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cDNAs Generated from Individual Epidermal Cells Reveal that Differential Gene Expression Predicting Subsequent Resistance or Susceptibility to Rust Fungal Infection Occurs Prior to the Fungus Entering the Cell Lumen

September 2003 , Volume 16 , Number  9
Pages  835 - 845

Michael J. R. Mould , 1 Tao Xu , 1 Mary Barbara , 2 Norman N. Iscove , 2 and Michèle C. Heath 1

1Department of Botany, University of Toronto, 25 Willcocks St., Toronto, Ontario, Canada M5S 3B2; 2Department of Cell and Molecular Biology, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9

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Accepted 4 June 2003.

As the cowpea rust fungus penetrates the wall of a cowpea epidermal cell, resistant and susceptible plants exhibit different ultrastructural and cytochemical changes within the epidermal protoplast. To examine plant gene expression at this stage of infection, cytoplasm was extracted from individual inoculated or uninoculated epidermal cells before the fungal penetration peg reached the cell lumen. Initial differential colony hybridization screening of an expressed sequence tag library constructed from globally amplified cDNAs generated from the inoculated resistant cells resulted in 80 clones (out of 835) with a differential hybridization pattern. Further slot-blot screening and screening of the amplified cDNAs generated from inoculated or uninoculated, resistant or susceptible cells revealed 28 separate genes, mostly with no matching sequences in the databases, that were up-regulated in response to the growth of the fungus through the wall of resistant or susceptible cells. Five genes, including those coding for β- and α-tubulin, were found to be down-regulated specifically in inoculated, susceptible cells, and five were specifically up-regulated in inoculated, resistant cells, including a PR-10 homolog and a phenylalanine ammonialyase gene. Probing the amplified cDNAs from each cell type for the expression of cell death-related genes revealed that an LLS1 homolog (vuLLS1), cloned from cowpea, was up-regulated by infection in both resistant and susceptible cells and that a homolog of HSR203J was differentially up-regulated in resistant cells. These data show that changes in gene expression predicting the subsequent expression of susceptibility or hypersensitive resistance to fungal infection occur prior to the fungus entering the cell lumen.

Additional keywords: defense, hypersensitive response, programmed cell death.

© 2003 The American Phytopathological Society