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Abstract 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. The chi1 gene was introduced into tobacco by the Agrobacterium 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 the chi1 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 pathogens Sclerotinia sclerotiorum and Botrytis cinerea revealed that the symptoms observed with these two were remarkably suppressed as compared with the control leaves.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.

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 ß-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.