Yasuo Itoh

AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration

ABSTRACT Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.

Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli

A high frequency transformation system has been developed for Penicillium paxilli using pAN7-1. Up to 44% of the primary transformants were heterokaryons. Loss of hygromycin resistance was observed in primary transformants that were sub-cultured on non-selective media, but single spores of these primary transformants were mitotically stable on both selective and non-selective media. A molecular analysis of the transformants generated showed that 78% had single-site integrations, with half of these containing a single copy of pAN7-1. CHEF-gel electrophoresis showed that P. paxilli has at least six chromosomes with a total genome size of about 23.4 Mb.

Effect of de-phosphorylation of linearized pAN7-1 and of addition of restriction enzyme on plasmid integration in Penicillium paxilli

Abstract As part of a study to investigate the pathways of plasmid pAN7-1 integration in Penicillium paxilli, a molecular analysis of 90 different integration events was carried out. Twenty out of forty five integration events analyzed from transformants obtained without the addition of restriction enzyme to the transformation reaction mixture were single-copy integrations, whereas the remaining 25 were tandem-repeat integrations. The addition of restriction enzyme resulted in a shift in this ratio in favour of single-copy integration events. Analysis of the 33 tandem-repeat integration events showed that the orientation of the plasmid copies was not random, with 88% organized as tandem head-to-tail arrays. De-phosphorylation of linearized pAN7-1 did not affect the frequency with which multiple copies were integrated. This suggests that the predominant mechanism for the generation of tandem repeats in P. paxilli is by homologous recombination rather than in vivo ligation of linearized plasmids.

Hyphal anastomosis and complementary growth of fused cells in Alternaria alternata

Hyphal anastomosis and complementary growth of fused cells inAlternaria alternata were investigated. Sixty-four experimental isolates were divided into anastomosis-positive (A+) and anastomosis-negative (A−) groups based on their self-anastomosing ability. Nonself-anastomoses (interisolate) were readily distinguished from self-anastomoses (intraisolate) by using a mixed culture of conidia and hyphal fragments prepared from the respective isolates. Nonself-anastomosis occurred only between the A+ isolates irrespective of their pathogenicity and geographic origin. The breakdown of cell walls and the establishment of cytoplasmic continuity between fused cells were microscopically observed only in the self-anastomoses. The frequency of the nonself-anastomosis was, in general, lower than that of the self-anastomosis. For analysis of complementation between the fused cells, mutants doubly marked with auxotrophy and hygromycin B (Hyg) resistance were prepared from wild-type isolates. The identity of the mutants was confirmed by RAPD analysis using three arbitrary primers. Complementary growth occurred only between an A+ isolate and its mutant(s) on a minimal medium containing Hyg, demonstrating that the self-anastomoses resulted in perfect cell fusions and the nonself-anastomoses were contact or imperfect fusions.

Heterologous and homologous plasmid integration at a spore-pigment locus in Penicillium paxilli generates large deletions.

Mutations in a spore pigmentation locus (brs; brown spore) in Penicillium paxilli were isolated at a relatively-high frequency (0.17%) following integrative transformation of the hygromycin-resistance plasmid pAN7-1. A molecular analysis of four independently-isolated Brs- mutants showed that all contained pAN7-1 integrated at a single-site that was unique for each mutant. A previously-described Brs- mutant, YI-34 (Itoh et al. 1994), was a two-site integration. Three of the mutants had multiple copies of pAN7-1 arranged in head-to-tail tandem arrays. A 9.6-kb BamHI junction fragment was cloned from one of these, YI-33, by plasmid rescue and used to isolate two overlapping lambda clones, λWB33-1 and λWB33-2, that span about 30 kb in the region of the wild-type locus. When genomic digests of the five Brs- mutants were probed with these lambda clones all of them were found to contain an extensive deletion through a common region of the P. paxilli genome. Subsequent attempts to generate one-step gene replacements within a 4.5-kb EcoRI fragment at the wild-type locus resulted in the isolation of Brs- mutants at a frequency of 1.6%, but all mutants with this phenotype were also found to contain an extensive genomic deletion. Therefore, a common outcome of both heterologous and homologous plasmid integration at this locus is deletion formation.

