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.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).

The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.

:The e †ect of phenylpyrroles on glycerol synthesis in Neurospora crassa has been investigated and compared with the mode of action of vinclozolin (a dicarboximide). The results indicated that fenpiclonil, Ñudioxonil and vinclozolin at concentrations which inhibit growth by 50% induce accumulation of glycerol in the mycelium of N. crassa. Furthermore a protein kinase (PK-III) possibly involved in the regulation of the glycerol synthesis is inhibited by phenylpyrroles, whereas vinclozolin is without e †ect. This implies that the target sites of phenylpyrroles and dicarboximides in the osmosensing signal transmission pathway are di †erent. Comparative experiments with enzymes from human and animal sources revealed that PK-III could be a protein kinase Cd. It is suggested that inhibition of PK-III activity may result in an increased concentration of a non-phosphorylated regulatory protein which may activate a MAP-kinase cascade of reactions resulting in increased glycerol synthesis.

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Three cinnamyl alcohol dehydrogenase (CAD) isoenzymes were separated from etiolated wheat seedlings (Triticum aestivum L.) and examined by native gel electrophoresis. Two of these enzymes (CAD-1 and CAD-2) were purified to apparent homogeneity. They exhibited a marked difference in substrate affinity. On sodium dodecyl sulphate-acrylamide gel the isolated isoenzymes showed only one protein band each with an M r 45 000 and 40 000 daltons, respectively, whereas on native gel two bands were identified for each protein. Isoenzymes from a variety of diploid, tetraploid, and hexaploid wheats were compared. The results indicated that the CAD polymorphism could be genetically determined.