Differences in the transcriptional immune response to Albugo candida between white rust resistant and susceptible cultivars in Brassica rapa L.

Albugo candida causing white rust disease decreases the yield of Brassica rapa vegetables greatly. Resistant and susceptible cultivars in B. rapa vegetables have different immune responses against A. candida inoculation, however, the mechanism of how host plants respond to A. candida is still unknown. Using RNA-sequencing, we identified differentially expressed genes (DEGs) between A. candida inoculated [48 and 72 h after inoculation (HAI)] and non-inoculated samples in resistant and susceptible cultivars of komatsuna (B. rapa var. perviridis). Functional DEGs differed between the resistant and susceptible cultivars in A. candida inoculated samples. Salicylic acid (SA) responsive genes tended to be changed in their expression levels by A. candida inoculation in both resistant and susceptible cultivars, but different genes were identified in the two cultivars. SA-dependent systemic acquired resistance (SAR) involving genes were upregulated following A. candida inoculation in the resistant cultivar. Particular genes categorized as SAR that changed expression levels overlapped between A. candida and Fusarium oxysporum f. sp. conglutinans inoculated samples in resistant cultivar, suggesting a role for SAR in defense response to both pathogens particularly in the effector-triggered immunity downstream pathway. These findings will be useful for understanding white rust resistance mechanisms in B. rapa.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum.

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

Pathogenicity and Distribution of Fusarium solani Isolates Associated with Erythrina Decline in Japan.

Erythrina spp. trees have been declining since the 2000s worldwide, and fungi belonging to Fusarium solani species complex (FSSC) have been suggested to be a causal factor of decline and mortality of Erythrina variegata trees in Okinawa Island, Japan. In addition to the FSSC isolate grouped as "it-1" based on ITS sequence data (previously called strain A), we conducted an inoculation experiment with two isolates grouped as "it-2" (previously strain B), which is genetically close to it-1. Two it-2 isolates originating from two islands showed pathogenicity to E. variegata with the same symptoms as those caused by it-1 isolate. We also found the isolates of it-1 and it-2 were widely distributed, including on Ishigaki Island, ∼400 km south of Okinawa Island across the ocean. All isolates of it-1 and it-2 belong to the ambrosia Fusarium clade of the FSSC, a group of symbionts of ambrosia beetles, including the pathogens of Fusarium dieback in avocados and teas. The detection of ambrosia beetles Euwallacea spp. from our specimens provided information on the vectors of the pathogens. Our present results suggest the fungi of the FSSC could be responsible for the Erythrina decline in other areas with damage.

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance.

A New Resistance Gene in Combination with Rmg8 Confers Strong Resistance Against Triticum Isolates of Pyricularia oryzae in a Common Wheat Landrace.

The wheat blast fungus (Triticum pathotype of Pyricularia oryzae) first arose in Brazil in 1985 and has recently spread to Asia. Resistance genes against this new pathogen are very rare in common wheat populations. We screened 520 local landraces of common wheat collected worldwide with Br48, a Triticum isolate collected in Brazil, and found a highly resistant, unique accession, GR119. When F2 seedlings derived from a cross between GR119 and Chinese Spring (CS, susceptible control) were inoculated with Br48, resistant and susceptible seedlings segregated in a 15:1 ratio, suggesting that GR119 carries two resistance genes. When the F2 seedlings were inoculated with Br48ΔA8 carrying a disrupted allele of AVR-Rmg8 (an avirulence gene corresponding to a previously reported resistance gene, Rmg8), however, the segregation fitted a 3:1 ratio. These results suggest that one of the two genes in GR119 was Rmg8. The other, new gene was tentatively designated as RmgGR119. GR119 was highly resistant to all Triticum isolates tested. Spikes of GR119 were highly resistant to Br48, moderately resistant to Br48ΔA8 and a hybrid culture carrying avr-Rmg8 (nonfunctional allele), and highly resistant to its transformant carrying AVR-Rmg8. The strong resistance of GR119 was attributed to the combined effects of Rmg8 and RmgGR119.

Rmg8 and Rmg7, wheat genes for resistance to the wheat blast fungus, recognize the same avirulence gene AVR-Rmg8.

