Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue.
Journal: 2002/March - Applied and Environmental Microbiology
ISSN: 0099-2240
PUBMED: 11823187
Abstract:
Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.
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Appl Environ Microbiol 68(2): 532-538

Osmoregulation and Fungicide Resistance: the <em>Neurospora crassa os-2</em> Gene Encodes a <em>HOG1</em> Mitogen-Activated Protein Kinase Homologue<sup><a href="#fn1" rid="fn1" class=" fn">†</a></sup>

Syngenta Agribusiness Biotechnology Research Inc., Research Triangle Park, North Carolina 27709, Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 479072
Corresponding author. Mailing address: Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907. Phone: (765) 496-6918. Fax: (765) 494-0363. E-mail: ude.eudrup.yntb@ux.
Received 2001 Aug 21; Accepted 2001 Nov 14.

Abstract

Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

Abstract

Wild-type Neurospora crassa strains can grow on media with different osmotic strengths. The members of one class of N. crassa mutants, known as os (osmosensitive) mutants, however, are sensitive to hyperosmotic pressure and are unable to grow on media supplemented with 4% NaCl (wt/vol) or 1 M sorbitol (25). Several os mutants, including os-1, os-2, os-3, os-4, os-5, os-6, and cut mutants, and the sorbose-resistant sor (T9) mutant have been described (25). Most os mutants have aberrant colony morphology on regular Vogel's medium N and form sticky, close-cropped aerial hyphae. The aggregated hyphae are intensely pigmented and have a tendency to rupture and bleed (25). In addition, os mutants have reduced conidiation and altered cell wall compositions (10, 16).

Only one of the N. crassa os genes, the os-1 gene (also designated the nik-1 gene), has been identified (1, 30). os-1 encodes a putative two-component histidine kinase that is homologous to the osmosensing Sln1p histidine kinase of Saccharomyces cerevisiae. The osmoregulation mediated by the yeast Sln1p, known as the high-osmolarity glycerol response (HOG), is well characterized (for a review, see reference 11). The major component of the HOG pathway is a mitogen-activated protein (MAP) kinase module consisting of one MAP kinase (Hog1p), one MAP kinase kinase (Pbs2p), and three MAP kinase kinase kinases (Ste11p, Ssk2p, and Ssk22p). The HOG1 MAP kinase can be activated by either of two branches of upstream osmosensing pathways that converge at Pbs2p. One branch involves a two-component histidine kinase phosphorelay system comprised of Sln1p (24), Ypd1p (27), and Ssk1p (17). The other upstream osmosensing branch involves a putative membrane protein, Sho1p, that activates Pbs2p via Ste11p (17, 28). Hyperosmotic stresses activate the HOG pathway, which in turn increases the transcription of key enzymes involved in glycerol synthesis, including glycerol-3-phosphate dehydrogenase (GPD1) and glycerol-3-phosphatase (HOR2). Hyperosmotic stresses also reduce the permeability of the plasma membrane to glycerol, primarily by inhibiting the activity of the Fps1p glycerol transporter. The resulting cytosolic glycerol accumulation leads to increased internal osmolarity and restores the osmotic gradient between the cells and their environment. Yeast mutants defective in HOG1, PBS2, or GPD1 are osmosensitive (12).

Fludioxonil and fenpiclonil are phenylpyrrole fungicides derived from the antibiotic pyrrolnitrin (9, 21). They are broad-spectrum fungicides used to control a variety of important plant-pathogenic fungi. Previous studies with Fusarium sulphureum Schlecht indicated that fenpiclonil inhibited the transport of monosaccharides and caused intracellular accumulation of polyols, such as glycerol and mannitol (13, 14). Wild-type N. crassa strains treated with fenpiclonil and fludioxonil at concentrations that inhibited fungal growth by 50% accumulated high concentrations of intracellular glycerol (26). Some Ustilago maydis and F. sulphureum mutants resistant to phenylpyrrole fungicides also were sensitive to high osmotic pressure (13, 14). These studies suggested that there is a relationship between osmoregulation and phenylpyrrole fungicide resistance.

To determine the relationship between osmoregulation and resistance to phenylpyrrole fungicides in filamentous fungi, in this study we cloned the N. crassa os-2 gene because os-2 mutants are resistant to phenylpyrrole fungicides. The os-2 gene encodes a putative MAP kinase highly homologous to yeast Hog1p. We found that null mutations in os-2 confer resistance to phenylpyrroles but result in sensitivity to high osmotic stresses in N. crassa. In germinating conidia or germ tubes treated with fludioxonil, the fungicide stimulates glycerol accumulation and causes fungal cells to swell and burst. Our data suggest that os-2 is critical for osmoregulation in N. crassa and that phenylpyrrole fungicides may exert their fungicidal effects by interfering with the os-2 MAP kinase pathway.

Acknowledgments

We thank Dan Ebbole of Texas A&amp;M University for providing a list of N. crassa chromosome-specific cosmids and performing restriction fragment length polymorphism mapping of G18G04, Kevin McCluskey of the Fungal Genetics Stock Center for supplying the N. crassa strains and libraries, and Michael Gustin of Rice University for providing yeast strains YPH102 and JBY10. We also thank Larry Dunkle, John Leslie, and Thomas Gaffney for critically reading the manuscript and Li Zheng and Jennifer Hunt for assistance during preparation of the manuscript.

Acknowledgments

Footnotes

Journal article no. 16663 of the Purdue University Agricultural Experiment Station.

Footnotes

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