Motoaki Kusaba

Multiple Translocation of the AVR-Pita Effector Gene among Chromosomes of the Rice Blast Fungus Magnaporthe oryzae and Related Species

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.

Analysis of Host Species Specificity of Magnaporthe grisea Toward Wheat Using a Genetic Cross Between Isolates from Wheat and Foxtail Millet.

ABSTRACT A genetic cross was performed between a Setaria isolate (pathogenic on foxtail millet) and a Triticum isolate (pathogenic on wheat) of Magnaporthe grisea to elucidate genetic mechanisms of its specific parasitism toward wheat. A total of 80 F(1) progenies were obtained from 10 mature asci containing 8 ascospores. Lesions on wheat leaves produced by the F(1) progenies were classified into four types, which segregated in a 1:1:1:1 ratio. This result suggested that the pathogenicity of the F(1) population on wheat was controlled by two genes located at different loci. This idea was supported by backcross analyses. We designated these loci as Pwt1 and Pwt2. Cytological analyses revealed that Pwt1 and Pwt2 were mainly associated with the hypersensitive reaction and papilla formation, respectively.

Genetic Constitution and Pathogenicity of Lolium Isolates of Magnaporthe oryzae in Comparison with Host Species-Specific Pathotypes of the Blast Fungus.

ABSTRACT Fungal isolates from gray leaf spot on perennial ryegrass (prg isolates) were characterized by DNA analyses, mating tests, and pathogenicity assays. All of the prg isolates were interfertile with Triticum isolates and clustered into the crop isolate group (CC group) on a dendrogram constructed from rDNA-internal transcribed spacer 2 sequences. Since the CC group corresponded to a newly proposed species, Magnaporthe oryzae, all of the prg isolates were designated M. oryzae. However, DNA fingerprinting with MGR586, MGR583, and Pot2 showed that the prg isolates are divided into two distinct populations, i.e., TALF isolates and WK isolates. The TALF isolates were virulent only on Lolium species, whereas the WK isolates were less specific, suggesting that gray leaf spot can be caused not only by Lolium-specific isolates but also by less specific isolates. We designated the TALF isolates as Lolium pathotype. The TALF isolates showed diverse karyotypes in spite of being uniform in DNA fingerprints, suggesting that theyare unstable in genome organization.

Comparative analyses of the distribution of various transposable elements in Pyricularia and their activity during and after the sexual cycle.

Abstract. We examined the distribution and activity of six transposable elements found in the blast fungus, Pyricularia spp. Sixty-eight isolates from various gramineous plants were used for the survey, and the elements were plotted on a dendrogram constructed on the basis of their rDNA-ITS2 sequences. MGR586 and Pot2 (Class II elements), Mg-SINE (SINE-like element) and MGR583 (LINE-like retrotransposon) were widely distributed among the Pyricularia isolates, suggesting that they are old elements which arose in, or invaded, the Pyricularia population at very early stages in its evolution. By contrast, the distribution of the LTR-retrotransposons MAGGY and Grasshopper was limited or sporadic, suggesting that they are relatively new elements which recently invaded the Pyricularia population by means of horizontal transfer events. The activity of these elements was evaluated by Southern analysis in progenies derived from a cross between a Setaria isolate and a Triticum isolate. Many new MAGGY signals were observed, which were absent in the parental isolates, at various stages of the sexual cycle and following vegetative growth. In contrast, the other elements yielded few, if any, such signals. Analysis of the sequences flanking the new MAGGY insertions revealed that they were each associated with a 5-bp target-site duplication at both ends of the insertion. These data suggested that MAGGY was the most active of the elements tested for transposition in Pyricularia.

Genetic Mapping and Chromosomal Assignment of Magnaporthe oryzae Avirulence Genes AvrPik, AvrPiz, and AvrPiz-t Controlling Cultivar Specificity on Rice

ABSTRACT A genetic map including three avirulence (Avr) genes, AvrPik, AvrPiz, and AvrPiz-t, was constructed in a genetic cross of two rice field isolates, 84R-62B and Y93-245c-2. The chromosomal locations of the Avr genes were determined by using selected markers to probe Southern blots of the parental chromosomes that had been separated by contour-clamped homogenous electric fields electrophoresis. Electrophoretic karyotyping showed that both parental isolates 84R-62B and Y93-245c-2 contained seven chromosomes greater than 3.5 megabases (Mb) in size and 84R-62B possessed a small chromosome of approximately 1.6 Mb. The linkage groups containing AvrPiz and AvrPiz-t were assigned to chromosomes 3 and 7, respectively. Some markers from the linkage group that contained AvrPik hybridized with chromosome 1 and the 1.6-Mb chromosome, yet all of the cloned RAPD markers that were closely linked to AvrPik hybridized exclusively to the 1.6-Mb chromosome in 84R-62B, the parent that possesses AvrPik. Thus, we conclude that AvrPik is located on the 1.6-Mb chromosome in 84R-62B.

