Host-selective toxins (HSTs) produced by fungal plant pathogens are generally low-molecular-weight secondary metabolites with a diverse range of structures that function as effectors controlling pathogenicity or virulence in certain plant-pathogen interactions. There are now seven known diseases caused by Alternaria alternata in which HSTs are responsible for fungal pathogenesis. The pathogens have been defined as pathotypes of A. alternata because of morphological similarity but pathological differences. Chemical structures of HSTs from six pathotypes have been determined. The role of A. alternata HSTs in pathogenesis has been studied extensively, and discovery of the release of HSTs from germinating conidia prior to penetration aids in understanding the early participation of HSTs to induce susceptibility of host cells by suppressing their defence reactions. Many attempts have been made to find the target sites of A. alternata HSTs, and four cellular components, plasma membrane, mitochondrion, chloroplast and a metabolically important enzyme, have been identified as the primary sites of each HST action, leading to elucidation of the molecular mechanisms of HST sensitivity in host plants. Studies of the molecular genetics of HST production have identified supernumerary chromosomes encoding HST gene clusters and have provided new insights into the evolution of A. alternata pathotypes.

The aromatic polymer lignin represents a possible renewable source of aromatic chemicals, if biocatalytic routes for lignin breakdown can be developed. The availability of a genome sequence for Rhodococcus jostii RHA1, a bacterium that breaks down lignin, has allowed the application of a targeted pathway engineering strategy to lignin breakdown to produce vanillin, a valuable food/flavor chemical. A gene deletion strain of R. jostii RHA1 in which the vanillin dehydrogenase gene had been deleted, when grown on minimal medium containing 2.5% wheat straw lignocellulose and 0.05% glucose, was found to accumulate vanillin with yields of up to 96 mg/L after 144 h, together with smaller amounts of ferulic acid and 4-hydroxybenzaldehyde.

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.

pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

The Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide, host-specific toxin, AM-toxin. We recently cloned a cyclic peptide synthetase gene, AMT, whose product catalyzes the production of AM-toxin and showed that it resides on chromosomes of 1.8 Mb or less, depending on the A. alternata apple pathotype strain. Reverse transcriptase (RT)-PCR, using primers specific to AMT, on laboratory sub-cultured strains previously shown to produce AM-toxin, identified one isolate that did not express the gene. A leaf necrosis bioassay confirmed an AM-toxin-minus phenotype. However, an original isolate of this strain which had not undergone sub-culture gave a positive result by both RTPCR and bioassay. Contour-clamped homogeneous electric field electrophoresis and Southern hybridization demonstrated the loss of a 1.1-Mb chromosome in the non-toxin-producing isolate. Since this chromosome can be entirely lost without affecting growth, but is necessary for pathogenicity, we propose it is a conditionally dispensable chromosome.

A reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 axis is associated with an extension of lifespan in laboratory rodents. Several phenotypes of such animal models resemble those induced by caloric restriction (CR). Using a transgenic male Wistar rat model whose GH-IGF-1 axis was moderately suppressed by overexpression of the antisense GH transgene (tg), we elucidated a relationship between the effects of a reduced GH-IGF-1 axis and CR for some biomarkers of aging, lifespan, and pathologies. Heterozygous (tg/-) rats fed ad libitum (AL) had a dwarf phenotype similar to that of control nontransgenic (-/-) rats subjected to 30% CR from 6 wk of age. Both the reduced GH-IGF-1 axis and CR extended lifespan to a similar extent, although the effect of CR seemed to be greater. There was an additive effect of CR to lifespan extension when tg/- rats were subjected to CR. Pathologic analyses indicated that the preventive effect of CR on selected diseases was greater than that of the reduced GH-IGF-1 axis. The present study suggests that CR affects aging and longevity by mechanisms other than suppression of the GH-IGF-1 axis, although CR might exhibit its effects partly through the reduced GH-IGF-1 axis.

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.