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.

Tomato as a model plant for plant-pathogen interactions

“…In contrast to interactions conforming to the gene-for-gene concept, described later, the toxin molecule is extremely selective for susceptible tomato cultivars such as First (Takii Seed, Kyoto, Japan) and causes severe necrosis on stems and leaves, whereas resistant cultivars are tolerant and show no symptoms. In AAL-tomato interactions, a major factor in pathogenicity is the production of AAL-toxins (Akamatsu et al 1997;Brandwagt et al 2000).…”

Integração de pAN7-1 no genoma de Magnaporthe grisea mediada por enzima de restrição

“…Optou-se por esta enzima de restrição devido aos resultados satisfatórios obtidos em outros sistemas envolvendo o mesmo vetor de transformação (Akamatsu Akamatsu et al, 1997;Linnemannstöns et al, 1999)…”

“…patótipo do tomate e Gibberella fujikuroi (Sawada) Wollenw. também foi alcançada utilizando pAN7-1 na presença da enzima de restrição HindIII (Akamatsu et al, 1997;Linnemannstöns et al, 1999).…”

“…Quando 600 U de HindIII ou BamHI, por mL de reação, foram utilizadas para mediar a transformação do isolado Guy 11 com pAN7-2, a recuperação de transformantes foi menor do que a obtida com o vetor circular (Shi et al, 1995). Respostas semelhantes têm sido obtidas com outros fungos fitopatogênicos (Akamatsu et al, 1997;Linnemannstöns et al, 1999). Por exemplo, a reação de transformação de reação de transformação de A. alternata Keissler patótipo do tomate com mais de 20 U de HindIII ou XbaI promoveu redução significativa no número de transformantes (Akamatsu et al, 1997).…”

“…Respostas semelhantes têm sido obtidas com outros fungos fitopatogênicos (Akamatsu et al, 1997;Linnemannstöns et al, 1999). Por exemplo, a reação de transformação de reação de transformação de A. alternata Keissler patótipo do tomate com mais de 20 U de HindIII ou XbaI promoveu redução significativa no número de transformantes (Akamatsu et al, 1997).…”