Cautionary Notes on Use of the MoT3 Diagnostic Assay for Magnaporthe oryzae Wheat and Rice Blast Isolates.

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat, and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the United Kingdom. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.

Pathogenicities of Rice Blast (Pyricularia oryzae Cavara) Isolates From Kenya.

A total of 99 isolates of rice blast (Pyricularia oryzae Cavara) were collected from 2010 to 2015 from four regions in Kenya: Kirinyaga County and Embu County, Kisumu County, Tana River County, and Mombasa County. The pathogenicities of these isolates were clarified based on the reaction patterns of Lijiangxintuanheigu and differential varieties (DVs) targeting 23 resistance genes. The frequency of virulent isolates was high for DVs for Pib, Pia, Pii, Pi3, Pi5(t), Pik-s, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Pi19(t), and Pi20(t); low for DVs for Pish, Pi9(t), Piz-5, and Piz-t; and intermediate for the remaining DVs for Pit, Piz, Pita-2, Pita, and Pi12(t). These blast isolates were classified into three cluster groups: Ia, Ib, and II. The frequencies of virulent isolates to DVs for Pit, Pii, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Piz, and Pi12(t) differed markedly between clusters I and II, and those of DVs for Pib, Pit, Pia, Pi3, Pita-2, Pita, and Pi20(t) differed between Ia and Ib. The frequencies of cluster groups in the four geographical regions were different. A total of 62 races were found, with 19 blast isolates categorized into one race (U63-i7-k177-z00-ta003), whereas the other races included only some isolates in each.

Dynamics of Race Structures of the Rice Blast Pathogen Population in Heilongjiang Province, China From 2006 Through 2015.

Rice blast caused by the fungus Magnaporthe oryzae is one of the most destructive diseases of rice. Its control through the deployment of host resistance genes would be facilitated by understanding the pathogen's race structure. Here, dynamics of race structures in this decade in Heilongjiang province were characterized by Chinese differential cultivars. Two patterns of dynamics of the race structures emerged: both race diversity and population-specific races increased gradually between 2006 and 2011, but they increased much more sharply between 2011 and 2015, with concomitant falls in both the population-common races and dominant races. Four races (ZD1, ZD3, ZD5, and ZE1) were among the top three dominant races over the whole period, indicating that the core of the race structure remained stable through this decade. On the host side, the composition of resistance in the cultivar differential set could be divided in two: the three indica-type entries of the differential set expressed a higher level of resistance to the population of M. oryzae isolates tested than did the four japonica-type entries. The cultivars Tetep and Zhenlong 13 as well as two additional resistance genes α and ε were confirmed as the most promising donors of blast resistance for the local rice improvement programs.[Formula: see text]Copyright © 2019 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass.

BACKGROUND: A number of Pyricularia species are known to infect different grass species. In the case of Pyricularia oryzae (syn. Magnaporthe oryzae), distinct populations are known to be adapted to a wide variety of grass hosts, including rice, wheat and many other grasses. The genome sizes of Pyricularia species are typical for filamentous ascomycete fungi [~ 40 Mbp for P. oryzae, and ~ 45 Mbp for P. grisea]. Genome plasticity, mediated in part by deletions promoted by recombination between repetitive elements [Genome Res 26:1091-1100, 2016, Nat Rev Microbiol 10:417-430,2012] and transposable elements [Annu Rev Phytopathol 55:483-503,2017] contributes to host adaptation. Therefore, comparisons of genome structure of individual species will provide insight into the evolution of host specificity. However, except for the P. oryzae subgroup, little is known about the gene content or genome organization of other Pyricularia species, such as those infecting Pennisetum grasses. RESULTS: Here, we report the genome sequence of P. penniseti strain P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. Phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species indicated that P1609 belongs to a Pyricularia subclade, which is genetically distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires had diverged between P1609 and the P. oryzae strain 70-15, including the known avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements relative to P. oryzae. CONCLUSION: Our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species and provide new insight into evolution of pathogen genomes during host adaptation.

Genetic Structure of the Rice Blast Pathogen (Magnaporthe oryzae) over a Decade in North Central California Rice Fields.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future.

Selection of Differential Isolates of Magnaporthe oryzae for Postulation of Blast Resistance Genes.

