Thomas J. Wolpert

Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.

Summary: In this study, we determined the timing of events associated with cell death induced by the host‐selective toxin, victorin. We show that the victorin‐induced collapse in mitochondrial transmembrane potential (Δψ_m), indicative of a mitochondrial permeability transition (MPT), on a per cell basis, did not occur simultaneously in the entire mitochondrial population. The loss of Δψ_m in a predominant population of mitochondria preceded cell shrinkage by 20–35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage. During and following cell shrinkage, tonoplast rupture did not occur, and membranes, including the plasma membrane and tonoplast, retained integrity. Ethylene signaling was implicated upstream of a victorin‐induced loss in mitochondrial motility and the collapse in Δψ_m. Results suggest that the victorin‐induced collapse in Δψ_m is a consequence of an MPT and that the timing of the victorin‐induced MPT is poised to influence the cell death response. The retention of plasma membrane and tonoplast integrity during cell shrinkage supports the interpretation that victorin induces an apoptotic‐like cell death response.

▪ Abstract Host-selective toxins, a group of structurally complex and chemically diverse metabolites produced by plant pathogenic strains of certain fungal species, function as essential determinants of pathogenicity or virulence. Investigations into the molecular and biochemical responses to these disease determinants reveal responses typically associated with host defense and incompatibility induced by avirulence determinants. The characteristic responses that unify these disparate disease phenotypes are numerous, yet the evidence implicating a causal relationship of these responses, whether induced by host-selective toxins or avirulence factors, in determining the consequences of the host-pathogen interaction is equivocal. This review summarizes some examples of the action of host-selective toxins to illustrate the similarity in responses with those to avirulence determinants.

Summary: The mitochondrion has emerged as a key regulator of apoptosis, a form of animal programmed cell death (PCD). The mitochondrial permeability transition (MPT), facilitated by a pore‐mediated, rapid permeability increase in the inner membrane, has been implicated as an early and critical step of apoptosis. Victorin, the host‐selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats, has been demonstrated to bind to the mitochondrial P‐protein and also induces a form of PCD. Previous results suggest that a MPT may facilitate victorin's access to the mitochondrial matrix and binding to the P‐protein: (i) victorin‐induced cell death displays features similar to apoptosis; (ii) in vivo, victorin binds to the mitochondrial P‐protein only in toxin‐sensitive genotypes whereas victorin binds equally well to P‐protein isolated from toxin‐sensitive and insensitive oats; (iii) isolated, untreated mitochondria are impermeable to victorin. The data implicate an in vivo change in mitochondrial permeability in response to victorin. This study focused on whether oat mitochondria can undergo a MPT. Isolated oat mitochondria demonstrated high‐amplitude swelling when treated with spermine or Ca²⁺ in the presence of the Ca²⁺‐ionophore A23187, and when treated with mastoparan, an inducer of the MPT in rat liver mitochondria. In all cases, swelling demonstrated size exclusion in the range 0.9–1.7 kDa, similar to that found in animal mitochondria. Further, MPT‐inducing conditions permitted victorin access to the mitochondrial matrix and binding to the P‐protein. In vivo, victorin treatment induced the collapse of mitochondrial transmembrane potential within 2 h, indicating a MPT. Also, the victorin‐induced collapse of membrane potential was clearly distinct from that induced by uncoupling respiration, as the latter event prevented the victorin‐induced PCD response and binding to P‐protein. These results demonstrate that a MPT can occur in oat mitochondria in vitro, and are consistent with the hypothesis that an MPT, which allows victorin access to the mitochondrial matrix and binding to the P‐protein, occurs in vivo during victorin‐induced PCD.

Victorin is a host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Previously, victorin was shown to be bound specifically by two proteins of the mitochondrial glycine decarboxylase complex, at least one of which binds victorin only in toxin-sensitive genotypes in vivo. This enzyme complex is involved in the photorespiratory cycle and is inhibited by victorin, with an effective concentration for 50% inhibition of 81 pM. The photorespiratory cycle begins with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and victorin was found to induce a specific proteolytic cleavage of the Rubisco large subunit (LSU). Leaf slices incubated with victorin for 4 hr in the dark accumulated a form of the LSU that is cleaved after the 14th amino acid. This proteolytic cleavage was prevented by the protease inhibitors E-64 and calpeptin. Another primary symptom of victorin treatment is chlorophyll loss, which along with the specific LSU cleavage is suggestive of a victorin-induced, senescence-like response. DNA from victorin-treated leaf slices showed a pronounced laddering effect, which is typical of apoptosis. Calcium appeared to play a role in mediating the plant response to victorin because LaCl₃ gave near-complete protection against victorin, preventing both leaf symptoms and LSU cleavage. The ethylene inhibitors aminooxyacetic acid and silver thiosulfate also gave significant protection against victorin-induced leaf symptoms and prevented LSU cleavage. The symptoms resulting from victorin treatment suggest that victorin causes premature senescence of leaves.