Resolving the polyphyletic nature of Pyricularia (Pyriculariaceae).

Species of Pyricularia (magnaporthe-like sexual morphs) are responsible for major diseases on grasses. Pyricularia oryzae (sexual morph Magnaporthe oryzae) is responsible for the major disease of rice called rice blast disease, and foliar diseases of wheat and millet, while Pyricularia grisea (sexual morph Magnaporthe grisea) is responsible for foliar diseases of Digitaria. Magnaporthe salvinii, M. poae and M. rhizophila produce asexual spores that differ from those of Pyricularia sensu stricto that has pyriform, 2-septate conidia produced on conidiophores with sympodial proliferation. Magnaporthe salvinii was recently allocated to Nakataea, while M. poae and M. rhizophila were placed in Magnaporthiopsis. To clarify the taxonomic relationships among species that are magnaporthe- or pyricularia-like in morphology, we analysed phylogenetic relationships among isolates representing a wide range of host plants by using partial DNA sequences of multiple genes such as LSU, ITS, RPB1, actin and calmodulin. Species of Pyricularia s. str. belong to a monophyletic clade that includes all P. oryzae/P. grisea isolates tested, defining the Pyriculariaceae, which is sister to the Ophioceraceae, representing two novel families. These clades are clearly distinct from species belonging to the Gaeumannomyces pro parte/Magnaporthiopsis/Nakataea generic complex that are monophyletic and define the Magnaporthaceae. A few magnaporthe- and pyricularia-like species are unrelated to Magnaporthaceae and Pyriculariaceae. Pyricularia oryzae/P. grisea isolates cluster into two related clades. Host plants such as Eleusine, Oryza, Setaria or Triticum were exclusively infected by isolates from P. oryzae, while some host plant such as Cenchrus, Echinochloa, Lolium, Pennisetum or Zingiber were infected by different Pyricularia species. This demonstrates that host range cannot be used as taxonomic criterion without extensive pathotyping. Our results also show that the typical pyriform, 2-septate conidium morphology of P. grisea/P. oryzae is restricted to Pyricularia and Neopyricularia, while most other genera have obclavate to more ellipsoid 2-septate conidia. Some related genera (Deightoniella, Macgarvieomyces) have evolved 1-septate conidia. Therefore, conidium morphology cannot be used as taxonomic criterion at generic level without phylogenetic data. We also identified 10 novel genera, and seven novel species. A re-evaluation of generic and species concepts within Pyriculariaceae is presented, and novelties are proposed based on morphological and phylogenetic data.

Microsatellite markers for population studies of Phytophthora megakarya (Pythiaceae), a cacao pathogen in Africa.

PREMISE OF THE STUDY: Phytophthora megakarya is the agent of black pod disease of cacao and is the main pathogen of this crop in Africa. Population genetic studies are required to investigate how this pathogen emerged. To this end, we developed 12 novel polymorphic microsatellite markers for P. megakarya. METHODS AND RESULTS: Microsatellite sequences were obtained by pyrosequencing of multiplex-enriched libraries. Candidate loci with di- or trinucleotide motifs were selected, and primer pairs were tested with nine P. megakarya isolates. The 12 most polymorphic and unambiguous loci were selected to develop three multiplex PCR pools. The total number of alleles varied from two to nine, depending on loci, and higher than expected heterozygosity was observed. CONCLUSIONS: These markers were used for population genetic studies of P. megakarya in Cameroon and for comparison with reference strains from West Africa. This is the first time that microsatellite markers have been developed for P. megakarya.

Preformed expression of defense is a hallmark of partial resistance to rice blast fungal pathogen Magnaporthe oryzae.

BACKGROUND: Partial resistance to plant pathogens is extensively used in breeding programs since it could contribute to resistance durability. Partial resistance often builds up during plant development and confers quantitative and usually broad-spectrum resistance. However, very little is known on the mechanisms underlying partial resistance. Partial resistance is often explained by poorly effective induction of plant defense systems. By exploring rice natural diversity, we asked whether expression of defense systems before infection could explain partial resistance towards the major fungal pathogen Magnaporthe oryzae. The constitutive expression of 21 defense-related genes belonging to the defense system was monitored in 23 randomly sampled rice cultivars for which partial resistance was measured. RESULTS: We identified a strong correlation between the expression of defense-related genes before infection and partial resistance. Only a weak correlation was found between the induction of defense genes and partial resistance. Increasing constitutive expression of defense-related genes also correlated with the establishment of partial resistance during plant development. Some rice genetic sub-groups displayed a particular pattern of constitutive expression, suggesting a strong natural polymorphism for constitutive expression of defense. Constitutive levels of hormones like salicylic acid and ethylene cannot explain constitutive expression of defense. We could identify an area of the genome that contributes to explain both preformed defense and partial resistance. CONCLUSION: These results indicate that constitutive expression of defense-related genes is likely responsible for a large part of partial resistance in rice. The finding of this preformed defense system should help guide future breeding programs and open the possibility to identify the molecular mechanisms behind partial resistance.

ARCHIPELAGO: a dedicated resource for exploiting past, present, and future genomic data on disease resistance regulation in rice.

Large amounts of expression data dealing with biotic stresses in rice have been produced in the past 5 years. Here, we extensively review approximately 70 publications and gather together information on more than 2,500 genes of the rice defense arsenal. This information was integrated into the OryGenesDB database. Several genes (e.g., metallothioneins and PBZ1) appear to be hallmarks of rice-pathogen interactions. Cross-referencing this information with the rice kinome highlighted some defense genes and kinases as possible central nodes of regulation. Cross referencing defense gene expression and quantitative trait loci (QTL) information identified some candidate genes for QTL. Overall, pathogenesis-related genes and disease regulators were found to be statistically associated with disease QTL. At the genomic level, we observed that some regions are richer than others and that some chromosomes (e.g., 11 and 12), which contain a lot of resistance gene analogs, have a low content of defense genes. Finally, we show that classical defense genes and defense-related genes such as resistance genes are preferentially organized in clusters. These clusters are not always coregulated and individual paralogs can show specific expression patterns. Thus, the rice defense arsenal has an ARCHIPELAGO-like genome structure at the macro and micro level. This resource opens new possibilities for marker-assisted selection and QTL cloning.

Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus.

* Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. * Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. * One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. * Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.

Identification and fine mapping of Pi33, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene ACE1.

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) x Azucena (susceptible) and Azucena x Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [ Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F(2) populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.

Identification of five new blast resistance genes in the highly blast-resistant rice variety IR64 using a QTL mapping strategy.

Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 x Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24( t) to Pi-32( t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea.

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers.

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.

RAPD-based screening of genomic libraries for positional cloning.

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.