Solution NMR structures of Pyrenophora tritici-repentis ToxB and its inactive homolog reveal potential determinants of toxin activity.

Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a ß-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent ß-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.

RNAi silencing of a cytochrome P450 monoxygenase disrupts the ability of a filamentous fungus, Graphium sp., to grow on short-chain gaseous alkanes and ethers.

Graphium sp. (ATCC 58400), a filamentous fungus, is one of the few eukaryotes that grows on short-chain alkanes and ethers. In this study, we investigated the genetic underpinnings that enable this fungus to catalyze the first step in the alkane and ether oxidation pathway. A gene, CYP52L1, was identified, cloned and functionally characterized as an alkane-oxidizing cytochrome P450 (GSPALK1). Analysis of CYP52L1 suggests that it is a member of the CYP52 cytochrome P450 family, which is comprised of medium- and long-chain alkane-oxidizing enzymes found in yeasts. However, phylogenetic analysis of GSPALK1 with other CYP52 members suggests they are not closely related. Post-transcriptional ds-RNA-mediated gene silencing of CYP52L1 severely reduced the ability of this fungus to oxidize alkanes and ethers, however, downstream metabolic steps in these pathways were unaffected. Collectively, the results of this study suggest that GSPALK1 is the enzyme that catalyzes the initial oxidation of alkanes and ethers but is not involved in the later steps of alkane or ether metabolism.

Characterization of the LOV1-mediated, victorin-induced, cell-death response with virus-induced gene silencing.

Victoria blight, caused by Cochliobolus victoriae, is a disease originally described on oat and recapitulated on Arabidopsis. C. victoriae pathogenesis depends upon production of the toxin victorin. In oat, victorin sensitivity is conferred by the Vb gene, which is genetically inseparable from the Pc2 resistance gene. Concurrently, in Arabidopsis, sensitivity is conferred by the LOCUS ORCHESTRATING VICTORIN EFFECTS1 (LOV1) gene. LOV1 encodes a nucleotide-binding site leucine-rich repeat protein, a type of protein commonly associated with disease resistance, and LOV1 "guards" the defense thioredoxin, TRX-h5. Expression of LOV1 and TRX-h5 in Nicotiana benthamiana is sufficient to confer victorin sensitivity. Virus-induced gene silencing was used to characterize victorin-induced cell death in N. benthamiana. We determined that SGT1 is required for sensitivity and involved in LOV1 protein accumulation. We screened a normalized cDNA library and identified six genes that, when silenced, suppressed LOV1-mediated, victorin-induced cell death and cell death induced by expression of the closely related RPP8 resistance gene: a mitochondrial phosphate transporter, glycolate oxidase, glutamine synthetase, glyceraldehyde 3-phosphate dehydrogenase, and the P- and T-protein of the glycine decarboxylase complex. Silencing the latter four also inhibited cell death and disease resistance mediated by the PTO resistance gene. Together, these results provide evidence that the victorin response mediated by LOV1 is a defense response.

A host-selective toxin of Pyrenophora tritici-repentis, Ptr ToxA, induces photosystem changes and reactive oxygen species accumulation in sensitive wheat.

Ptr ToxA (ToxA) is a proteinaceous necrotizing host-selective toxin produced by Pyrenophora tritici-repentis, a fungal pathogen of wheat (Triticum aestivum). In this study, we have found that treatment of ToxA-sensitive wheat leaves with ToxA leads to a light-dependent accumulation of reactive oxygen species (ROS) that correlates with the onset of necrosis. Furthermore, the accumulation of ROS and necrosis could be inhibited by the antioxidant N-acetyl cysteine, providing further evidence that ROS production is required for necrosis. Microscopic evaluation of ToxA-treated whole-leaf tissue indicated that ROS accumulation occurs in the chloroplasts. Analysis of total protein extracts from ToxA-treated leaves showed a light-dependent reduction of the chloroplast protein RuBisCo. In addition, Blue native-gel electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that ToxA induces changes in photosystem I (PSI) and photosystem II (PSII) in the absence of light, and therefore, the absence of ROS. When ToxA-treated leaves were exposed to light, all proteins in both PSI and PSII were extremely reduced. We propose that ToxA induces alterations in PSI and PSII affecting photosynthetic electron transport, which subsequently leads to ROS accumulation and cell death when plants are exposed to light.

Characterization of natural and induced variation in the LOV1 gene, a CC-NB-LRR gene conferring victorin sensitivity and disease susceptibility in Arabidopsis.

