Genetic Analysis of Victorin Sensitivity and Identification of a Causal Nucleotide-Binding Site Leucine-Rich Repeat Gene in Phaseolus vulgaris.

Cochliobolus victoria, the causal agent of Victoria blight, is pathogenic due to its production of a toxin called victorin. Victorin sensitivity in oats, barley, Brachypodium spp., and Arabidopsis has been associated with nucleotide-binding site leucine-rich repeat (NLR) genes, a class of genes known for conferring disease resistance. In this work, we investigated the sensitivity of Phaseolus vulgaris to victorin. We found that victorin sensivity in Phaseolus vulgaris is a developmentally regulated, quantitative trait. A single quantitative trait locus (QTL) accounted for 34% of the phenotypic variability in victorin sensitivity among Stampede × Red Hawk (S×R) recombinant inbred lines. We cloned two NLR-encoding genes within this QTL and showed one, Phvul05G031200 (PvLOV), confers victorin-dependent cell death when overexpressed in Nicotiana benthamiana. Protein sequences of PvLOV from victorin-sensitive and the victorin-resistant bean parents differ by two amino acids in the leucine-rich repeat region, but both proteins confer victorin-dependent cell death when overexpressed in N. benthamiana.

Purification and immunological characterization of toxic components from cultures of Pyrenophora tritici-repentis.

To facilitate the genetic analysis of pathogenicity in the wheat-Pyrenophora tritici-repentis interaction, a host-selective toxic protein, designated ToxA, was purified from culture filtrates of this fungus. ToxA was shown to be a 13.2-kDa heat-stable protein which induced visible necrosis in sensitive wheat cultivars at an average minimum concentration of 60 nM. Polyclonal antibodies raised against ToxA were shown by Western analysis and indirect immunoprecipitation to be specific for this protein. Bioassays of immunoprecipitated protein and ToxA protein eluted from polyacrylamide gels indicated that ToxA protein is the toxic agent. Other less abundant necrosis-inducing components that are chromatographically and immunologically distinct from ToxA were also detected in culture filtrates of P. tritici-repentis. These components were found in cationic and anionic protein fractions and, like ToxA, induced cultivar-specific necrosis.

Heterologous expression of functional Ptr ToxA.

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.

Sensitivity of Wheat Genotypes to a Toxic Fraction Produced by Cephalosporium gramineum and Correlation with Disease Susceptibility.

ABSTRACT Cephalosporium stripe is an important disease of winter wheat (Triticum aestivum) in several areas of the world, especially where stubble mulch and early seeding are practiced to maintain soil moisture and prevent erosion. We developed a procedure to mass-produce a toxic fraction produced by Cephalosporium gramineum through a modification of the method of Kobayashi and Ui. Exposure of excised wheat leaves to a concentration of 60 mul/ml of the toxic fraction for 72 h produced distinct wilting symptoms that allowed us to distinguish toxin-sensitive wheat genotypes in a repeatable manner. Twenty wheat genotypes belonging to four distinct germ plasm groups (common, club, durum, and synthetic) were evaluated. Variation in toxin sensitivity of wheat genotypes was mostly at the level of the germ plasm group, and all differences among the four germ plasm groups were highly significant (P < 0.001) based on linear contrasts. Seventeen winter wheat genotypes representing the common, club, and durum germ plasm groups were planted in C. gramineum-infested fields at two locations. The logarithm of the percentage of tillers showing whitehead symptoms at each of the two locations was significantly (P < 0.0001) correlated with wilting symptoms measured by the toxin assay (r = 0.80 and 0.84). The common wheat genotypes were all sensitive to the toxic fraction, but showed a substantial range of disease reactions in the field. However, we found no case of a toxin-insensitive genotype being susceptible in the field. These results suggest that toxin insensitivity may be an important mechanism of resistance to Cephalosporium stripe, but that other mechanisms are operative as well. The toxin assay may be useful as an initial screening procedure to reduce the number of genotypes to be tested in the field.

Tricking the guard: exploiting plant defense for disease susceptibility.

Typically, pathogens deploy virulence effectors to disable defense. Plants defeat effectors with resistance proteins that guard effector targets. We found that a pathogen exploits a resistance protein by activating it to confer susceptibility in Arabidopsis. The guard mechanism of plant defense is recapitulated by interactions among victorin (an effector produced by the necrotrophic fungus Cochliobolus victoriae), TRX-h5 (a defense-associated thioredoxin), and LOV1 (an Arabidopsis susceptibility protein). In LOV1's absence, victorin inhibits TRX-h5, resulting in compromised defense but not disease by C. victoriae. In LOV1's presence, victorin binding to TRX-h5 activates LOV1 and elicits a resistance-like response that confers disease susceptibility. We propose that victorin is, or mimics, a conventional pathogen virulence effector that was defeated by LOV1 and confers virulence to C. victoriae solely because it incites defense.

A plant alternative to animal caspases: subtilisin-like proteases.

Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not revealed clear orthologues of caspase genes, indicating that enzyme(s) structurally unrelated yet possessing caspase specificity have functions in plant PCD. Here, we review recent data showing that some caspase-like activities are attributable to the plant subtilisin-like proteases, saspases and phytaspases. These proteases hydrolyze a range of tetrapeptide caspase substrates following the aspartate residue. Data obtained with saspases implicate them in the proteolytic degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during biotic and abiotic PCD, whereas phytaspase overproducing and silenced transgenics provide evidence that phytaspase regulates PCD during both abiotic (oxidative and osmotic stresses) and biotic (virus infection) insults. Like caspases, phytaspases and saspases are synthesized as proenzymes, which are autocatalytically processed to generate a mature enzyme. However, unlike caspases, phytaspases and saspases appear to be constitutively processed and secreted from healthy plant cells into the intercellular space. Apoplastic localization presumably prevents enzyme-mediated protein fragmentation in the absence of PCD. In response to death-inducing stimuli, phytaspase has been shown to re-localize to the cell interior. Thus, plant PCD-related proteases display both common (D-specific protein fragmentation during PCD) and distinct (enzyme structure and activity regulation) features with animal PCD-related proteases.

Alterations in gene expression in sorghum induced by the host-specific toxin from Periconia circinata.

Susceptibility of sorghum to the fungal pathogen Periconia circinata and sensitivity to its host-specific toxin are determined by the semidominant allele at the pc locus. Pretreatment of susceptible seedlings with cycloheximide or cordycepin for 4 hr before treatment with the toxin protected the seedlings against toxin-induced loss of electrolytes and prevented development of disease symptoms. In vivo incorporation of [(3)H]leucine into protein was inhibited 91% and 47% by cycloheximide and cordycepin, respectively, but was not affected by the toxin. Gel electrophoresis and fluorography of in vivo-labeled proteins extracted from non-treated and toxin-treated root tips of near-isogenic susceptible and resistant lines revealed a selective increase in radioactivity of a protein band at M(r) 16,000 only in preparations from toxin-treated susceptible root tips. Two-dimensional gel electrophoresis separated the M(r) 16,000 band into four proteins and confirmed the increased rate of synthesis. Products of in vitro translation were substantially enriched with the four M(r) 16,000 proteins when total RNA from toxin-treated susceptible root tips was used in a cell-free protein-synthesizing system. Because the proteins that increase are common to both susceptible and resistant genotypes, the toxin apparently interferes with a regulatory function, perhaps a function of the pc locus, and thereby alters gene expression in the susceptible genotype. The data suggest but do not establish that phytotoxicity results from the increased rate of synthesis of the specific proteins.

Immunological comparison of the in vitro and in vivo labeled victorin binding protein from susceptible oats.

The fungus Cochliobolus victoriae causes victoria blight of oats and produces the host-specific toxin victorin. The reaction of oats to the fungus and its toxin is controlled by a single dominant gene whose product has been hypothesized to function as the site of action (receptor) of the toxin in susceptible oat genotypes. Previously, using a biologically active (125)I derivative of the toxin, we identified a 100 kilodalton victorin-binding protein (VBP) which binds victorin in a ligand-specific manner and binds in vivo only in susceptible oat genotypes. However, a VBP in both the susceptible and resistant oat genotypes was identified by in vitro binding experiments. One interpretation of the lack of genotype-specific binding in vitro is that the 100 kilodalton protein detected in vitro is not the same 100 kilodalton protein detected in vivo. To clarify the relationship between the 100 kilodalton protein(s) labeled in vivo and in vitro, we developed antisera to the in vitro-labeled VBP from the susceptible genotype and demonstrated that these preparations react with the in vivo-labeled VBP from the susceptible genotype. This finding coupled with previous observations strongly suggest that the VBP observed in vivo is the same protein detected in vitro. Furthermore, the results support our previous observations which suggest that the VBPs labeled in vitro in susceptible and resistant genotypes are closely related or identical.

Inhibition of the glycine decarboxylase multienzyme complex by the host-selective toxin victorin.

Victoria blight of oats is caused by the fungus Cochliobolus victoriae. This fungus is pathogenic due to its ability to produce the host-selective toxin victorin. We previously identified a 100-kD protein that binds victorin in vivo only in susceptible genotypes and a 15-kD protein that binds victorin in vivo in both susceptible and resistant genotypes. Recently, we determined that the oat 100-kD victorin binding protein is the P protein of the glycine decarboxylase complex (GDC). In this study, we examined the effect of victorin on glycine decarboxylase activity (GDA). Victorin was a potent in vivo inhibitor of GDA. Leaf slices pretreated for 2 hr with victorin displayed an effective concentration for 50% inhibition (EC50) of 81 pM for GDA. Victorin inhibited the glycine-bicarbonate exchange reaction in vitro with an EC50 of 23 microM. We also identified a 15-kD mitochondrial protein that bound victorin in a ligand-specific manner. Based on amino acid sequence analysis, we concluded that the 15-kD mitochondrial protein is the H protein component of the GDC. Thus, victorin specifically binds to two components of the GDC. GDA in resistant tissue treated with 100 micrograms/mL victorin for 5 hr was inhibited 26%, presumably as a consequence of the interaction of victorin with the H protein. Victorin had no detectable effect on GDA in isolated mitochondria, apparently due to the inability of isolated mitochondria to import victorin. These results suggest that the interaction of victorin with the GDC is central to victorin's mode of action.

