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Published on: April 23, 2012
The relationship between the mitochondrial gene T-urf13 and fungal pathotoxin sensitivity in maize
J N Siedow1, D M Rhoads, G C Ward
1Duke University, Durham, NC 27708-1000, USA.
Abstract:
Mitochondria isolated from maize containing cms-T cytoplasm are specifically sensitive to pathotoxins (T-toxins) produced by the fungi Bipolaris maydis race T and Phyllosticta maydis. T-toxins interact with a 13 kDa membrane-bound toxin receptor protein, URF13, to produce hydrophillic pores in the membrane. Expression of URF13 in Escherichia coli produces bacterial cells that form hydrophillic pores in the plasma membrane when exposed to T-toxin or methomyl. Topological studies have established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and may contribute to pore formation. URF13 specifically binds T-toxin in a cooperative manner. Oligonucleotide-directed mutagenesis of URF13 led to the isolation of methomyl/T-toxin-resistant mutations at 39 separate positions throughout the URF13 primary sequence. Chemical cross-linking of URF13 demonstrated the presence of URF13 oligomers and established that the pore-forming species is oligomeric. The ability of the carboxylate-specific reagent, dicyclohexycarbodiimide to cross-link URF13 has been used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle that may undergo a conformational change after T-toxin or methomyl binding. Experimental evidence indicates that URF13 acts as a ligand-gated, pore-forming T-toxin receptor.
Insights
Mitochondria in T-cytoplasm maize are sensitive to fungal toxins. The URF13 protein forms pores in membranes upon binding toxins or methomyl, acting as a ligand-gated receptor.
Area of Science:
- Plant-pathogen interactions
- Molecular biology
- Biochemistry
Background:
- Mitochondria from maize with T-cytoplasm are susceptible to pathotoxins from Bipolaris maydis race T and Phyllosticta maydis.
- These pathotoxins (T-toxins) interact with the URF13 protein, a 13 kDa membrane-bound receptor.
Purpose of the Study:
- To investigate the mechanism of T-toxin interaction with the URF13 protein.
- To elucidate the structure and function of URF13 in pore formation and toxin binding.
Main Methods:
- Expression of URF13 in Escherichia coli.
- Topological studies to determine membrane-spanning regions.
- Oligonucleotide-directed mutagenesis to identify resistant mutations.
- Chemical cross-linking to study URF13 oligomerization.
- Site-directed mutagenesis combined with chemical cross-linking.
Main Results:
- URF13 forms hydrophilic pores in bacterial plasma membranes upon exposure to T-toxin or methomyl.
- URF13 contains three membrane-spanning alpha-helices, with two potentially involved in pore formation.
- URF13 binds T-toxin cooperatively.
- Mutagenesis identified 39 positions conferring resistance to methomyl/T-toxin.
- URF13 exists as oligomers, and the pore-forming species is oligomeric.
- The URF13 tetramer forms a four-alpha-helical bundle that may change conformation upon ligand binding.
Conclusions:
- URF13 functions as a ligand-gated, pore-forming receptor for T-toxin.
- The structural and functional studies provide insights into the molecular basis of T-toxin sensitivity in maize.
