BINDING AND CYTOTOXICITY OF CERATO-ULMIN TO ELM CELLS

G. Del Sorbo, P. Ambrosino, C. Comparini, S. Tegli, A. Scala, F. Scala

Abstract


In vitro cultivated elm cells were used to study the cytotoxicity of cerato-ulmin (= CU), a hydrophobin produced by Ophiostoma ulmi and O. novo-ulmi, two causal agents of Dutch elm disease (= DED). Purified CU was found to bind to elm cells and cause a dose-dependent death both on cells derived from a DED susceptible (C06) and a moderately resistant (196/6) elm clone. Indirect immunofluorescence (IF) tests, using a rabbit anti-CU antiserum, indicated that homogeneous CU binds to elm but not to cells of fennel and pear, two non-host species of Ophiostoma. The binding was demonstrated by quantitative ELISA on extracts from elm cells incubated with purified CU and by measuring the decrease in CU concentration in the incubation mixture. The equation of the saturation binding curve indicated that the maximum quantity of bound CU for elm clones C06 and 196/6 were similar (351.92 and 355.59 pmoles mg-1 d.wt. elm cells, respectively), as similar were the equilibrium dissociation constants (7.09 and 8.08 µM CU, respectively). The increase in bound CU correlated with the intensity of the fluorescence signal on elm cells and a higher mortality. Elm cell-Ophiostoma spp. dual cultures in Takai or Murashige & Skoogderived media were used to test the ability of elm cells to support growth of Ophiostoma fungi and CU production. Cells derived from two elm clones supported both abundant fungal growth and CU production. Fungal growth was supported by cells of the Ophiostoma nonhosts fennel and pear, whereas CU was only produced in the presence of fennel cells. The binding and direct cytotoxicity of CU on elm cells support its role as a pathogenicity factor in DED.

Keywords


Ophiostoma spp.; Ulmus spp.; toxin binding; in vitro fungus-plant cells dual cultures

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DOI: http://dx.doi.org/10.4454/jpp.v85i3.1027

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EDIZIONI ETS, Pisa, Italy