Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea.

An effective way to study the infection mechanisms of fungal pathogens is to disrupt their genes via transformation in both targeted and random manners. This isolates the mutants that exhibit altered virulence. In this paper, we report the successful transformation of Magnaporthe grisea, the causal agent for rice blast, that is mediated by Agrobacterium tumefaciens. Employing the binary vector pBHt2, which carries the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 500 to > 1,000 hygromycin B-resistant transformants per 1 x 10(6) conidia of M. grisea. The transformation efficiency is correlated with the number of A. tumefaciens cells used, pre-treating bacterial cells with acetosyringone prior to co-cultivation with fungal spores, and the duration of co-cultivation. All of the transformants tested remained mitotically stable, maintaining their hygromycin B resistance after several generations of growth in the absence of hygromycin B. A genomic Southern blot analysis showed that over 60% of the transformants contained a single T-DNA insert on their genome. Considering the efficiency and flexibility of A. tumefaciens-mediated transformation (ATMT), this technique offers highly efficient means for characterizing the genes that are important for the pathogenicity of M. grisea.     

Cellular differentiation and host invasion by the rice blast fungus Magnaporthe grisea

This review describes current advances in understanding the biology of plant infection by the rice blast fungus Magnaporthe grisea. Development of the specialized infection structure, the appressorium, in M. grisea has recently been shown to be controlled by cell cycle progression and initiation of autophagic, programmed cell death in the fungal spore. Re-cycling of the contents of the fungal spore and peroxisomal fatty acid beta-oxidation are therefore important processes for appressorium function. Following entry to the host plant, new evidence suggests that M. grisea grows biotrophically within rice cells, bounded by the plant plasmalemma, and the fungus moves from cell-to-cell by means of plasmodesmata. Biotrophic proliferation of the fungus is likely to require secretion of effector proteins and suppression of host defences. Consistent with this, a component of the polarized exocytosis machinery of M. grisea is necessary for pathogenicity and also for induction of host defences in an incompatible interaction. Large-scale insertional mutagenesis is now allowing the rapid analysis of gene function in M. grisea, heralding a new approach to the study of this important fungal pathogen.     

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.     

Transformation of the rice blast fungus Magnaporthe grisea to benomyl resistance

A transformation method based on a dominant selectable marker (benomyl resistance) was developed for the rice blast fungus Magnaporthe grisea. The heterologous gene for -tubulin from Neurospora crassa (pBT3) was used to obtain benomyl-resistant M. grisea transformants at a frequency of 20 to 30/g of DNA. Control transformations carried out with a plasmid conferring hygromycin resistance or a derivative of pBT3 containing a repetitive DNA sequence, yielded the same frequency of transformation as that of pBT3. Molecular analysis of the transformants indicated multiple integration of the vector DNA.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

PDE1 encodes a P-type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea

Plant infection by the rice blast fungus Magnaporthe grisea is brought about by the action of specialized infection cells called appressoria. These infection cells generate enormous turgor pressure, which is translated into an invasive force that allows a narrow penetration hypha to breach the plant cuticle. The Magnaporthe pde1 mutant was identified previously by restriction enzyme-mediated DNA integration mutagenesis and is impaired in its ability to elaborate penetration hyphae. Here we report that the pde1 mutation is the result of an insertion into the promoter of a P-type ATPase-encoding gene. Targeted gene disruption confirmed the role of PDE1 in penetration hypha development and pathogenicity but highlighted potential differences in PDE1 regulation in different Magnaporthe strains. The predicted PDE1 gene product was most similar to members of the aminophospholipid translocase group of P-type ATPases and was shown to be a functional homolog of the yeast ATPase gene ATC8. Spatial expression studies showed that PDE1 is expressed in germinating conidia and developing appressoria. These findings implicate the action of aminophospholipid translocases in the development of penetration hyphae and the proliferation of the fungus beyond colonization of the first epidermal cell.     

Microsatellite markers for population studies of the rice blast fungus,Magnaporthe grisea

We developed nine new microsatellite markers for rice blast (Magnaporthe grisea) population studies. These markers were used in addition to nine microsatellite markers previously developed by our group for mapping purpose. Altogether, the 18 markers were used in multiplex PCR (polymerase chain reaction) to characterize six populations from different geographical origins. The average number of alleles per locus across populations ranged from 1.2 to 7 and the total number of alleles detected from 2 to 19. Based on this large range of polymorphism, this set of markers is expected to be useful for different kind of population studies at different geographical scales.     

Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus,Magnaporthe grisea

We isolated a neuronal calcium sensor 1/frequenin-like gene, Mg-NCS-1, from Magnaporthe grisea and evaluated the phenotypes of null-mutants of the gene. The putative Mg-NCS-1 protein showed high similarity to the other NCS-1 proteins. The null-mutants had normal growth and pathogenicity similar to the parental strain, but their growth was suppressed in high concentrations of Ca2+ or acidic conditions.     

Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea.

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.     

Identification of isobavachalcone as a potential drug for rice blast disease caused by the fungus Magnaporthe grisea

The rice blast disease caused by the fungus Magnaporthe grisea is one of the most devastating rice diseases, but there is no effective fungicide toward chitinase which is a key enzyme of M. grisea. In this study, we observed that distortion and cell-wall damage of M. grisea hyphae were significantly under the scanning electron micrograph after a 24-h treatment with 10?mg/L isobavachalcone (IBC) extracted from Psoralea corylifolia L. To further explore the effect of IBC on the cell wall of M. grisea, we examined changes in enzymes associated with cell wall degradation by enzyme activity experiments, treated liquid culture mycelia with 10?mg/L IBC for 1?h. Results displayed that chitinase was obviously more active than control group. To illustrate the interactions between IBC and chitinase, the studies of homology modeling and molecular docking were carried out successively. The results revealed that IBC had hydrogen bonds with residues ASP267 and ARG276 of chitinase. Besides, these nonpolar residues TYR270, PRO271, VAL272, LEU310, PRO311, TYR316, and LEU317 were able to form strong hydrophobic interactions. Binding energies of the chitinase-IBC complexes were calculated by MM-GBSA showed that the ΔG_bind score of molecular dynamics had lower binding energy and more stable than docking complexes. All above, IBC owns significant agonistic activity in chitinase and would be a potent fungicide to inhibit the growth of M. grisea. We hope the above information provides an important insight for understanding the interactions between IBC and chitinase, which may be useful in the discovery of a novel potent agonist.

Communicated by Ramaswamy H. Sarma     

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus,Magnaporthe grisea

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.     

Analysis of the structure of the AVR1-CO39 avirulence locus in virulent rice-infecting isolates of Magnaporthe grisea

The AVR1-CO39 gene that came from a Magnaporthe grisea isolate from weeping lovegrass controls avirulence on the rice cultivar CO39. AVR1-CO39 was not present in the genome of the rice-infecting M. grisea isolate Guyll from French Guyana, suggesting that the gene had been deleted. Molecular analysis of the deletion breakpoints in the AVR1-CO39 locus revealed the presence of a truncated copy of a previously unknown retrotransposon at the left-hand border. At the right-hand border was a truncated copy of another repetitive element that is present at multiple locations in the genome of Guyll. The structures of avr1-CO39 loci were further examined in 45 rice-infecting isolates collected in Brazil, China, Japan, India, Indonesia, Mali, and the Philippines. Most isolates showed no hybridization signal with the AVR1-CO39 probe and had the same locus structure as Guyll. Some isolates from Japan showed a signal with the AVR1-CO39 probe, but the region specifying avirulence activity was rearranged. These findings suggest that widespread virulence to 'CO39' among rice-infecting M. grisea isolates is due to ancestral rearrangements at the AVR1-CO39 locus that may have occurred early in the evolution of pathogenicity to rice.     

Sequences homologous to the human D1S1 locus present on human chromosome 3.

We have investigated the segregation, in somatic cell hybrids, of the human D1S1 locus, previously assigned to 1p36 by in situ hybridization. We have shown that the clone which defines this locus, lambda Ch4A-H3, originates from human chromosome 3, but contains a 1.7-kilobase (kb) PstI-HindIII repetitive element that is also present on chromosome 1, probably distal to PGD. The clone recognizes restriction fragment length polymorphisms within the single-copy sequence on chromosome 3 and one for the enzyme StuI in the repeated sequence on chromosome 1. These experiments thus expose a level of complexity in the D1S1 locus not revealed by earlier in situ hybridization studies.     

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.