A set of differential isolates of Magnaporthe oryzae is needed for the postulation of blast resistance genes in numerous rice varieties and breeding materials. In this study, the pathotypes of 1,377 M. oryzae isolates from different regions of China were determined by inoculating detached rice leaves of 24 monogenic lines. Among them, 25 isolates were selected as differential isolates based on the following characteristics: they had distinct responses on the monogenic lines, contained the minimum number of avirulence genes, were stable in pathogenicity and conidiation during consecutive culture, were consistent colony growth rate, and, together, could differentiate combinations of the 24 major blast resistance genes. Seedlings of rice cultivars were inoculated with this differential set of isolates to postulate whether they contain 1 or more than 1 of the 24 blast resistance genes. The results were consistent with those from polymerase chain reaction analysis of target resistance genes. Establishment of a standard set of differential isolates will facilitate breeding for blast resistance and improved management of rice blast disease.

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.

MoCps1 is important for conidiation, conidial morphology and virulence in Magnaporthe oryzae.

Conidia play important roles in primary and secondary infections of airborne fungal pathogens. In this study, an insertional mutant with reduced capacity for conidiation was isolated from the rice blast fungus Magnaporthe oryzae. The mutant has a T-DNA insertion that disrupts a gene named MoCPS1. The deduced MoCps1 protein contains three AMP-binding domains. Gene complementation and gene knockout assays confirmed that MoCPS1 is important for conidiation. Conidia produced by the MoCPS1 deletion mutants are much more slender and longer than those produced by the wild-type strain. The Mocps1 mutants are less efficient in both appressorial penetration and invasive growth of infection hyphae, resulting in attenuated virulence toward host plants. MoCPS1 is highly expressed in a mature appressorium. Interestingly, the expression levels of several genes related to conidiation and pathogenicity have been significantly altered in the MoCPS1 deletion mutants. Taken together, our results indicated that MoCPS1 is important for conidiogenesis, conidial morphogenesis, and pathogenesis in the rice blast fungus.

Systematic characterization of the bZIP transcription factor gene family in the rice blast fungus, Magnaporthe oryzae.

Regulatory roles of the basic leucine zipper (bZIP) transcription factors (TFs) in fungi have been identified in diverse cellular processes such as development, nutrient utilization and various stress responses. In this study, the 22 Magnaporthe oryzae genes encoding bZIP TFs were systematically characterized. Phylogenetic analysis of fungal bZIP TFs revealed that seven MobZIPs are Magnaporthe-specific, while others belongs to 15 clades of orthologous Ascomycota genes. Expression patterns of MobZIPs under various conditions showed that they are highly stress responsive. We generated deletion mutants for 13 MobZIPs: nine with orthologues in other fungal species and four Magnaporthe-specific ones. Seven of them exhibited defects in mycelial growth, development and/or pathogenicity. Consistent with the conserved functions of the orthologues, MobZIP22 and MobZIP13 played a role in sulfur metabolism and iron homeostasis respectively. Along with MobZIP22 and MobZIP13, one Magnaporthe-specific gene, MobZIP11 is essential for pathogenicity in a reactive oxygen species-dependent manner. Taken together, our results will contribute to understanding the regulatory mechanisms of the bZIP TF gene family in fungal development, adaptation to environmental stresses and pathogenicity in the rice blast fungus.

Comparative analysis of the genomes of two field isolates of the rice blast fungus Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus.

Transposable elements impact the population divergence of rice blast fungus Magnaporthe oryzae.

Dynamic transposition of transposable elements (TEs) in fungal pathogens has significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae, genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here, we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/Japonica and Xian/Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage. IMPORTANCE: Magnaporthe oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward the two rice subspecies, Xian/Indica and Geng/Japonica. Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remain largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/Indica-infecting and Geng/Japonica-infecting populations. Our results revealed a TE insertion-mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies.

Magnaporthiopsis, a new genus in Magnaporthaceae (Ascomycota).

The phylogenetic relationships among taxa in the Magnaporthaceae are investigated based on DNA sequences of multiple genes including SSU, ITS, LSU, MCM7, RPB1 and TEF1. The genera Magnaporthe and Gaeumannomyces are shown to be polyphyletic and their members are divided into four major groups based on the phylogenetic analyses. Considering morphological, biological and molecular data, we establish a new genus, Magnaporthiopsis. It is characterized by black and globose perithecia with a cylindrical neck, two-layered perithecial wall, clavate asci with a refractive apical ring, fusiform to fusoid and septate ascospores, simple hyphopodia, and Phialophora-like anamorph. Species in this genus are necrotrophic parasites infecting roots of grasses. Three new combinations, Magnaporthiopsis poae, M. rhizophila and M. incrustans, are proposed accordingly. Pyricularia is suggested as the generic name for the rice blast fungus over Magnaporthe, following Article 59.1 of the International Code of Nomenclature for algae, fungi and plants. A new combination, Nakataea oryzae, is proposed for the rice stem rot fungus.

