AAL Toxin TD1

The plant disease resistance gene Asc-1 prevents disruption of sphingolipid metabolism during AAL-toxin-induced programmed cell death.[Pubmed:12445127]

Plant J. 2002 Nov;32(4):561-72.

The nectrotrophic fungus Alternaria alternata f.sp. lycopersici infects tomato plants of the genotype asc/asc by utilizing a host-selective toxin, AAL-toxin(include AAL Toxin TD1), that kills the host cells by inducing programmed cell death. Asc-1 is homologous to genes found in most eukaryotes from yeast to humans, suggesting a conserved function. A yeast strain with deletions in the homologous genes LAG1 and LAC1 was functionally complemented by Asc-1, indicating that Asc-1 functions in an analogous manner to the yeast homologues. Examination of the yeast sphingolipids, which are almost absent in the lag1Deltalac1Delta mutant, showed that Asc-1 was able to restore the synthesis of sphingolipids. We therefore examined the biosynthesis of sphingolipids in tomato by labeling leaf discs with l-[3-3H]serine. In the absence of AAL-toxin(include AAL Toxin TD1), there was no detectable difference in sphingolipid labeling between leaf discs from Asc/Asc or asc/asc leaves. In the presence of pathologically significant concentrations of AAL-toxin(include AAL Toxin TD1) however, asc/asc leaf discs showed severely reduced labeling of sphingolipids and increased label in dihydrosphingosine (DHS) and 3-ketodihydrosphingosine (3-KDHS). Leaf discs from Asc/Asc leaves responded to AAL-toxin(include AAL Toxin TD1) treatment by incorporating label into different sphingolipid species. The effects of AAL-toxin on asc/asc leaflets could be partially blocked by the simultaneous application of AAL-toxin(include AAL Toxin TD1) and myriocin. Leaf discs simultaneously treated with AAL-toxin(include AAL Toxin TD1) and myriocin showed no incorporation of label into sphingolipids or long-chain bases as expected. These results indicate that the presence of Asc-1 is able to relieve an AAL-toxin-induced block on sphingolipid synthesis that would otherwise lead to programmed cell death.

AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration.[Pubmed:18945069]

Phytopathology. 1997 Sep;87(9):967-72.

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(include AAL Toxin TD1), 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.