Takashi Tsuge

Mutation of an Arginine Biosynthesis Gene Causes Reduced Pathogenicity in Fusarium oxysporum f. sp. melonis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.

Phologeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA

The internal transcribed spacer regions (ITS1 and ITS2) of ribosomal DNA from Alternaria species, including seven fungi known to produce host-specific toxins, were analyzed by polymerase chain reaction-amplification and direct sequencing. Phylogenetic analysis of the sequence data by the Neighbor-joining method showed that the seven toxin-producing fungi belong to a monophyletic group together with A. alternata. In contract, A. dianthi, A. panax, A. dauci, A. bataticola, A. porri, A. sesami and A. solani, species that can be morphologically distinguished from A. alternata, could be clearly separated from A. alternata by phylogenetic analysis of the ITS variation. These results suggest that Alternaria pathogens which produce host-specific toxins are pathogenic variants within a single variable species, A. alternata.

Plant Colonization by the Vascular Wilt Fungus Fusarium oxysporum Requires FOW1, a Gene Encoding a Mitochondrial Protein

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme–mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1–green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum.

The Melanin Biosynthesis Genes of Alternaria alternata Can Restore Pathogenicity of the Melanin-Deficient Mutants of Magnaporthe grisea

The phytopathogenic fungi Magnaporthe grisea and Alternaria alternata produce melanin via the polyketide biosynthesis, and both fungi form melanized colonies. However, the site of melanin deposition and the role of melanin in pathogenicity differ between these two fungi. M. grisea accumulates melanin in appressoria, and their melanization is essential for host penetration. On the other hand, A. alternata produces colorless appressoria, and melanin is not relevant to host penetration. We examined whether the melanin biosynthesis genes of A. alternata could complement the melanin-deficient mutations of M. grisea. Melanin-deficient, nonpathogenic mutants of M. grisea, albino (Alb-), rosy (Rsy-), and buff (Buf-), were successfully transformed with a cosmid clone pMRB1 that carries melanin biosynthesis genes ALM, BRM1, and BRM2 of A. alternata. This transformation restored the melanin synthesis of the Alb- and Buf- mutants, but not that of the Rsy- mutant. The melanin-restored transformants regained mycelial melanization, appressorium melanization, and pathogenicity to rice. Further, transformation of Alb- and Buf- mutants with subcloned ALM and BRM2 genes, respectively, also produced melanin-restored transformants. These results indicate that the Alternaria genes ALM and BRM2 can restore pathogenicity to the mutants Alb- and Buf-, respectively, due to their function during appressorium development in M. grisea.

Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host‐specific toxins and cause diseases on different plants. The strawberry pathotype produces host‐specific AF‐toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK‐toxin. The strawberry pathotype produces two related molecular species, AF‐toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT‐1 from the strawberry pathotype that contains three genes involved in AF‐toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT‐1. AFTS1 encodes a protein with similarity to enzymes of the aldo‐ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: ΔaftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF‐toxin II, but not AF‐toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.

FoSTUA, encoding a basic helix-loop-helix protein, differentially regulates development of three kinds of asexual spores, macroconidia, microconidia, and chlamydospores, in the fungal plant pathogen Fusarium oxysporum.

ABSTRACT The soil-borne fungus Fusarium oxysporum causes vascular wilt of a wide variety of plant species. F. oxysporum produces three kinds of asexual spores, macroconidia, microconidia, and chlamydospores. Falcate macroconidia are formed generally from terminal phialides on conidiophores and rarely from intercalary phialides on hyphae. Ellipsoidal microconidia are formed from intercalary phialides on hyphae. Globose chlamydospores with thick walls are developed by the modification of hyphal and conidial cells. Here we describe FoSTUA of F. oxysporum, which differentially regulates the development of macroconidia, microconidia, and chlamydospores. FoSTUA encodes a basic helix-loop-helix protein with similarity to Aspergillus nidulans StuA, which has been identified as a transcriptional regulator controlling conidiation. Nuclear localization of FoStuA was verified by using strains expressing FoStuA-green fluorescent protein fusions. The FoSTUA-targeted mutants exhibited normal microconidium formation in cultures. However, the mutants lacked conidiophores and produced macroconidia at low frequencies only from intercalary phialides. Thus, FoSTUA appears to be necessary to induce conidiophore differentiation. In contrast, chlamydospore formation was dramatically promoted in the mutants. These data demonstrate that FoStuA is a positive regulator and a negative regulator for the development of macroconidia and chlamydospores, respectively, and is dispensable for microconidium formation in cultures. The disease-causing ability of F. oxysporum was not affected by mutations in FoSTUA. However, the mutants produced markedly fewer macroconidia and microconidia in infected plants than the wild type. These results suggest that FoSTUA also has an important role for microconidium formation specifically in infected plants.

An Isolate of Alternaria alternata That Is Pathogenic to Both Tangerines and Rough Lemon and Produces Two Host-Selective Toxins, ACT- and ACR-Toxins.