An appropriate increase in the transcription of Aspergillus nidulans uvsC improved gene targeting efficiency

Gene targeting to knock out the activity of specific genes has become important due to recent progress in genomics research. But this technique is still unavailable for many organisms, including economically important microorganisms, due to the high background of ectopic integration during genetic transformation. Strategies to improve targeting efficiency have included manipulating the expression of genes that are involved in homologous recombination. In this study, transcription of Aspergillus nidulans uvsC was elevated using the promoter sequences of the glyceraldehyde-3-phosphate dehydrogenase and Taka-amylase A genes from A. nidulans and A. oryzea respectively. Although a several-fold increase in the efficiency of targeting was observed at 3 loci, mycelial growth was suppressed in strains that had higher levels of uvsC transcription. These results suggest that uvsC is a rate-limiting factor in gene targeting, and that the increased efficiency of this targeting is hindered by a negative effect of increased transcription on cell proliferation.

Suppression of tandem-multimer formation during genetic transformation of the mycotoxin-producing fungus Penicillium paxilli by disrupting an orthologue of Aspergillus nidulans uvsC

An orthologue of Aspergillus nidulans uvsC and Saccharomyces cerevisiae RAD51 was cloned from the filamentous fungus, Penicillium paxilli. A mutation in uvsC causes UV sensitivity during germination. The product of RAD51 is involved in meiotic recombination and DNA damage repair. The deduced amino acid sequence of the product of this gene (Pprad51) shared 92% identity with UVSC. Site-specific disruption of pprad51 showed a significant effect for extra-cellular DNA integration. Transformation of the null mutant with pII99, which confers geneticin resistance, resulted in a shift from a predominance of direct repeats at a single site to single copies when compared with a control strain. A copy-number effect of integrated pII99 for geneticin selection was suggested as the frequency of direct repeat formation was less when selected at a lower concentration in the control strain. However, such an effect was not observed in the null mutant, further supporting an involvement of Pprad51 in direct repeat formation.

Host-Specific Toxin Deficient Mutants of the Tomato Pathotype of Alternaria Alternata Obtained by Restriction Enzyme-Mediated Integration

Gene tagging utilizing the heterologous integration of plasmids is now widely used to clone genes where little biochemical information is available. The development of the so-called REMI (restriction enzyme mediated integration) method has substantially improved this technique and some genes important for fungal pathogenicity have already been tagged and cloned. We demonstrate here the isolation of host-specific toxin deficient mutants from Alternaria alternata by the REMI method. The apple and tomato pathotypes of A. alternata that are producers of AM- and AAL-toxin, respectively, were first examined for the effect of addition of restriction enzyme to the transformation. A significant increase in transformation frequency by addition of enzyme was observed in these pathotypes, and a simple integration profile was confirmed in the tomato pathotypes. AAL-toxin deficient mutants were isolated from a library of transformants obtained with the addition of enzymes. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity. Detailed molecular analysis of these mutants, rescue of flanking genomic sequence, and gene disruption experiments are now in progress.

Improvement of gene targeting in Aspergillus nidulans with excess non-homologous fragments.

Efficiency of gene targeting was increased more than 10-fold when an excess molar ratio of non-homologous fragments was added in the transformation of Aspergillus nidulans. In the targeted transformants, integration of such fragments into the host chromosome was a rare event. Hence, our approach proved practical in terms of producing successful targeting events without disturbing the host chromosomes.

A Catalytic Domain of a Cyclic Peptide Synthetase that is Specific for the Apple Pathotype of Alternaria Alternata and its Possible Involvement in Host-Specific AM-Toxin Production

PCR products were obtained from the apple pathotype of Alternaria alternata with two sets of primers whose design was based on the aligned amino acid activating domains of cyclic peptide synthetase genes. Some of these products were subcloned and sequenced and all sequences showed considerable homology to the corresponding region of other CPSs. These were used as probes against genomic digests of saprophytic and other A. alternata pathotypes. Hybridization at low stringency produced several signals of varying size from all the strains examined, but only A. alternata apple pathotype hybridized at high stringency. Two of the sequences were then subcloned into a vector and used for gene targeting. Three out of seventy transformants appeared to lack AM-toxin production, based on both a leaf necrosis assay and HPLC analysis. However, analysis of these transformants showed that the subcloned sequence hybridized to a fragment that was the same size in the wild type, although the signal intensity was significantly lower. This may indicate a possible multi-copy nature for the AM-toxin synthetase gene. The integration profile of the transforming vector in these transformants has not been solved, so the loss of AM-toxin production is not conclusively due to a gene disruption event.