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Alternaria alternata includes seven pathogenic variants, called pathotypes, which produce host-selective toxins (HSTs) as determinant factors for pathogenicity. The gene clusters for HST biosynthesis were identified from six pathotypes (Japanese pear, strawberry, tangerine, apple, tomato and rough lemon) and were found to reside on small chromosomes of

G protein-coupled receptors (GPCRs) are a large transmembrane receptor superfamily that is involved in many cellular signaling pathways. In the present study, GPCR-family genes from the toxigenic and necrotrophic plant pathogen Alternaria alternata were cloned and characterized. Three GPCR-encoding genes, AaGPR1, AaGPR2, and AaGPR3 were identified in the draft genomic data of the A. alternata tomato pathotype, which produces the host-specific AAL-toxin. AaGPR1, AaGPR2, and AaGPR3 each encodes a protein containing a seven transmembrane domain that is characteristic of GPCRs. Targeted deletion of AaGPR1, AaGPR2, or AaGPR3 in the A. alternata tomato pathotype was conducted to understand the influence of G-protein signaling mechanisms on developmental processes and virulence of this pathogen. No changes in colony morphology or AAL-toxin production were observed for the deletion strain ΔAaGPR1, 2, and 3, compared with the wild-type strain. However, one deletion strain, ΔAaGPR3, exhibited aberrant conidial morphology including decreased conidial length and beak formation. The ability to induce the formation of necrotic lesions on susceptible leaves also significantly decreased in ΔAaGPR3, indicating a reduction in virulence. These defects are similar to the phenotypes found for the Gα gene (AGA1) mutant of A. alternata. These results indicate that the G-protein signal transduction pathway appears to be involved in conidial development and virulence of A. alternata.

Trichoderma harzianum, a biocontrol agent for various plant pathogens, is known to degrade fungal cell walls; this mycoparasitism is believed to require secretion of cell-wall-degrading enzymes against host pathogens. In this study, we identified a homologue of yeast SNF1 (sucrose nonfermenting 1) encoding protein kinase in T. harzianum (ThSNF1) by draft genome sequencing of strain T36. Targeted gene disruption of ThSNF1 was performed using the PEG method with fusion PCR products. Growth of mutant ΔThSNF1 was markedly less than for the wild-type strain on minimal medium with chitin as a carbon source. The mutant exhibited reduced expression of the genes encoding chitinase and polygalacturonase and markedly reduced spore production. Mycoparasitism against plant pathogens such as Fusarium oxysporum f. sp. cubense (Panama disease) and Fusarium graminearum (Fusarium head blight) was clearly impaired in the mutant. The results suggest that ThSNF1 is critical for asexual development, utilization of certain carbon sources and virulence on fungi, and is therefore important for the biocontrol ability of T. harzianum.

Alternaria alternata tomato pathotypeは，宿主特異的AAL毒素生産によりトマトアルターナリア茎枯病を引き起こす．AAL毒素はマイコトキシン フモニシンの構造類縁体であり，その生物活性も類似する．罹病植物組織内における病原菌の伸長と産生された毒素の確認は，毒素の病理学的重要性とリスクの評価のために重要である．抵抗性植物品種上において，病原菌および毒素は接種部位のみに局在していた．一方，感受性品種上では，毒素の広範な分布と病原菌の伸長が確認された．本研究の結果から，AAL毒素の病理学的重要性が確認されるとともに，罹病トマトにおける毒素による汚染リスクの可能性が示唆された．

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.

The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1 , which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato 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.

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity.

The tomato pathotype of Alternaria alternata produces a host-specific AAL-toxin and causes Alternaria stem canker on susceptible tomato cultivars. AAL-toxin is a sphinganine-analog mycotoxin which induces apoptotic cell death in tomato cells and mammalian cells by inhibiting ceramide biosynthesis. Insensitivity to the AAL-toxin in resistant tomatoes and other plants is conferred by the Asc1 gene, a homolog of the yeast ceramide synthase gene Lag1 . The ALT7 gene, a putative acyl-CoA-dependent ceramide synthase, was found to be located in the AAL-toxin biosynthetic ( ALT ) gene cluster of the tomato pathotype of A. alternata . ALT7 and Asc1 have the TLC (TRAM/Lag1/ CLN8) domain characteristic of proteins involved in ceramide biosynthesis and are members of the LASS/Lag family. To test the hypothesis that ALT7 and Asc1 , both of which are Lag1 ceramide synthase gene homologs, might share a common biological function as toxin tolerance genes, we have cloned and characterized ALT7. ALT7 -deleted mutants were generated to investigate the effects of the deletion on vegetative growth, sporulation, toxin-sensitivity, toxin-production and pathogenicity. The deletion of ALT7 has no deleterious effect on the toxin-producing pathogen, indicating that the gene does not act as a resistance/self-tolerance factor against the toxin in the toxin biosynthetic gene cluster.

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.