ABSTRACT Two different pathotypes of Alternaria alternata cause Alternaria brown spot of tangerines and Alternaria leaf spot of rough lemon. The former produces the host-selective ACT-toxin and the latter produces ACR-toxin. Both pathogens induce similar symptoms on leaves or young fruits of their respective hosts, but the host ranges of these pathogens are distinct and one pathogen can be easily distinguished from another by comparing host ranges. We isolated strain BC3-5-1-OS2A from a leaf spot on rough lemon in Florida, and this isolate is pathogenic on both cv. Iyokan tangor and rough lemon and also produces both ACT-toxin and ACR-toxin. Isolate BC3-5-1-OS2A carries both genomic regions, one of which was known only to be present in ACT-toxin producers and the other was known to exist only in ACR-toxin producers. Each of the genomic regions is present on distinct small chromosomes, one of 1.05 Mb and the other of 2.0 Mb. Alternaria species have no known sexual or parasexual cycle in nature and populations of A. alternata on citrus are clonal. Therefore, the ability to produce both toxins was not likely acquired through meiotic or mitotic recombination. We hypothesize that a dispensable chromosome carrying the gene cluster controlling biosynthesis of one of the host-selective toxins was transferred horizontally and rearranged by duplication or translocation in another isolate of the fungus carrying genes for biosynthesis of the other host-selective toxin.

Tfo1: an Ac-like transposon from the plant pathogenic fungus Fusarium oxysporum.

Abstract A transposable element from a plant pathogenic fungus, Fusarium oxysporum, was isolated and characterized. Four clones carrying moderately repetitive DNA were selected from a genomic library of the strain MAFF305118 of F. oxysporum f. sp. lagenariae, which causes wilt of bottle gourd. One the four clones was found to include a transposable element, which we have named Tfo1. This element is 2763 bp in size and appears to contain a long ORF that can encode a polypeptide of 777 amino acids. The amino acid sequence shows significant similarity to transposases from the hAT family of transposons, such as the maize transposon Activator (Ac). The element has 15-bp terminal inverted repeats and causes an 8-bp target site duplication upon insertion, as expected for an hAT-family transposon. Northern analysis detected a transcript, which hybridized to the putative transposase-encoding region of Tfo1. The size of this transcript (about 2.3 kb) corresponds to that of the ORF. A Southern analysis using pulsed-field gel electrophoresis showed that multiple chromosomal bands carry Tfo1 elements. PCR amplification of the Tfo1 elements with a 15-base inverted repeat primer produced a single DNA fragment of about 2.8 kb in all bottle gourd-infecting strains used. The element was found in multiple copies in the genome of all these strains and also in strains from other formae speciales tested. The sequence similarity of the Tfo1 element to other transposons, together with its transcriptional expression and genomic distribution, strongly suggests that Tfo1 is a member of the hAT transposon family.

REN1 is required for development of microconidia and macroconidia, but not of chlamydospores, in the plant pathogenic fungus Fusarium oxysporum.

The filamentous fungus Fusarium oxysporum is a soil-borne facultative parasite that causes economically important losses in a wide variety of crops. F. oxysporum exhibits filamentous growth on agar media and undergoes asexual development producing three kinds of spores: microconidia, macroconidia, and chlamydospores. Ellipsoidal microconidia and falcate macroconidia are formed from phialides by basipetal division; globose chlamydospores with thick walls are formed acrogenously from hyphae or by the modification of hyphal cells. Here we describe rensa, a conidiation mutant of F. oxysporum, obtained by restriction-enzyme-mediated integration mutagenesis. Molecular analysis of rensa identified the affected gene, REN1, which encodes a protein with similarity to MedA of Aspergillus nidulans and Acr1 of Magnaporthe grisea. MedA and Acr1 are presumed transcription regulators involved in conidiogenesis in these fungi. The rensa mutant and REN1-targeted strains lack normal conidiophores and phialides and form rod-shaped, conidium-like cells directly from hyphae by acropetal division. These mutants, however, exhibit normal vegetative growth and chlamydospore formation. Nuclear localization of Ren1 was verified using strains expressing the Ren1-green fluorescent protein fusions. These data strongly suggest that REN1 encodes a transcription regulator required for the correct differentiation of conidiogenesis cells for development of microconidia and macroconidia in F. oxysporum.

Fow2, a Zn(II)2Cys6-type transcription regulator, controls plant infection of the vascular wilt fungus Fusarium oxysporum

The filamentous fungus Fusarium oxysporum is a soil‐borne parasite that causes vascular wilts in a wide variety of crops by directly penetrating roots and colonizing the vascular tissue. In previous work, we generated the non‐pathogenic mutant B137 of the melon wilt pathogen F. oxysporum f. sp. melonis by using restriction enzyme‐mediated integration (REMI) mutagenesis. Molecular characterization of B137 revealed that this mutant has a single‐copy plasmid insertion in a gene, designated FOW2, which encodes a putative transcription regulator belonging to the Zn(II)2Cys6 family. The REMI mutant B137 and other FOW2‐targeted mutants completely lost pathogenicity, but were not impaired in vegetative growth and conidiation in cultures. Microscopic observation of infection behaviours of green fluorescent protein (GFP)‐marked wild‐type and mutant strains revealed that the mutants were defective in their abilities to invade roots and colonize plant tissues. FOW2 is conserved in F. oxysporum pathogens that infect different plants. The FOW2‐targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also lost pathogenicity. Nuclear localization of Fow2 was verified using strains expressing Fow2‐GFP and GFP‐Fow2 fusion proteins. These data strongly suggest that FOW2 encodes a transcription regulator controlling the plant infection capability of F. oxysporum pathogens.