Yukio Tosa

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Cellulases belonging to glycoside hydrolase families 6 and 7 contribute to the virulence of Magnaporthe oryzae.

Upon infection, phytopathogenic fungi secrete an array of hydrolytic enzymes that can degrade components of the host epidermis, including waxes, the cuticle, and cell walls. Cellulases, which can hydrolyze crystalline cellulose in the plant cell wall, are among these hydrolytic enzymes. Here, we provide RNAi-based evidence to show that cellulases belonging to glycosyl hydrolase (GH) families 6 and 7 contribute to the penetration of the host epidermis and further invasion by the phytopathogenic fungus Magnaporthe oryzae. The GH6 and GH7 cellulases likely include all members of the cellobiohydrolase family and some endoglucanases in M. oryzae. Quantitative reverse-transcriptase polymerase chain reaction analysis indicated that more than half of the cellulases were highly induced during infection. We constructed knock-down (KD) mutants of these cellulases using the building blocks method we reported previously. The transcript levels of the target genes and cellulase activity were considerably reduced in the KD mutants. The KD mutants resulted in fewer lesions, less penetration, and infection of fewer cells compared with the parent strain. Cytological analyses showed that a high rate of papilla formation blocked invasion of the KD mutants into host cells. These results suggest that the GH6 and GH7 cellulases play roles in the virulence of M. oryzae.

Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements

BackgroundMagnaporthe oryzae (anamorph Pyricularia oryzae) is the causal agent of blast disease of Poaceae crops and their wild relatives. To understand the genetic mechanisms that drive host specialization of M. oryzae, we carried out whole genome resequencing of four M. oryzae isolates from rice (Oryza sativa), one from foxtail millet (Setaria italica), three from wild foxtail millet S. viridis, and one isolate each from finger millet (Eleusine coracana), wheat (Triticum aestivum) and oat (Avena sativa), in addition to an isolate of a sister species M. grisea, that infects the wild grass Digitaria sanguinalis.ResultsWhole genome sequence comparison confirmed that M. oryzae Oryza and Setaria isolates form a monophyletic and close to another monophyletic group consisting of isolates from Triticum and Avena. This supports previous phylogenetic analysis based on a small number of genes and molecular markers. When comparing the host specific subgroups, 1.2–3.5xa0% of genes showed presence/absence polymorphisms and 0–6.5xa0% showed an excess of non-synonymous substitutions. Most of these genes encoded proteins whose functional domains are present in multiple copies in each genome. Therefore, the deleterious effects of these mutations could potentially be compensated by functional redundancy. Unlike the accumulation of nonsynonymous nucleotide substitutions, gene loss appeared to be independent of divergence time. Interestingly, the loss and gain of genes in pathogens from the Oryza and Setaria infecting lineages occurred more frequently when compared to those infecting Triticum and Avena even though the genetic distance between Oryza and Setaria lineages was smaller than that between Triticum and Avena lineages. In addition, genes showing gain/loss and nucleotide polymorphisms are linked to transposable elements highlighting the relationship between genome position and gene evolution in this pathogen species.ConclusionOur comparative genomics analyses of host-specific M. oryzae isolates revealed gain and loss of genes as a major evolutionary mechanism driving specialization to Oryza and Setaria. Transposable elements appear to facilitate gene evolution possibly by enhancing chromosomal rearrangements and other forms of genetic variation.

Evolution of the wheat blast fungus through functional losses in a host specificity determinant

Genetic analysis of disease emergence In the 1980s, wheat crops began to fall to the fungal pathogen that causes blast disease. First seen in Brazil, wheat blast last year caused devastating crop losses in Bangladesh. Inoue et al. tracked down the shifting genetics that have allowed the emergence of this potentially global threat to wheat crops (see the Perspective by Maekawa and Schulze-Lefert). Wheat varieties with a disabled resistance gene were susceptible to pathogen strains that affected oat and ryegrass crops. Subsequent genetic changes in the pathogen amped up the virulence in wheat. Science, this issue p. 80; see also p. 31 Removal of a gene-based resistance system allowed blast fungus to jump to a new host—common wheat. Wheat blast first emerged in Brazil in the mid-1980s and has recently caused heavy crop losses in Asia. Here we show how this devastating pathogen evolved in Brazil. Genetic analysis of host species determinants in the blast fungus resulted in the cloning of avirulence genes PWT3 and PWT4, whose gene products elicit defense in wheat cultivars containing the corresponding resistance genes Rwt3 and Rwt4. Studies on avirulence and resistance gene distributions, together with historical data on wheat cultivation in Brazil, suggest that wheat blast emerged due to widespread deployment of rwt3 wheat (susceptible to Lolium isolates), followed by the loss of function of PWT3. This implies that the rwt3 wheat served as a springboard for the host jump to common wheat.

Various species of Pyricularia constitute a robust clade distinct from Magnaporthe salvinii and its relatives in Magnaporthaceae

In a phylogenetic analysis of species of Magnaporthaceae based on nucleotide sequences of rDNA-ITS and the RPB1 gene, isolates of the tested species were divided into two clusters with high bootstrap support. One group was composed of Pyricularia spp.; the other was composed of Magnaporthe salvinii, M. rhizophila, M. poae, Gaeumannomyces graminis, and G. incrustans. On the basis of this result, we concluded that Pyricularia spp. constitute a large but distinct phylogenetic species group that is not congeneric with Magnaporthe salvinii, the type species of Magnaporthe.

Generic names in Magnaporthales.

