The Japanese pear pathotype of Alternaria alternata causes black spot of Japanese pear by producing a host-specific toxin known as AK-toxin. Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for toxin biosynthesis. Protoplasts of a wild-type strain were treated with a linearized plasmid along with the restriction enzyme used to linearize the plasmid. Of 984 REMI transformants recovered, three produced no detectable AK-toxin and lost pathogenicity on pear leaves. Genomic DNA flanking the integrated plasmid was recovered from one of the mutants. With the recovered DNA used as a probe, a cosmid clone of the wild-type strain was isolated. Structural and functional analyses of an 8.0-kb region corresponding to the tagged site indicated the presence of two genes. One, designated AKT1, encodes a member of the class of carboxyl-activating enzymes. The other, AKT2, encodes a protein of unknown function. The essential roles of these two genes in both AK-toxin production and pathogenicity were confirmed by transformation-mediated gene disruption experiments. DNA gel blot analysis detected AKT1 and AKT2 homologues not only in the Japanese pear pathotype strains but also in strains from the tangerine and strawberry pathotypes. The host-specific toxins of these two pathotypes are similar in structure to AK-toxin. Homologues were not detected in other pathotypes or in non-pathogenic strains of A. alternata, suggesting acquisition of AKT1 and AKT2 by horizontal transfer.

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Strains of the plant-pathogenic bacterium Xanthomonas axonopodis pv. citri are differentiated into two groups with respect to aggressiveness (normal and weak) on Citrus grandis cultivars but not on other Citrus species such as Citrus sinensis. Random mutagenesis using the transposon Tn5 in X. axonopodis pv. citri strain KC21, which showed weak aggressiveness on a C. grandis cultivar, was used to isolate mutant KC21T46, which regained a normal level of aggressiveness on the cultivar. The gene inactivated by the transposon, hssB3.0, was shown to be responsible for the suppression of virulence on C. grandis. Sequence analysis revealed it to be a new member of the pthA homologs, which was almost identical in sequence to the other homologs except for the number of tandem repeats in the central region of the gene. hssB3.0 appears to be a chimera of other pthA homologs, pB3.1 and pB3.7, and could have been generated by recombination between these two genes. Importantly, in X. axonopodis pv. citri, hssB3.0 was found in all of the tested isolates belonging to the weakly aggressive group but not in the isolates of the normally aggressive group. Isolation of the virulence-deficient mutant KC21T14 from KC21, in which the pathogenicity gene pthA-KC21 was disrupted, showed that hssB3.0 induces a defense response on the host but partially interrupts canker development elicited by the pathogenicity gene in this bacterium.

To characterize homologous recombination of transforming DNA in the filamentous fungus Alternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. The A. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus. BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Mel-) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of the BRM1 segment in the transforming DNA. Restriction enzyme digestion within the BRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of the BRM1 segments. Plasmids carrying both BRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Mel- transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within the BRM1 segment was used, almost all transformants were Mel-. These results indicate that homologous integration of circular molecules in A. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.

Susceptibility to Xanthomonas axonopodis pv. citri of a citrus cybrid, in which the nuclear and cytoplasmic genomes were derived from Citrus sinensis and C. unshiu, respectively, was evaluated. Bacterial growth in the leaves of the cybrid was similar to that in C. sinensis after pin-prick inoculation throughout the experiment, whereas growth was greater than that in C. unshiu from 8 days after inoculation. Lesion expansion and pustules that later developed from the lesions on the cybrid and on C. sinensis also appeared to be greater than those on C. unshiu. The incidence of citrus canker disease caused by the bacteria on the cybrid trees was in the field was equivalent to that on C. sinensis but was severer than on C. unshiu. These results indicate that the nuclear genome of the cybrid plays a critical role in susceptibility to citrus canker disease. However, the pathogenicity gene pthA of the bacteria is not likely to be involved in the difference in susceptibility to the bacteria between C. unshiu and C. sinensis because their susceptibility to a pthA-deficient mutant of the bacteria also differs.