Specific binding of victorin to a 100-kDa protein from oats.

Susceptibility of oats to victoria blight, caused by the fungus Cochliobolus victoriae, and sensitivity to the host-specific toxin victorin, produced by the fungus, are controlled by the dominant allele at the Vb locus. It has been postulated that the Vb locus encodes a toxin receptor, although direct evidence for such a receptor is not available. Our recent studies on structure-activity relationships of the toxin established a methodology for producing (125)I-labeled victorin. Electrophoretic analysis of proteins from isogenic susceptible and resistant oat genotypes following treatment of leaves with radiolabeled victorin showed that victorin binds in a covalent and a genotype-specific manner to a 100-kDa protein only in susceptible oat leaf slices. This in vivo binding was competitively displaced by reduced victorin, a nontoxic protective compound, and appeared to be correlated with biological activity. In vitro binding to the 100-kDa protein in leaf extracts showed several differences from in vivo binding. Binding was not genotype specific and required a reducing agent that was not required for in vivo binding. Differential centrifugation showed that the 100-kDa victorin binding protein was not a cytosolic protein but was enriched in a high-speed particulate fraction. The data support the hypothesis that the 100-kDa protein is the victorin receptor.

Victorin induction of an apoptotic/senescence-like response in oats.

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.

Molecular Features Affecting the Biological Activity of the Host-Selective Toxins from Cochliobolus victoriae.

The structures of the toxins produced by Cochliobolus victoriae, victorin B, C, D, E, and victoricine, have recently been established. These toxins and modified forms of victorin C were tested for their effect on dark CO(2) fixation in susceptible oat (Avena sativa) leaf slices. Half-maximal inhibition of dark CO(2) fixation occurred with the native toxins in the range of 0.004 to 0.546 micromolar. An essential component for the inhibitory activity of victorin is the glyoxylic acid residue, particularly its hydrated aldehyde group. Removal of glyoxylic acid completely abolished the inhibitory activity of victorin, and the reduction of the aldehydo group transformed the toxin into a protectant. Conversion of victorin to its methyl ester resulted in diminution of inhibitory activity to 10% of the original activity of the toxin, whereas derivatization of the epsilon-amino group of the beta-hydroxylysine moiety resulted in a decrease of inhibitory activity to 1% of that of victorin C. However, the derivatized toxin retained its host selectivity. In addition, the opening of the macrocyclic ring of the toxin drastically reduced the inhibitory activity.

Identification of the 100-kD victorin binding protein from oats.

The fungus Cochliobolus victoriae, the causal agent of victoria blight of oats, produces the host-specific toxin victorin. Sensitivity of oats to victorin, and thus susceptibility to the fungus, is controlled by a single dominant gene. This gene is believed to also confer resistance to the crown rust pathogen Puccinia coronata. In the case of victoria blight, the gene has been hypothesized to condition susceptibility by encoding a toxin receptor. A 100-kD victorin binding protein (VBP) has been identified; it binds radiolabeled victorin derivatives in a ligand-specific manner and in a genotype-specific manner in vivo. The VBP may function as a toxin receptor. In vitro translation coupled with indirect immunoprecipitation was used to identify the mRNA for the 100-kD VBP, and fractionated mRNAs were used to prepare cDNA libraries enriched in the relative abundance of cDNA for the 100-kD VBP. A 3.4-kb cDNA clone was isolated that, when subjected to a 400-bp 5' deletion, was capable of directing the synthesis of a protein in Escherichia coli, which reacted to an antibody specific for the 100-kD VBP. Peptide mapping, by limited proteolysis, indicated that the protein directed by the cDNA is the 100-kD VBP. Nucleotide sequence analysis of the cDNA revealed extensive homology to a previously cloned cDNA for the P protein component of the multienzyme complex glycine decarboxylase. Glycine decarboxylase is a nuclear-encoded, mitochondrial enzyme complex. Protein gel blot analysis indicated that the 100-kD VBP copurifies with mitochondria. Based on analysis of in vitro translation products, nucleotide sequence homology, mitochondrial localization, and the widespread species distribution of the 100-kD VBP, we concluded that the 100-kD VBP is the P protein component of glycine decarboxylase.

Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form.

The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. This conversion was dilution independent and thought to be intramolecular. Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form. The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.

Structure of the host-specific toxins produced by the fungal pathogen Periconia circinata.

Four metabolites named peritoxins A and B and periconins A and B have been isolated together with the known metabolite circinatin from culture filtrates of the fungal pathogen Periconia circinata. Peritoxins A and B, which correspond to the P. circinata toxins Ia and IIa partially characterized in previous work, are selectively toxic to genotypes of Sorghum bicolor susceptible to the pathogen, whereas periconins A and B are biologically inactive. Combination of instrumental analysis and chemical degradation has led to structural assignments for each of the four compounds; only the configuration at some of the chiral centers remains undefined. Structural comparison suggests a precursor role for circinatin in the formation of the peritoxins and the periconins.