The fungus Cochliobolus victoriae, the causal agent of Victoria blight, produces a compound called victorin that is required for pathogenicity of the fungus. Victorin alone reproduces disease symptoms on sensitive plants. Victorin sensitivity and susceptibility to C. victoriae were originally described on oats but have since been identified on Arabidopsis thaliana. Victorin sensitivity and disease susceptibility in Arabidopsis are conferred by LOV1, a coiled-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR) protein. We sequenced the LOV1 gene from 59 victorin-insensitive mutants and found that the spectrum of mutations causing LOV1 loss of function was similar to that found to cause loss of function of RPM1, a CC-NB-LRR resistance protein. Also, many of the mutated residues in LOV1 are in conserved motifs required for resistance protein function. These data indicate that LOV1 may have a mechanism of action similar to resistance proteins. Victorin sensitivity was found to be the prevalent phenotype in a survey of 30 Arabidopsis ecotypes, and we found very little genetic variation among LOV1 alleles. As selection would not be expected to preserve a functional LOV1 gene to confer victorin sensitivity and disease susceptibility, we propose that LOV1 may function as a resistance gene to a naturally-occurring pathogen of Arabidopsis.

Plant disease susceptibility conferred by a "resistance" gene.

The molecular nature of many plant disease resistance (R) genes is known; the largest class encodes nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins that are structurally related to proteins involved in innate immunity in animals. Few genes conferring disease susceptibility, on the other hand, have been identified. Recent identification of susceptibility to the fungus Cochliobolus victoriae in Arabidopsis thaliana has enabled our cloning of LOV1, a disease susceptibility gene that, paradoxically, is a member of the NBS-LRR resistance gene family. We found LOV1 mediates responses associated with defense, but mutations in known defense response pathways do not prevent susceptibility to C. victoriae. These findings demonstrate that NBS-LRR genes can condition disease susceptibility and resistance and may have implications for R gene deployment.

Thioredoxin h5 is required for victorin sensitivity mediated by a CC-NBS-LRR gene in Arabidopsis.

The fungus Cochliobolus victoriae causes Victoria blight of oats (Avena sativa) and is pathogenic due to its production of victorin, which induces programmed cell death in sensitive plants. Victorin sensitivity has been identified in Arabidopsis thaliana and is conferred by the dominant gene LOCUS ORCHESTRATING VICTORIN EFFECTS1 (LOV1), which encodes a coiled-coil-nucleotide binding site-leucine-rich repeat protein. We isolated 63 victorin-insensitive mutants, including 59 lov1 mutants and four locus of insensitivity to victorin1 (liv1) mutants. The LIV1 gene encodes thioredoxin h5 (ATTRX5), a member of a large family of disulfide oxidoreductases. To date, very few plant thioredoxins have been assigned specific, nonredundant functions. We found that the victorin response was highly specific to ATTRX5, as the closely related ATTRX3 could only partially compensate for loss of ATTRX5, even when overexpressed. We also created chimeric ATTRX5/ATTRX3 proteins, which identified the central portion of the protein as important for conferring specificity to ATTRX5. Furthermore, we found that ATTRX5, but not ATTRX3, is highly induced in sensitive Arabidopsis following victorin treatment. Finally, we determined that only the first of the two active-site Cys residues in ATTRX5 is required for the response to victorin, suggesting that ATTRX5 function in the victorin pathway involves an atypical mechanism of action.

Identification and characterization of victorin sensitivity in Arabidopsis thaliana.

Cochliobolus victoriae is a necrotrophic fungus that produces a host-selective toxin called victorin. Victorin is considered to be host selective because it has been known to affect only certain allohexaploid oat cultivars containing the dominant Vb gene. Oat cultivars containing Vb are also the only genotypes susceptible to C. victoriae. Assays were developed to screen the "nonhost" plant of C. victoriae, Arabidopsis thaliana, for victorin sensitivity. Sensitivity to victorin was identified in six of 433 bulk populations of Arabidopsis. In crosses of Col-4 (victorin-insensitive) x victorin-sensitive Arabidopsis ecotypes, victorin sensitivity segregated as a single dominant locus, as it does in oats. This Arabidopsis locus was designated LOV, for locus orchestrating victorin effects. Allelism tests indicate that LOV loci are allelic or closely linked in all six victorin-sensitive ecotypes identified. LOV was localized to the north arm of Arabidopsis thaliana chromosome I. The victorin-sensitive Arabidopsis line LOV1 but not the victorin-insensitive line Col-4 was susceptible to C. victoriae infection. Consequently, the LOV gene appears to be a genetically dominant, disease susceptibility gene.

The victorin-induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity.

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 (Deltapsi(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 Deltapsi(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 Deltapsi(m). Results suggest that the victorin-induced collapse in Deltapsi(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.

Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa.

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.

Host-selective toxins and avirulence determinants: what's in a name?

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.

The oat mitochondrial permeability transition and its implication in victorin binding and induced cell death.

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.