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 (u = 8.2 3 10 25 ), 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.

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 and, whose gene products elicit defense in wheat cultivars containing the corresponding resistance genes and 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 wheat (susceptible to isolates), followed by the loss of function of This implies that the wheat served as a springboard for the host jump to common wheat.

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.

We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. In M. oryzae, a silencing vector with the cutinase intron spacer (147 bp) showed a higher efficiency in silencing of the eGFP gene than did those with a spacer of a GUS gene fragment or a longer intron (850 bp) of a chitin binding protein gene. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene, resulted in various degrees of silencing of the genes in 70-90% of the resulting transformants. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.

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, 1.2–3.5 % 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.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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2690-6) contains supplementary material, which is available to authorized users.