Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance

Leung, Hei; Lehtinen, Ulla; Karjalainen, Reijo; Skinner, Daniel Z.; Tooley, Paul W.; Leong, Sally A.; Ellingboe, Albert H.

doi:10.1007/bf00334519

Cited by 72 publications

(29 citation statements)

References 13 publications

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“…Transformation of M. grisea was carried out by an adaptation of the previously described methods (Parsons et al, 1987;Leung et al, 1990). Briefly, a 2 5 cm2 square of M. grisea strain Guy-11 mycelium was cut from the surface of an oatmeal agar plate, macerated in 50 mL of CM liquid media, and incubated overnight at 23OC on a rotary shaker at 120 rpm.…”

Section: Funga1 Transformationsmentioning

confidence: 99%

Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea.

1993

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Differential cDNA cloning was used to identify genes expressed during infectious growth of the fungal pathogen Magnaporthe grisea in its host, the rice plant. We characterized one of these genes, MPG1, in detail. Using a nove1 assay to determine the proportion of fungal biomass present in the plant, we determined that the MPGl transcript was 60-fold more abundant during growth in the plant than in culture. Mpgl mutants have a reduced ability to cause disease symptoms that appears to result from an impaired ability to undergo appressorium formation. MPGl mRNA was highly abundant very early in plant infection concomitant with appressorium formation and was also abundant at the time of symptom development. The MPGl mRNA was also expressed during conidiation and i n mycelial cultures starved for nitrogen or carbon. MPGl potentially encodes a small, secreted, cysteine-rich, moderately hydrophobic protein with the characteristics of a fungal hydrophobin. Consistent with the role of the MPGl gene product as a hydrophobin, Mpgl mutants show an basily wettable" phenotype. Our results suggest that hydrophobins may have a role in the elaboration of infective structures by fungi and may fulfill other functions in fungal phytopathogenesis.

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Section: Funga1 Transformationsmentioning

confidence: 99%

Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea.

1993

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“…Transformation protocols were adapted from established procedures (Leung et al, 1990;Sweigard et al, 1992). Conidia (1.5 x 106) were germinated in liquid medium overnight at room temperature with agitation.…”

Section: Transformation Of M Griseamentioning

confidence: 99%

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

1995

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Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primem designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares >45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant W and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.

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“…Protocols for protoplast formation and transformation were adapted from established procedures (Leung et al, 1990;Sweigard et al, 1992). Mycelia were prepared as described above and harvested using sterile Miracloth (Calbiochem-Novabiochem Corp., La Jolla, CA).…”

Section: Transformationmentioning

confidence: 99%

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

1997

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Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MACl) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacteria1 artificial chromosome library. Sequence characterization showed that MACl has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MACl were unable t o form appressoria on an inductive surface and were unable t o penetrate susceptible rice leaves. m a c l -transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.

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Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance

Abstract: Low frequency, integrative transformation of three fertile hermaphroditic strains of Magnaporthe grisea has been achieved using plasmid pAN7-1 and cosmid pAN7-2, which contain an Escherichia coli hygromycin B phosphotransferase gene linked to Aspergillus nidulans regulatory sequences.

Cited by 72 publications

References 13 publications

Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea.

Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea.

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

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