Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by self-inhibitors and requires surface attachment.
Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in Colletotrichum gloeosporioides and Colletotrichum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment. (+info)
Population structure and dynamics of Magnaporthe grisea in the Indian Himalayas.
The population genetics of Magnaporthe grisea, the rice blast pathogen, were analyzed in a center of rice diversity (the Uttar Pradesh hills of the Indian Himalayas) using multilocus and single-, or low-copy, DNA markers. Based on DNA fingerprinting with the multilocus probe MGR586 and single-locus probes, 157 haplotypes clustered into 56 lineages (at >/=70% MGR586 band similarity, each with unique single-locus profiles) and high diversity indices were detected among 458 isolates collected from 29 sites during 1992-1995. Most valleys sampled had distinct populations (73% of the lineages were site specific) with some containing one or a few lineages, confirming the importance of clonal propagation, and others were very diverse. Widely distributed lineages suggested that migration occurs across the region and into the Indo-Gangetic plains. Repeated sampling at one site, Matli, (170 isolates, 1992-1995) yielded 19 lineages and diversity significantly greater than that reported from similar samples from Colombia and the Philippines. Analysis of allelic associations using pairwise comparisons and multilocus variance analysis failed to reject the hypothesis of gametic phase equilibrium. The Matli population shifted from highly diverse in 1992 to almost complete dominance by one lineage in 1995. Such population dynamics are consistent with recombination followed by differential survival of clonal descendants of recombinant progeny. At another site, Ranichauri, population (n = 84) composition changed from 2 to 11 lineages over 2 yr and yielded additional evidence for equilibrium. Sexually fertile and hermaphrodite isolates of both mating types were recovered from rice in both Matli and Ranichauri. We demonstrate that Himalayan M. grisea populations are diverse and dynamic and conclude that the structure of some populations may be affected to some extent by sexual recombination. (+info)
Physical map and organization of chromosome 7 in the rice blast fungus, Magnaporthe grisea.
The rice blast fungus Magnaporthe grisea is a highly destructive plant pathogen and one of the most important for studying various aspects of host-plant interactions. It has been widely adopted as a model organism because it is ideally suited for genetic and biological studies. To facilitate map-based cloning, chromosome walking, and genome organization studies of M. grisea, a complete physical map of chromosome 7 was constructed using a large-insert (130 kb) bacterial artificial chromosome (BAC) library. Using 147 chromosome 7-specific single-copy BAC clones and 20 RFLP markers on chromosome 7, 625 BAC clones were identified by hybridization. BAC clones were digested with HindIII, and fragments were size separated on analytical agarose gels to create DNA fingerprints. Hybridization contigs were constructed using a random cost algorithm, whereas fingerprinting contigs were constructed using the software package FPC. Results from both methods were generally in agreement, but numerous anomalies were observed. The combined data produced five robust anchored contigs after gap closure by chromosomal walking. The genetic and physical maps agreed closely. The final physical map was estimated to cover >95% of the 4.2 Mb of chromosome 7. Based on the contig maps, a minimum BAC tile containing 42 BAC clones was created, and organization of repetitive elements and expressed genes of the chromosome was investigated. (+info)
Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues.
Mutagenesis of Magnaporthe grisea strain 4091-5-8 led to the identification of PTH11, a pathogenicity gene predicted to encode a novel transmembrane protein. We localized a Pth11-green fluorescent protein fusion to the cell membrane and vacuoles. pth11 mutants of strain 4091-5-8 are nonpathogenic due to a defect in appressorium differentiation. This defect is reminiscent of wild-type strains on poorly inductive surfaces; conidia germinate and undergo early differentiation events, but appressorium maturation is impaired. Functional appressoria are formed by pth11 mutants at 10 to 15% of wild-type frequencies, suggesting that the protein encoded by PTH11 (Pth11p) is not required for appressorium morphogenesis but is involved in host surface recognition. We assayed Pth11p function in multiple M. grisea strains. These experiments indicated that Pth11p can activate appressorium differentiation in response to inductive surface cues and repress differentiation on poorly inductive surfaces and that multiple signaling pathways mediate differentiation. PTH11 genes from diverged M. grisea strains complemented the 4091-5-8 pth11 mutant, indicating functional conservation. Exogenous activation of cellular signaling suppressed pth11 defects. These findings suggest that Pth11p functions at the cell cortex as an upstream effector of appressorium differentiation in response to surface cues. (+info)
Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea.
The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase-encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea deltaosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Deltaosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol. (+info)
Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly.
Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60-meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5-nitroso-6-ribitylamino-2,4-pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 A resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four-stranded parallel beta-sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N-terminal beta-strand interacts with the beta-sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand beta1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the beta-hairpin connecting helices alpha4 and alpha5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface. (+info)
BWMK1, a novel MAP kinase induced by fungal infection and mechanical wounding in rice.
The activation of the mitogen-activated protein (MAP) kinases by different environmental stresses has been previously observed in several dicot plant species. Here, we report the isolation of a novel MAP kinase in rice that is induced during infection by the blast fungus Magnaporthe grisea or upon mechanical wounding. The gene is designated as BWMK1 for blast- and wound-induced MAP kinase. The cDNA of BWMK1 was isolated from rice leaves challenged by the blast pathogen. Transcripts of the corresponding gene accumulated in rice leaves 4 h after blast inoculation and 30 min after mechanical wounding. This gene encodes a 506 amino acid protein that contains a new dual-phosphorylation activation motif TDY and about 150 unique amino acids on its C terminus. In-gel kinase activity and immunoprecipitation assays confirmed that BWMK1 is a functional MAP kinase. These results show that BWMK1 is a new member of the plant MAP kinase family and may mediate both defense and wound signaling in rice. (+info)
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. (+info)