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Eukaryotic Cell, December 2004, p. 1525-1532, Vol. 3, No. 6
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.6.1525-1532.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cellular Localization and Role of Kinase Activity of PMK1 in Magnaporthe grisea

Kenneth S. Bruno,1 Fernando Tenjo,2 Lei Li,1 John E. Hamer,2,{ddagger} and Jin-Rong Xu1*

Department of Botany and Plant Pathology,1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana2

Received 9 June 2004/ Accepted 13 August 2004

A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.

* Corresponding author. Mailing address: Department of Botany and Plant Pathology, 915 W. State St., Lilly Hall, Purdue University, West Lafayette, IN 47907. Phone: (765) 496-6918. Fax: (765) 494-0363. E-mail: jinrong{at}purdue.edu.

{dagger} These authors contributed equally to this work.

Eukaryotic Cell, December 2004, p. 1525-1532, Vol. 3, No. 6
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.6.1525-1532.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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