Tip Growth of Plant-Pathogenic Fungi and Tip-Chitin Metabolism

日本方面研究了几丁质合成酶在丝状真菌顶端生长中的作用。从子囊菌丝状真菌中分离到4个几丁质合成酶基因（chsA、chsB、chsC和chsD），从少孢根霉丝状真菌中分离到2个基因（chs1和chs2）。当我们对拟南芥基因组中的4个几丁质合成酶基因进行破坏时，我们发现除了chsB基因破坏物外，这些破坏物的表型与野生型菌株没有差异。chsB基因干扰物的尖端生长出现严重缺陷。我们从chsA、chsC和chsD基因干扰物中构建了每一种双基因干扰物。chsA和chsC双基因干扰物和chsA和chsC双基因干扰物的出现频率分别为野生型菌株的0.002%和10%。为检验chsB基因产物在条件下的功能。chsB基因的开放阅读框与alcA基因启动子融合较多，alcA基因启动子的表达受培养基中碳源的调控。在分生阶段抑制chsB基因的表达，分生次数减少约为野生型菌株的十分之一。因此，所有chs基因产物在分生阶段都是必需的。chsA和chsC双基因干扰物的菌丝密度非常罕见。说明chsA和chsC基因产物也参与了菌丝的生长。用PCR方法分离出一段几丁质酶基因（chiA），并对该基因进行了破坏。chiA基因的干扰物生长并形成分生孢子和野生型菌株。为了研究该基因的表达期，我们将大肠杆菌lacZ基因与chiA基因融合，并将其引入到拟南芽孢杆菌中。chiA基因在分生期的表达强度是菌丝生长期的2倍。因此，chiA基因产物在这两个阶段都起作用。在韩国，为了构建植物病原真菌稻瘟病菌（pyricaria oryzae）的宿主-载体体系，分离出了稻瘟病菌的一些营养不良突变体。他们还从P.oryzae的基因组DNA中分离出了几丁质合成酶基因的PCR片段，该基因在国内正在研究其在植物致病性中的作用。这些工作是我们在日本的团队和韩国团队合作完成的。少

In Japanese side, we investigated the functions of chitin synthases in tip growth of filamentous fungi. We have isolated four chitin synthase genes (chsA,chsB,chsC,and chsD) from Aspergillus nidulans which belongs to Ascomycete filamentous fungi and two genes (chs1 and chs2) from Rhizopus oligosporus which belongs to Zygomycete filamentous fungi. When we disrupted each one of four chitin synthase genes in the genome of A.nidulans, we detected no difference of the phenotype of these disruptants from that of wild type strain except for the chsB gene disruptant. The tip growth of chsB gene disruptant was severely defected. We constructed each kind of double gene disruptant from the chsA,chsC,and chsD gene disruptants. The frequency of conidiation of the chsA and chsC double gene disruptant and chsA and chsC double gene disruptant were 0.002% and 10% of that of wild type strain, respectively. To test the function of chsB gene product in the conidiation, .open reading frame of chsB gene was … More fused to alcA gene promoter whose expression was regulated by the carbon source in the medium. When the expression of chsB gene was repressed at the conidiation stage, the frequency of conidiation decreased about one-tenth of that of wild type strain. Thus, all of chs gene products are required at the stage of conidiation. The density of hyphae of chsA and chsC double gene disruptant was very rare. It suggests that the gene products of chsA and chsC also function in the hyphae growth.We have isolated a chitinase gene (chiA) from A.nidulans by PCR method and disrupted the gene. The disruptant of chiA gene grew and formed conidia as well as wild type strain. To investigate the phase of expression of the gene, we fused lacZ gene of Escherichia coli to chiA gene and introduced it into A.nidulans. The expression of chiA gene at the conidiation stage was twice as strong as the hyphae growth stage. Thus, it is suggested that chiA gene product function at the both stage.In Korean side, to construct the host-vector system in the plant pathogenic fungi, Pyricularia oryzae, they have isolated some auxotrophic mutants of P.oryzae. They have also isolated a PCR fragment of chitin synthase gene from genomic DNA of P.oryzae whose function in plant pathogenicity is being investigated in Korea.These works have been done in a cooperative manner between our group in Japan and the Korean group. Less