Naoki Takaya

A chitinase gene, chiB, involved in the autolytic process of Aspergillus nidulans.

Chitinases are thought to be involved in the morphogenesis and autolysis of filamentous fungi. We cloned a gene (chiB) encoding a class V chitinase from Aspergillus nidulans. ChiB expressed in Escherichia coli had chitin-hydrolyzing activity, indicating that chiB encoded a chitinase. Deletion of chiB affected neither germination efficiency nor hyphal growth rate, but considerably reduced the intracellular and extracellular chitinase activities. The decrease in hyphal dry weight during autolytic phase was slower in the mutant than in the wild-type strain. Western blot analysis indicated that the quantity of ChiB significantly increased when the wild-type mycelia were starved for carbon sources, a condition that induced hyphal autolysis. These results suggest that chiB plays an important role in the autolytic process in A. nidulans.

A fungal chitinase gene from Rhizopus oligosporus confers antifungal activity to transgenic tobacco

We have studied whether a chitinase involved in cell autolysis of a filamentous fungus,Rhizopus oligosporus, can operate as an antifungal defense system in tobacco. Thechi1 gene was introduced into tobacco by theAgrobacterium tumefaciens leaf disc system. Among 22 transgenic tobacco plants, 2 were selected and their individual homozygous progeny, Tch1-1 and Tch2-1, were studied. Chitinase activity in the extracts of young leaves from Tch1-1 or Tch2-1, in which thechi1 gene product was detected by Western blot analysis, was three- to four-fold higher than that from the control plants. A fungal infection assay on the leaves infected with the discomycete pathogensSclerotinia sclerotiorum andBotrytis cinerea revealed that the symptoms observed with these two were remarkably suppressed as compared with the control leaves.

Cloning and characterization of two 3-phosphoglycerate kinase genes of Rhizopus niveus and heterologous gene expression using their promoters

Two 3-phosphoglycerate kinase genes (pgk1 and pgk2) were cloned from Rhizopus niveus. It was deduced that both pgk genes have two introns. They have open reading frames of 1355 bp and 1356 bp, and code for proteins of 417 and 416 amino acids, respectively. The first introns of both genes are located at similar positions as those of pgk genes from other fungi based on the deduced amino-acid sequences of PGK proteins. The position of their second introns was similar to that of the seventh intron of the human pgk gene. The deduced amino-acid sequences of PGK proteins show high identity (64.8–72.2%) to those of PGKs of other filamentous fungi. When the promoters of each of the pgk genes were fused to the E. coli β-glucuronidase (GUS) gene and introduced into R. niveus, significant GUS activities were detected in the cell lysates of the transformants, suggesting that GUS protein was expressed under the control of both pgk gene promoters in R. niveus. GUS activity was induced by glucose but not by glycerol, indicating that expression of R. niveus pgk genes was regulated by the carbon source.

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5′-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli β-glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.

Analysis of the 3-phosphoglycerate kinase 2 promoter in Rhizopus niveus

Promoter analysis was performed on the Rhizopus niveus 3-phosphoglycerate kinase 2-encoding gene (pgk2), one of the two pgk genes (pgk1 and pgk2) from this filamentous fungus sequenced so far. Deletion mutants of the promoter region were fused to the Escherichia coli uidA gene (which codes for beta-glucuronidase; GUS), and introduced into R. niveus to measure the intracellular GUS activities of the transformants. Deletion of the sequence between nt -174 to -133 (numbers indicate the position from the putative translation start codon) caused a significant decrease in the ratio of the GUS activity of the transformant cultured in glucose medium compared to that in glycerol medium. In this region, a 21-nt sequence which is well conserved between pgk1 and pgk2 is present. When it was inserted into the promoter region of the uninducible gene encoding RNase Rh of R. niveus, ligated in front of uidA and introduced into R. niveus, the GUS activity of the transformant was greatly induced by glucose, but less by glycerol. We therefore suggest that the 21-nt sequence is a glucose-inducible transcriptional activator of R. niveus. This is the first report on a transcriptional activator in zygomycetes.