A carnitine-acylcarnitine carrier protein, MoCrc1, is essential for pathogenicity in Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae forms a specialized infection structure called an appressorium to breach the host-plant epidermis for successful infection. In this study, a mutant defective in appressorial penetration was isolated by a mutagenesis approach, in which an exogenous DNA fragment was found to be inserted into the first exon of MoCRC1. This gene encodes a putative carnitine-acylcarnitine carrier protein that is widely conserved among eukaryotic organisms. Deletion of MoCRC1 severely reduces appressorium turgor generation, appressorial penetration, and development of infection hyphae. The null mutant of MoCRC1 lost pathogenicity on intact and abraded host leaves. MoCRC1 was also found to be required for growth on minimal medium containing sodium acetate or olive oil. Moreover, the transformed MoCrc1-eGFP fusion protein was expressed throughout the infection process. Our results suggest that the carnitine-acylcarnitine carrier protein plays vital roles in appressorium-mediated infection and is essential for pathogenesis of M. oryzae and perhaps other phytopathogenic fungi.

The genome sequence of the rice blast fungus Magnaporthe grisea.

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.

Convergent evolution: gene sharing by eukaryotic plant pathogens.

Oomycetes and filamentous parasitic fungi are plant pathogens that have undergone convergent evolution. A recent study has shown that these microbial eukaryotes have exchanged metabolic genes, which might explain some of their phenotypic similarities.

Identification of genes for resistance to a Digitaria isolate of Magnaporthe grisea in common wheat cultivars.

Common wheat cultivars are resistant to Magnaporthe grisea, a crabgrass (Digitaria sanguinalis)-specific species of the blast fungus. To dissect the genetic basis of this "nonhost" type of resistance, we need an exceptional cultivar that is susceptible to M. grisea. A screening under various conditions revealed that Triticum aestivum 'Chinese Spring' (CS) was susceptible to M. grisea isolate Dig41 when incubated at high temperature (26 degrees C) after inoculation. By contrast, T. aestivum 'P168', 'Shin-chunaga' (Sch), 'Norin 4' (N4), 'Norin 26' (N26), 'Norin 29' (N29), 'Red Egyptian' (RE), and 'Salmon' (Slm) and Triticum compactum 'No. 44' (Cmp) were highly resistant even at the high temperature. When F2 seedlings derived from crosses between the resistant cultivars and CS were inoculated with Dig41, they segregated in a 3:1 ratio of resistant to susceptible, suggesting that the resistance of each cultivar is controlled by one major gene. Crosses of N4 with P168, Sch, N26, N29, and Cmp yielded no susceptible F2 seedlings, suggesting that these six cultivars share the same gene. Similarly, a cross between RE and Slm yielded no susceptible F2 seedlings, suggesting that these two cultivars share the same gene. On the other hand, crosses between the N4 group and the RE group produced resistant and susceptible seedlings in a 15:1 ratio, indicating that these two groups carry different genes inherited independently. The gene in N4 was located on chromosome 4A by a monosomic analysis and designated Rmg4, while the gene in RE was located on chromosome 6D using a series of chromosome substitution lines and designated Rmg5. These results suggest that the resistance of common wheat to M. grisea, an inappropriate species of the blast fungus, is under a simple genetic control.

MADS-box transcription factor mig1 is required for infectious growth in Magnaporthe grisea.

Magnaporthe grisea is a model fungus for studying fungus-plant interactions. Two mitogen-activated protein (MAP) kinase genes, PMK1 and MPS1, have been implicated in regulating plant infection processes in M. grisea. However, transcription factors activated by these MAP kinases are not well studied. In this study we functionally characterized the MIG1 gene that encodes a MADS-box transcription factor homologous to Saccharomyces cerevisiae Rlm1. In yeast two-hybrid assays, MIG1 interacts with MPS1, suggesting that MIG1 may function downstream from the MPS1 pathway. The mig1 deletion mutant had a normal growth rate and formed melanized appressoria, but it was nonpathogenic and failed to infect rice leaves through wounds. Appressoria formed by the mig1 mutant developed penetration pegs and primary infectious hyphae, but further differentiation of the secondary infectious hyphae inside live plant cells was blocked. However, the mig1 mutant formed infectious hypha-like structures in heat-killed plant cells or cellophane membranes. In transformants expressing the MIG1-GFP fusion, green fluorescent protein (GFP) signals were not detectable in vegetative hyphae and conidiophores. Mig1-GFP was localized to nuclei in conidia, appressoria, and infectious hyphae. Deletion of the MADS box had no effect on the expression and localization of the MIG1-GFP fusion but eliminated its ability to complement the mig1 mutant. These results suggest that MIG1 may be required for overcoming plant defense responses and the differentiation of secondary infectious hyphae in live plant cells. The MADS-box domain is essential for the function of MIG1 but dispensable for its nuclear localization, which may be associated with the activation of MIG1 by MPS1 during conidiation and plant infection.

