Genetic and biochemical analysis of Victoria blight: Identification of AFLP markers and purification and characterization of the oat saspase.

摘要

Victoria blight of oats (Avena sativa) is caused by the fungus, Cochliobolus victoriae, which produces the toxin, victorin. Victorin production is required for pathogenicity of the fungus. In oats, sensitivity to the toxin and susceptibility to the pathogen is conditioned by a dominant allele at the Vb locus, while oats with a homozygous recessive allele are insensitive to victorin and resistant to the fungus. Vb is either tightly linked to or the same gene as Pc-2, a gene that confers crown rust resistance in oats. Therefore, the same gene may provide resistance to one pathogen while conferring susceptibility to another. To better understand the interaction of victorin with oats, genetic markers linked to the Vb locus were identified and proteases involved in victorin-induced programmed cell death (PCD) were purified and characterized.; Amplified fragment length polymorphism (AFLP) technology was used to identify two genetic markers that flank the Vb locus. We produced an F2 population segregating for victorin sensitivity. Over 51,000 markers generated by 512 different AFLP primer pairs were screened for polymorphisms between victorin-sensitive and insensitive genotypes. These results facilitated the production of a genetic map of the Vb locus.; Victorin induces a response in oats that has been characterized as a form of programmed cell death (PCD). One biochemical feature of victorin-induced PCD is the proteolytic cleavage of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Rubisco proteolysis had previously been shown to be inhibited by general protease inhibitors. Here, we describe inhibition of rubisco proteolysis by caspase-specific inhibitors and present evidence for a protease cascade. The first protease from plants that exhibits caspase specificity and involvement with PCD was purified and characterized. This protease, we have termed a “saspase”, contains amino acid sequence homology to plant subtilisin-like serine proteases and is found in the extracellular fluid (ECF) after victorin treatment. Heat shock-induced PCD is also described and displays characteristics similar to victorin-induced PCD, including DNA laddering, rubisco proteolysis, and localization of the saspase in the ECF.