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Journal of Bacteriology, June 1999, p. 3571-3577, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Early Expression of the Calmodulin Gene, Which Precedes Appressorium Formation in Magnaporthe grisea, Is Inhibited by Self-Inhibitors and Requires Surface Attachment

Zhi-Mei Liu and Pappachan E. Kolattukudy*

Departments of Biochemistry and Medical Biochemistry and Neurobiotechnology Center, The Ohio State University, Columbus, Ohio 43210

Received 16 February 1999/Accepted 24 March 1999

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in Colletotrichum gloeosporioides and Colletotrichum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.

* Corresponding author. Mailing address: Neurobiotechnology Center, The Ohio State University, 206 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210. Phone: (614) 292-5682. Fax: (614) 292-5379. E-mail: Kolattukudy.2{at}osu.edu.

Journal of Bacteriology, June 1999, p. 3571-3577, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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