1-c MAT1-2 left is a sequence corresponding to 1219-2754 bp of 880621a-1, and tomino1-c MAT1-2 right is a sequence corresponding to 7556-10302 bp of 880621a-1.

From the above, according to the present embodiment, the following effects can be obtained.
In the future, the full generation of Fusarium oxysporum can be named.
In Fusarium oxysporum, genetic functions (including plant pathogenicity and host specificity) can be analyzed by mating based on genetic techniques.
Genetic methods can be used to analyze pathogenic factors in the same way for other dysmorphic phytopathogenic fungal species.

A similar technique can be applied to non-phytopathogenic filamentous fungi (for example, Aspergillus oryzae), and it is possible to form a complete generation and to perform genetic analysis such as gene function by mating tests.
It is an industrially useful mating deficient bacterium (Aspergillus oryzae, Penicillium spp.), And it is possible to improve traits by breeding, that is, breeding.

  An industrially useful mating deficient bacterium (for example, Aspergillus oryzae, Penicillium spp.) Enables improvement of traits by mating, that is, breeding.

[0007]
Non-Patent Document 10: Sharon A. et al. , Yamaguchi K .; , Christiansen S .; Horwitz B. A. , Yoder O. C. , Turgeon B. G. , 1996. An sexual fungus has the potential for sex development. Mol Gen Genet 250: 11-122
Non-Patent Document 11: Yun, Sung-Hwan, Arie, Tsutomu, Kaneko, Isao, Yoder, O. C. Turgeon, B .; Gillian, 2000. Molecular Organization of Matching Type Loc in Heterothallic, Homothallic, and Axial Gibbellaella / Fusarium Species. Fungal Genet Biol 31: 7-20
Non-Patent Document 12: Shusuke Imai, Toru Teraoka, Tsutomu Arie 2007. “Creation of a replacement gene for the mating type gene region (MAT1) of Fusarium oxysporum” Abstracts of the 2007 Kanto Division Meeting of the Japanese Society of Plant Pathology 8
DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention As described above, in Fusarium oxysporum, which is a mating failure, there is no partner in the close population that can be mated, which may cause the mating failure Suggested sex. That is, it suggests the possibility of the above (3) in the background art section presumed as the cause.
Therefore, the development of a new method for mating deficient mating fungi is desired.
An object of the present invention is to provide a novel method for mating deficient mating fungi.
Means for Solving the Problems In order to solve the above-mentioned problems and achieve the object of the present invention, the present inventors have determined that the mating-deficient filamentous fungus that is a mating strain and the mating-type gene region of the parent strain, Crossing with a replacement strain having a different chromosomal type while having a substantially homologous chromosomal pattern by replacement with another hybrid-type gene region that is different from the parent strain but derived from the same type of hybrid deficiency strain By the method, the method of making a mating deficient filamentous fungus possible was discovered.
Here, although not necessarily limited, it is preferable that the filamentous fungus is a phytopathogenic filamentous fungus or a koji mold. Moreover, although not necessarily limited, it is preferable that a phytopathogenic filamentous fungus is Fusarium oxysporum. Moreover, although not necessarily limited, it is preferable that replacement | exchange is performed by homologous recombination.
The present invention includes a Fusarium oxysporum tomino 1-c strain, a Fusarium oxysporum tomino 1-c strain MAT1-2 and a Fusarium oxysporum 880621a-1 MAT1-1 strain transformed into a Fusarium oxysporum tom1R1 transformant (R) Is a method of mating.
The present invention is a method for culturing a mating deficient strain in which a mating type gene region derived from another mating deficient strain is inserted.
The present invention is a method for culturing a Fusarium oxysporum tomino1-c strain (referred to as transformant E1) into which MAT1-1 of Fusarium oxysporum 880621a-1 strain has been inserted.
The present invention is produced by replacing a parental mating deficient fungus and a mating type gene region of the parent strain with another mating type gene region different from the parent strain but derived from the same type of mating deficient strain. In addition, it is a method of analyzing gene function (including plant pathogenicity and host specificity) based on genetic methods by mating with strains that have substantially homologous chromosome patterns but different mating types. .
The present invention relates to a Fusarium oxysporum tomino 1-c strain, a Fusarium oxysporum tomino 1-c strain MAT1-2 and a Fusarium oxysporum 880621a-1 strain MAT1-1, and an inherited strain of Fusarium oxysporum tominoc 1 to 1 This is a method for analyzing gene function (including plant pathogenicity and host specificity) based on a genetic approach.

Claims (5)

  A method of mating an incompetent filamentous fungus, wherein the mating incompetent filamentous fungus that is a parent strain and another mating type gene region of the parent strain are different from the parent strain but derived from the same incompatibility strain Said method comprising crossing strains produced by transposition in the type gene region, with strains having substantially different chromosomal patterns but different mating types.   The method according to claim 1, wherein the filamentous fungus is a phytopathogenic filamentous fungus, gonococcus, or other ascomycetes.   The method according to claim 1 or 2, wherein the phytopathogenic fungus is Fusarium oxysporum.   The method according to any one of claims 1 to 3, wherein the mating type gene region or another mating type gene region is either MAT1-1 or MAT1-2.   The method according to any one of claims 1 to 4, wherein the replacement of the mating type gene region is performed by homologous recombination.

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