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.
is composed of pathotypes that show host specificity at the plant genus level. To elucidate the genetic mechanisms of the incompatibility between the
pathotype (pathogenic on finger millet) and ...common wheat, an
isolate (MZ5-1-6) was crossed with a
isolate (Br48) pathogenic on wheat, and resulting F
cultures were sprayed onto common wheat cultivars Hope, Norin 4 (N4), and Chinese Spring (CS). On Hope, avirulent and virulent cultures segregated in a 3:1 ratio, suggesting that two avirulence genes are involved. They were tentatively designated as
and
. On N4 and CS, the segregation ratio was not significantly deviated from the 7:1, 15:1, or 31:1 ratios, suggesting that three or more genes are involved. A comparative analysis of the segregation patterns suggested that two of these genes were
and
. A complementation test indicated that the third gene (tentatively designated as
) was the Ao9 type of the PWT3 gene controlling the avirulence of
and
isolates on wheat. The fourth gene (tentatively designated as
) was detected by backcrossing 200R72, an F
culture lacking
, and
, with Br48. Comparative analyses of phenotypes and the presence and/or absence of molecular markers in the F
population revealed that some cultures were avirulent on N4/CS in spite of lacking
, and
, indicating the presence of the fifth gene (tentatively designated as
). Taken together, we conclude that at least five avirulence genes are involved in the incompatibility between MZ5-1-6 and N4/CS.
Summary
Wheat blast caused by the Triticum pathotype of Pyricularia oryzae poses a serious threat to wheat production in South America and Asia and is now becoming a pandemic disease. Here, we show ...that Rmg8, a promising wheat gene for resistance breeding, is suppressed by PWT4, an effector gene of P. oryzae, and in turn that the suppression is counteracted by Rwt4, a wheat gene recognizing PWT4.
When PWT4 was introduced into a wheat blast isolate carrying AVR‐Rmg8 (an avirulence gene corresponding to Rmg8), PWT4 suppressed wheat resistance conferred by Rmg8. PWT4 did not alter the expression of AVR‐Rmg8, but higher expression of PWT4 led to more efficient suppression. This suppression was observed in rwt4 carriers, but not in Rwt4 carriers, indicating that it is counteracted by Rwt4.
PWT4 was assumed to have been horizontally transferred from a weed‐associated cryptic species, P. pennisetigena, to an Avena isolate of P. oryzae in Brazil. This implies a potential risk of the acquisition of PWT4 by the wheat blast fungus and the ‘breakdown’ of Rmg8.
We suggest that Rmg8 should be introduced together with Rwt4 into a wheat cultivar when it is used for resistance breeding.
To subvert rice (Oryza sativa) host defenses, the devastating ascomycete fungus pathogen Magnaporthe oryzae produces a battery of effector molecules, including some with avirulence (AVR) activity, ...which are recognized by host resistance (R) proteins resulting in rapid and effective activation of innate immunity. To isolate novel avirulence genes from M. oryzae, we examined DNA polymorphisms of secreted protein genes predicted from the genome sequence of isolate 70-15 and looked for an association with AVR activity. This large-scale study found significantly more presence/absence polymorphisms than nucleotide polymorphisms among 1032 putative secreted protein genes. Nucleotide diversity of M. oryzae among 46 isolates of a worldwide collection was extremely low ({theta} = 8.2 x 10⁻⁵), suggestive of recent pathogen dispersal. However, no association between DNA polymorphism and AVR was identified. Therefore, we used genome resequencing of Ina168, an M. oryzae isolate that contains nine AVR genes. Remarkably, a total of 1.68 Mb regions, comprising 316 candidate effector genes, were present in Ina168 but absent in the assembled sequence of isolate 70-15. Association analyses of these 316 genes revealed three novel AVR genes, AVR-Pia, AVR-Pii, and AVR-Pik/km/kp, corresponding to five previously known AVR genes, whose products are recognized inside rice cells possessing the cognate R genes. AVR-Pia and AVR-Pii have evolved by gene gain/loss processes, whereas AVR-Pik/km/kp has evolved by nucleotide substitutions and gene gain/loss.
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.
Wheat blast caused by the Triticum pathotype of Pyricularia oryzae was first reported in 1985 in Brazil and recently spread to Bangladesh. We tested whether Rmg8 and RmgGR119, recently identified ...resistance genes, were effective against Bangladeshi isolates of the pathogen. Common wheat accessions carrying Rmg8 alone (IL191) or both Rmg8 and RmgGR119 (GR119) were inoculated with Brazilian isolates (Br48, Br5, and Br116.5) and Bangladeshi isolates (T-108 and T-109). Br48, T-108, and T-109 carried the eI type of AVR-Rmg8 (the avirulence gene corresponding to Rmg8) while Br5 and Br116.5 carried its variants, eII and eII’ types, respectively. Detached primary leaves of IL191 and GR119 were resistant to all isolates at 25°C. At a higher temperature (28°C), their resistance was still effective against the eI carriers but was reduced to a low level against the eII/eII’ carriers. A survey of databases and sequence analyses revealed that all Bangladeshi isolates carried the eI type which induced a higher level of resistance than the eII/eII’ types. The resistance of IL191 (Rmg8/−) to the eI carriers was maintained even at the heading stage and at the higher temperature. In addition, GR119 (Rmg8/RmgGR119) displayed higher levels of resistance than IL191 at this stage. These results suggest that Rmg8 combined with RmgGR119 will be useful in breeding for resistance against wheat blast in Bangladesh.
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.
Pyricularia oryzae, a blast fungus of gramineous plants, is composed of various host-genus specific pathotypes. The avirulence of an Avena isolate on wheat is conditioned by PWT3 and PWT4. We ...isolated the third avirulence gene from the Avena isolate and designated it as PWT7. PWT7 was effective as an avirulence gene only at the seedling stage or on leaves. PWT7 homologs were widely distributed in a subpopulation of the Eleusine pathotype and the Lolium pathotype but completely absent in the Triticum pathotype (the wheat blast fungus). The PWT7 homolog found in the Eleusine pathotype was one of the five genes involved in its avirulence on wheat. A comparative analysis of distribution of PWT7 and the other two genes previously identified in the Eleusine pathotype suggested that, in the course of parasitic specialization toward the wheat blast fungus, a common ancestor of the Eleusine, Lolium, Avena, and Triticum pathotypes first lost PWT6, secondly PWT7, and finally the function of PWT3. PWT7 or its homologs were located on core chromosomes in Setaria and Eleusine isolates but on supernumerary chromosomes in Lolium and Avena isolates. This is an example of inter-chromosomal translocations of effector genes between core and supernumerary chromosomes.
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.
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