Rmg8 and Rmg7 are genes for resistance to the wheat blast fungus (Pyricularia oryzae), located on chromosome 2B in hexaploid wheat and chromosome 2A in tetraploid wheat, respectively. AVR-Rmg8, an avirulence gene corresponding to Rmg8, was isolated from a wheat blast isolate through a map-based strategy. The cloned fragment encoded a small protein containing a putative signal peptide. AVR-Rmg8 was recognized not only by Rmg8, but also by Rmg7, suggesting that these two resistance genes are equivalent to a single gene from the viewpoint of resistance breeding.

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

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.

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).

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

BACKGROUND: Magnaporthe 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. RESULTS: Whole 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, 1.2-3.5 % of genes showed presence/absence polymorphisms and 0-6.5 % 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. CONCLUSION: Our 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.

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.

Loss of a 1.6 Mb chromosome in Pyricularia oryzae harboring two alleles of AvrPik leads to acquisition of virulence to rice cultivars containing resistance alleles at the Pik locus.

A small and extra chromosome of 1.6 Mb was previously identified in a Pyricularia oryzae strain, 84R-62B. To understand a role of the 1.6 Mb chromosome in the pathogenic changeability of P. oryzae, we performed experiments designed to characterize the 1.6 Mb chromosome in the present study. A gene family encoding secreted protein Pex31s in P. oryzae consists of five homologs, Pex31-A to -E. Among them, Pex31-A and -D are known to be recognized by Pik-m and Pik/Pik-m/Pik-p, respectively. In the present study, we identified Pex31-A and -D in the genome of 84R-62B. Segregation analyses using an F1 population between 84R-62B and another rice blast strain, Y93-245c-2, revealed a strong linkage between the two homologs and the 1.6 Mb chromosome of 84R-62B. A CHEF-Southern analysis revealed an association between the 1.6 Mb chromosome and the homologs, indicating that both homologs are located on the 1.6 Mb chromosome of 84R-62B. The loss of the 1.6 Mb chromosome was observed in subcultures of a F1 progeny, F1-327. These subcultures concomitantly acquired virulence on Pik, Pik-m, and Pik-p. The present study is the first report showing that loss of a small and extra chromosome leads to pathogenic mutation of P. oryzae and may provide a new insight into the mechanisms generating pathogenic variation of this fungus.

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.

Is the fungus Magnaporthe losing DNA methylation?

The long terminal repeat retrotransposon, Magnaporthe gypsy-like element (MAGGY), has been shown to be targeted for cytosine methylation in a subset of Magnaporthe oryzae field isolates. Analysis of the F1 progeny from a genetic cross between methylation-proficient (Br48) and methylation-deficient (GFSI1-7-2) isolates revealed that methylation of the MAGGY element was governed by a single dominant gene. Positional cloning followed by gene disruption and complementation experiments revealed that the responsible gene was the DNA methyltransferase, MoDMT1, an ortholog of Neurospora crassa Dim-2. A survey of MAGGY methylation in 60 Magnaporthe field isolates revealed that 42 isolates from rice, common millet, wheat, finger millet, and buffelgrass were methylation proficient while 18 isolates from foxtail millet, green bristlegrass, Japanese panicgrass, torpedo grass, Guinea grass, and crabgrass were methylation deficient. Phenotypic analyses showed that MoDMT1 plays no major role in development and pathogenicity of the fungus. Quantitative polymerase chain reaction analysis showed that the average copy number of genomic MAGGY elements was not significantly different between methylation-deficient and -proficient field isolates even though the levels of MAGGY transcript were generally higher in the former group. MoDMT1 gene sequences in the methylation-deficient isolates suggested that at least three independent mutations were responsible for the loss of MoDMT1 function. Overall, our data suggest that MoDMT1 is not essential for the natural life cycle of the fungus and raise the possibility that the genus Magnaporthe may be losing the mechanism of DNA methylation on the evolutionary time scale.

Identification and molecular mapping of a wheat gene for resistance to an unadapted isolate of Colletotrichum cereale.