Relationship between avirulence genes of the same family in rice blast fungus Magnaporthe grisea

A genetic cross between rice-field isolates of Magnaporthe grisea produced progeny segregating for avirulence/ virulence on six rice cultivars among nine race differentials, while on three other cultivars, Shin 2 (Pik-s), Aichi Asahi (Pia) and Ishikari Shiroke (Pii), parental and progeny isolates were all virulent. Based on segregation ratios in 115 progeny isolates, avirulence on Kanto 51 (Pik), Yashiro-mochi (Pita), Fukunishiki (Piz) and Toride 1 (Piz-t) is under monogenic control. On Tsuyuake (Pik-m) and Pi No. 4 (Pita-2), however, a disproportionate ratio in the segregation was observed, suggesting that avirulence on these two cultivars is controlled by two or more genes. Assuming that the avirulence gene AvrPik-m consists of at least two genes, AvrPik-m1 and AvrPik-m2, each of which functions in the whole gene AvrPik-m, and that one of AvrPik-m1 and AvrPik-m2 is AvrPik, we could account for the disproportion in the avirulence/virulence segregation of the progeny. This hypothesis would also be consistently applied for avirulence gene AvrPita-2. There seem to be two types of the avirulence genes : AvrPik-m, that is comprised of the tightly linked genes, AvrPik-ml (=AvrPik) and AvrPik-m2, and AvrPita-2, that is comprised of the loosely linked genes AvrPita-2A (=AvrPita) and AvrPita-2B. As one possible explanation of the rice resistant reaction to blast, multiple specificity was suggested for the first time for the blast fungus. On the contrary, the avirulence genes AvrPiz and AvrPiz-t were inherited independently, despite the corresponding genes for resistance (Piz and Piz-t) being located at the same locus. The cross of rice blast isolates (races 447 and 337) produced only 25 kinds of races in the progeny, although theoretically about 64 kinds of races should be produced if six avirulence genes segregated independently. Because no progeny are with AvrPik (or AvrPita) and without AvrPik-m (or AvrPita-2), the number of races theoretically should be 36 at most. A number of strains, such as races 377 and 737, with a single avirulence gene were obtained from this cross. These strains may be valuable for analysis of resistance genes in rice plant.

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.

Characterization of interactions between barley and various host-specific subgroups of Magnaporthe oryzae and M. grisea

The Magnaporthe oryzae–M. grisea species complex is composed of several host-specific subgroups, but does not contain a barley-specific subgroup. To characterize the relationship between barley and these subgroups, we inoculated 24 barley cultivars separately with each of 18 isolates from various hosts. The interactions between these cultivars and isolates included various reactions from nonhost-like immune responses to typical host responses. Evenly closely related isolates of the blast fungi caused such contrasting reactions. The immune responses of barley cultivars against a Setaria isolate, Si-1J, were examined in detail. An infection assay with near-isogenic fungal strains suggested that PWT1, which was first identified as a major gene conditioning the avirulence of Si-1J on wheat, was involved in the avirulence on two-thirds of the barley cultivars. At the cytological level, the immune responses were associated with both papilla formation and hypersensitive reaction (HR). Of these two, however, HR played a more critical role than papilla formation. Studying the interactions of barley with M. oryzae and M. grisea may reveal various steps in the process of host specialization of a parasite species and the concomitant evolution of host resistance.

Taxonomic characterization of Pyricularia isolates from green foxtail and giant foxtail, wild foxtails in Japan

Twenty-eight Pyricularia isolates from two wild foxtails—green foxtail (Setaria viridis) and giant foxtail (S. faberii)—in Japan were taxonomically characterized by DNA analyses, mating tests, and pathogenicity assays. Although most of the isolates failed to produce perithecia in mating tests with Magnaporthe oryzae, a diagnostic polymerase chain reaction-restriction fragment length polymorphism phenotype of M. oryzae was detected in the beta-tubulin genomic region in all isolates. The pathogenicity assays revealed that host ranges of the isolates were similar to those of isolates from foxtail millet (S.italica), which were exclusively pathogenic on foxtail millet. In addition to the 28 isolates from wild foxtails, 22 Pyricularia isolates from 11 other grasses were analyzed by RFLP using single-copy sequences as probes. In a dendrogram constructed from the RFLP data, isolates that were previously identified as M. oryzae formed a single cluster. All the wild foxtail isolates formed a subcluster with foxtail millet isolates within the M. oryzae cluster. From these results, we conclude that Pyricularia isolates from the wild foxtails are closely related to isolates from foxtail millet and should be classified into the Setaria pathotype of M. oryzae.

Population structure of rice blast isolates resistant to scytalone dehydratase inhibitors in Mie Prefecture and implications for their origin

In Mie Prefecture in Japan, rice blast isolates resistant to melanin biosynthesis inhibitors targeting scytalone dehydratase (SDH) were first observed in 2005. To analyze the distribution of the resistant isolates, 527 isolates were collected from wide areas in this prefecture during 2006 and 2007. Almost half of the isolates collected (233 of 527 isolates) carried a point mutation in the SDH gene conferring the resistant phenotype. To compare population structures of resistant and sensitive isolates, we analyzed the isolates with repetitive-element-based PCR DNA fingerprinting using a single primer complementary to a sequence in the terminal inverted repeat of transposable element Pot2. A majority of the resistant isolates were classified into a single DNA fingerprint haplotype, Mie1. Despite its prevalence in the resistant isolates, Mie1 was not found in the sensitive isolates. Furthermore, in a dendrogram constructed from the DNA fingerprint data, Mie1 and six other haplotypes formed a cluster composed of resistant isolates alone. These results suggest that the resistant isolates that belonged to the Mie1 haplotype had migrated from regions outside Mie Prefecture and selectively propagated in a short period in this prefecture.