The order Magnaporthales comprises about 200 species and includes the economically and scientifically important rice blast fungus and the take-all pathogen of cereals, as well as saprotrophs and endophytes. Recent advances in phylogenetic analyses of these fungi resulted in taxonomic revisions. In this paper we list the 28 currently accepted genera in Magnaporthales with their type species and available gene and genome resources. The polyphyletic Magnaporthe 1972 is proposed for suppression, and Pyricularia 1880 and Nakataea 1939 are recommended for protection as the generic names for the rice blast fungus and the rice stem rot fungus, respectively. The rationale for the recommended names is also provided. These recommendations are made by the Pyricularia/Magnaporthe Working Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF).

Rmg8, a New Gene for Resistance to Triticum Isolates of Pyricularia oryzae in Hexaploid Wheat

Blast, caused by Pyricularia oryzae, is one of the major diseases of wheat in South America. We identified a new gene for resistance to Triticum isolates of P. oryzae in common wheat S-615, and designated it resistance to Magnaporthe grisea 8 (Rmg8). Rmg8 was assigned to chromosome 2B through molecular mapping with simple-sequence repeat markers. To identify an avirulence gene corresponding to Rmg8, Triticum isolate Br48 (avirulent on S-615) was crossed with 200R29 (virulent on S-615), an F1 progeny derived from a cross between an Eleusine isolate (MZ5-1-6) and Br48. Segregation analysis of their progeny revealed that avirulence of Br48 on S-615 was conditioned by a single gene, which was designated AVR-Rmg8. AVR-Rmg8 was closely linked to AVR-Rmg7, which corresponded to Rmg7 located on chromosome 2A of tetraploid wheat.

Rmg7, a New Gene for Resistance to Triticum Isolates of Pyricularia oryzae Identified in Tetraploid Wheat

A single gene for resistance, designated Rmg7 (Resistance to Magnaporthe grisea 7), was identified in a tetraploid wheat accession, St24 (Triticum dicoccum, KU120), against Br48, a Triticum isolate of Pyricularia oryzae. Two other wheat accessions, St17 (T. dicoccum, KU112) and St25 (T. dicoccum, KU122), were also resistant against Br48 and showed a similar disease reaction pattern to St24. Crosses between these resistant accessions yielded no susceptible F2 seedlings, suggesting that St24, St17, and St25 carry the same resistance gene. Furthermore, a single avirulence gene corresponding to Rmg7 was detected in a segregation analysis of random F1 progenies between Br48 and MZ5-1-6, an Eleusine isolate virulent to St24 at a higher temperature. This avirulence gene was recognized not only by St24, but also by St17 and St25, thus supporting the preceding results indicating that all three accessions carry Rmg7. This resistance gene may have potential in future wheat breeding programs.

Accelerated Senescence and Enhanced Disease Resistance in Hybrid Chlorosis Lines Derived from Interspecific Crosses between Tetraploid Wheat and Aegilops tauschii

Hybrid chlorosis, a type of hybrid incompatibility, has frequently been reported in inter- and intraspecific crosses of allopolyploid wheat. In a previous study, we reported some types of growth abnormalities such as hybrid necrosis and observed hybrid chlorosis with mild or severe abnormalities in wheat triploids obtained in crosses between tetraploid wheat cultivar Langdon and four Ae. tauschii accessions and in their derived synthetic hexaploids. However, the molecular mechanisms underlying hybrid chlorosis are not well understood. Here, we compared cytology and gene expression in leaves to characterize the abnormal growth in wheat synthetics showing mild and severe chlorosis. In addition, we compared disease resistance to wheat blast fungus. In total 55 and 105 genes related to carbohydrate metabolism and 53 and 89 genes for defense responses were markedly up-regulated in the mild and severe chlorosis lines, respectively. Abnormal chloroplasts formed in the mesophyll cells before the leaves yellowed in the hybrid chlorosis lines. The plants with mild chlorosis showed increased resistance to wheat blast and powdery mildew fungi, although significant differences only in two, third internode length and maturation time, out of the examined agricultural traits were found between the wild type and plants showing mild chlorosis. These observations suggest that senescence might be accelerated in hybrid chlorosis lines of wheat synthetics. Moreover, in wheat synthetics showing mild chlorosis, the negative effects on biomass can be minimized, and they may show substantial fitness under pathogen-polluted conditions.

Identification of a Hidden Resistance Gene in Tetraploid Wheat Using Laboratory Strains of Pyricularia oryzae Produced by Backcrossing

In the process (BC3F1 generation) of backcrossing an Avena isolate of Pyricularia oryzae with a Triticum isolate, color mutants with white mycelia were obtained. These white mutants lacked virulence on all (31/31) hexaploid and most (28/32) tetraploid wheat lines tested. In a BC4F1 population, white and black cultures segregated in a 1:1 ratio, suggesting that the mutant phenotype is controlled by a single gene. Furthermore, the mycelial color was perfectly linked with avirulence in the BC4F1 population; white cultures were all avirulent on common wheat (Triticum aestivum) Norin 4 (N4) whereas black cultures were all virulent. White cultures in the BC3F1 and BC4F1 generations were also avirulent on tetraploid wheat (T. dicoccoides) accession KU109 (Tat4), which was susceptible to all cultures derived from the parental wild isolates through the BC2F1 generation. A cross between Tat4 and a susceptible tetraploid (T. paleocolchicum) accession KU196 (Tat14) produced resistant and susceptible F2 seedlings in a 3:1 ratio against the white cultures. In the F3 generation homozygous resistant/segregating/homozygous susceptible lines segregated in a 1:2:1 ratio. These results suggest that the resistance of Tat4 to the white cultures is controlled by a single major gene. This gene, tentatively designated as RmgTd(t), is considered to be a hidden resistance gene because it was not detected with the Br58, F1, BC1F1, or BC2F1 cultures. Cytological analysis revealed that the moderate resistance controlled by RmgTd(t) was associated with a hypersensitive reaction of mesophyll cells.