Abstract
Two novel elicitor peptides that are produced by the race 1 of the cowpea rust fungus Uromyces vignae and that specifically induce a hypersensitive response (a putative form of programmed cell death) in a resistant cultivar of cowpea (Vigna unguiculata L. Walp) have been purified to homogeneity. Purification steps included gel filtration, anion-exchange chromatography, continuous elution electrophoresis, and reversed-phase C18 high performance liquid chromatography. The relative molecular masses of the peptide elicitors as deduced from Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 5600 Da (major) and 5800 Da (minor), respectively. Peptide 1 (major) and the minor copurifying peptide (peptide 2) resolved at the final C18 high performance liquid chromatography step. The NH2 terminus of peptide 1 was deblocked with anhydrous trifluoroacetic acid prior to sequencing. However, the NH2 terminus of peptide 2 was free. The acidic and hydrophobic peptides show some homology between themselves but do not show any significant similarity with known proteins. The two specific elicitors may be products of two avirulence genes corresponding to the two genes for resistance in the resistant cultivar.
Highlights
The hypersensitive response (HR),1 a putative form of programmed cell death [1], is a nearly invariant marker of disease resistance in plants and is characterized by rapid localized pathogen-induced cell death and restriction of further pathogen growth
The hypersensitive response, which is the common result of such interactions in host cultivars resistant to the parasite, has been suggested to be a form of programmed cell death evolved as a defense against microbial attack
We have demonstrated here for the first time the existence of two novel race-cultivar specific HR-inducing elicitors from the cowpea-cowpea rust fungus interaction
Summary
Materials—The host-pathogen system used for the study was the cowpea-cowpea rust fungus. IWFs were obtained from the susceptible cultivar (California Blackeye) of cowpea plants Walp.) inoculated with basidiospores (monokaryotic stage) of the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) as described earlier [17]. Aqueous exudates from in vitro differentiated basidiospore germlings were collected as described previously [18]. Equipment for electrophoresis, electroblotting, and continuous elution electrophoresis was from Bio-Rad. Mark 12 wide range (2.5–200 kDa) standard markers were from Novex. All organic solvents for reversed-phase HPLC were of HPLC grade. Other reagents were of standard reagent grade. Purification of HR-inducing Specific Elicitors—Specific elicitors that elicit a HR were purified from IWFs of fungus-infected leaves.
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