To elucidate genetic mechanisms of host species specificity between graminicolous anthracnose fungi and gramineous plants, infection assays were performed with a Sorghum isolate (Colletotrichum sublineolum), a Digitaria isolate (C. hanaui), a Polypogon isolate (C. cereale), and an Avena isolate (C. cereale). They were specifically virulent on the plants from which they were isolated. When 72 wheat lines were inoculated with an unadapted isolate from Asia Minor bluegrass (Cgp29), however, some exceptional cultivars were recognized. Although most cultivars were resistant to Cgp29, 'Hope' was susceptible. In F2 populations derived from crosses between three resistant cultivars-'Norin 4' (N4), 'Chinese Spring' (CS), and 'Shin-chunaga' (Sch)-and the susceptible Hope, resistant and susceptible seedlings segregated in a 3:1 ratio, suggesting that a major gene is involved in the resistance of each cultivar to Cgp29. In F2 populations derived from crosses between the three resistant cultivars, all seedlings were resistant, suggesting that these three cultivars carry the same gene. This resistance gene was designated as "resistance to Colletotrichum cereale 1" (Rcc1). Analysis with the CS-Hope chromosome substitution lines and molecular mapping revealed that Rcc1 was located on the long arm of chromosome 5A. Cytologically, Rcc1 was mainly associated with hypersensitive reaction. These results suggest that major genes similar to those controlling cultivar specificity are involved in the resistance of wheat against the unadapted isolate of C. cereale.

Identification of a novel locus Rmo2 conditioning resistance in barley to host-specific subgroups of Magnaporthe oryzae.

Barley cultivars show various patterns of resistance against isolates of Magnaporthe oryzae and M. grisea. Genetic mechanisms of the resistance of five representative barley cultivars were examined using a highly susceptible barley cultivar, 'Nigrate', as a common parent of genetic crosses. The resistance of the five cultivars against Setaria, Oryza, Eleusine, and Triticum isolates of M. oryzae was all attributed to a single locus, designated as Rmo2. Nevertheless, the Rmo2 locus in each cultivar was effective against a different range of isolates. Genetic analyses of pathogenicity suggested that each cultivar carries an allele at the Rmo2 locus that recognizes a different range of avirulence genes. One allele, Rmo2.a, corresponded to PWT1, which conditioned the avirulence of Setaria and Oryza isolates on wheat, in a gene-for-gene manner. The other alleles, Rmo2.b, Rmo2.c, and Rmo2.d, corresponded to more than one avirulence gene. On the other hand, the resistance of those cultivars to another species, M. grisea, was conditioned by another locus, designated as Rmo3. These results suggest that Rmo2 is effective against a broad range of blast isolates but is specific to M. oryzae. Molecular mapping revealed that Rmo2 is located on the 7H chromosome.

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.

PWT1, an avirulence gene of Magnaporthe oryzae tightly linked to the rDNA Locus, is recognized by two staple crops, common wheat and barley.

The pathogenicity to wheat (Pwt1) locus conditions host species specificity of Magnaporthe oryzae on wheat. GFSI1-7-2 (Setaria isolate) carries the avirulence allele (PWT1) at this locus while Br48 (Triticum isolate) carries the virulence allele (pwt1). An F(1) culture derived from a cross between GFSI1-7-2 and Br48 was backcrossed with Br48 to produce a tester population in which PWT1 alone segregated. When hexaploid wheat lines were inoculated with the BC(1)F(1) testers, they were all resistant to all PWT1 carriers and susceptible to all pwt1 carriers, suggesting that they recognize PWT1. When barley cultivars were inoculated with the testers, they showed the same pattern of reactions as the hexaploid lines, suggesting that the barley cultivars also recognize PWT1. These results suggest that PWT1 is a fundamental gene that universally conditions the avirulence of Setaria isolates on two staple crops, hexaploid wheat and barley. Interestingly, tetraploid wheat lines did not recognize PWT1. Molecular mapping using the F(1) and BC(1)F(1) populations revealed that the Pwt1 locus is located on chromosome 2 and tightly linked to the ribosomal DNA locus and a telomere.

Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae.

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=8.2x10(-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.