Differentiation of Magnaporthe species complex by rep-PCR genomic fingerprinting.

Rice blast disease, caused by the fungus Magnoporthe grisea is responsible for considerable damages on rice and leaf spot on some weeds in Iran and in other parts of the world. Infected samples were collected from rice and weeds including Digitaria sanguinalis (crabgrass), Setaria italica (foxtail millet), Echinochloa crus-galli (barnyard millet), and some unknown weeds during 1997-2005 and were preserved in collection of Mycology at the University of Tehran, Iran. In this study, genetic diversity of Magnaporthe grisea species complex isolates was studied based on DNA fingerprinting by rep-PCR, using of two primers including ERIC and BOX. The total DNA of 75 isolates was extracted and DNA fragments were amplified in a thermal cycler program using mentioned primers. Therefore, DNA fragments from 400 bp to 3000 bp were amplified. Based on cluster analysis for two primers (ERIC and BOX), eight fingerprinting groups (ctonal lineages) and sixty haplotypes were identified. "A" clonal lineage was containing the highest number of isolates and became dominant clonal lineages with 35 isolates from rice and 3 isolates from S. italica, whereas the highest number of isolates obtained from D. sanguinalis belonged to "E" clonal lineage and was the second largest clonal lineage. Approximately all of the M. grisea species complex isolates from crabgrass and some of unknown weeds were separated from other isolates in 42% similarity. As a result, asexual fertility causes low diversity in populations of M. grisea species complex and speciation could be one of the reasons of differentiation between isolates from D. sanguinalis with other isolates. Overall, these data indicated a low level of genetic diversity in the Iranian M. grisea species complex population similar to that reported in other countries.

Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex.

The avirulence (AVR) gene AVR-Pita in Magnaporthe oryzae prevents the fungus from infecting rice cultivars containing the resistance gene Pi-ta. A survey of isolates of the M. grisea species complex from diverse hosts showed that AVR-Pita is a member of a gene family, which led us to rename it to AVR-Pita1. Avirulence function, distribution, and genomic context of two other members, named AVR-Pita2 and AVR-Pita3, were characterized. AVR-Pita2, but not AVR-Pita3, was functional as an AVR gene corresponding to Pi-ta. The AVR-Pita1 and AVR-Pita2 genes were present in isolates of both M. oryzae and M. grisea, whereas the AVR-Pita3 gene was present only in isolates of M. oryzae. Orthologues of members of the AVR-Pita family could not be found in any fungal species sequenced to date, suggesting that the gene family may be unique to the M. grisea species complex. The genomic context of its members was analyzed in eight strains. The AVR-Pita1 and AVR-Pita2 genes in some isolates appeared to be located near telomeres and flanked by diverse repetitive DNA elements, suggesting that frequent deletion or amplification of these genes within the M. grisea species complex might have resulted from recombination mediated by repetitive DNA elements.

Characterization of the model system rice--Magnaporthe for the study of nonhost resistance in cereals.

The best characterized form of resistance is gene-for-gene resistance. Less well characterized is nonhost resistance in which an entire plant species is resistant to an entire pathogen species. Here, different rice genotypes were inoculated with host and nonhost strains of Magnaporthe isolated from rice, wheat and crabgrass. The different types of interactions were characterized at a cytological level using a 3,3'-diaminobenzidine (DAB) stain to investigate the occurrence of reactive oxygen intermediates or by observing the occurrence of cellular autofluorescence. Gene expression of a set of selected PR-genes was analysed using quantitative real-time polymerase chain reaction. Inoculation with the isolate from crabgrass resulted in a lack of penetration. The wheat isolate induced a hypersensitive response with varying degrees of pathogen growth inside the invaded cell according to the rice genotype. Expression analysis of our PR-gene set revealed clear differences between the different types of interactions in both kinetic and magnitude of gene induction. Our integrated study opens the way to the dissection of molecular components leading to nonhost reactions to Magnaporthe grisea in rice and points to novel sources of durable resistance to fungal plant pathogens in other cereal crops.