ABSTRACT
Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.
Subject(s)
Drug Resistance , Fluorescent Dyes/pharmacology , Oryza/genetics , Plants, Genetically Modified/metabolism , Base Sequence , Biomarkers/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Models, Genetic , Molecular Sequence Data , Oryza/metabolism , Plants, Genetically Modified/genetics , Transformation, GeneticABSTRACT
Three groups of fungicides (phenylpyrroles, dicarboximides, aromatic hydrocarbons) are effective against filamentous fungi. The target of these fungicides is the osmotic stress signal transduction pathway, which is dependent on the Os-1 family of two-component histidine kinases. These fungicides usually have no fungicidal effect on the yeast Saccharomyces cerevisiae. In this report, we found that expression of Hik1, an Os-1 orthologue from rice blast fungus, can confer fungicide-sensitivity to yeast. This requires both the histidine kinase and the response regulator domains of Hik1. Analysis of yeast mutants indicated that this sensitivity is Hog1- and Ssk1-dependent. In addition, our studies revealed an interaction between Hik1 and Ypd1. These observations suggest that Hik1 is a direct target of the fungicides or is a mediator of fungicide action and that the fungicidal effect is transmitted to the Hog1 pathway via Ypd1.
Subject(s)
Drug Resistance, Fungal/genetics , Fungicides, Industrial/pharmacology , Magnaporthe/enzymology , Protein Kinases/genetics , Saccharomyces cerevisiae/drug effects , Base Sequence , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Histidine Kinase , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Magnaporthe/genetics , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Protein Kinases/drug effects , Protein Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/drug effects , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
We isolated and characterized a histidine kinase gene (HIK1) from the rice blast fungus, Pyricularia oryzae (Magnaporthe grisea). The deduced amino acid sequence of HIK1 showed highest similarity (85.7%) to a hybrid-type histidine kinase, Os-1/Nik-1 of Neurospora crassa. Disruption of HIK1 caused no defect in cell growth on normal media and in pathogenicity to rice plants. The Deltahik1 strain acquired resistance to three groups of fungicides (phenylpyrroles, dicarboximides, and aromatic hydrocarbons) similar to os-1 mutants of N. crassa. The Deltahik1 strain showed increased sensitivity to high concentrations of sugars although its salt sensitivity was not elevated, suggesting that the rice blast fungus can distinguish osmostresses caused by high sugar concentrations and high salt concentrations. In contrast, os-1 mutants of N. crassa are sensitive to high concentrations of both salts and sugars. These findings suggest that P. oryzae and N. crassa partially differ in their os (osmosensitive) signal transduction pathway.
