Host-specific Toxin Research Articles

Plants versus pathogens: an evolutionary arms race.

The analysis of plant-pathogen interactions is a rapidly moving research field and one that is very important for productive agricultural systems. The focus of this review is on the evolution of plant defence responses and the coevolution of their pathogens, primarily from a molecular-genetic perspective. It explores the evolution of the major types of plant defence responses including pathogen associated molecular patterns and effector triggered immunity as well as the forces driving pathogen evolution, such as the mechanisms by which pathogen lineages and species evolve. Advances in our understanding of plant defence signalling, stomatal regulation, R gene-effector interactions and host specific toxins are used to highlight recent insights into the coevolutionary arms race between pathogens and plants. Finally, the review considers the intriguing question of how plants have evolved the ability to distinguish friends such as rhizobia and mycorrhiza from their many foes.

Ubiquity of ToxA and absence of ToxB in Australian populations ofPyrenophora tritici-repentis

Pyrenophora tritici-repentis, the causal organism of the necrotrophic foliar wheat disease tan spot [also known as yellow (leaf) spot in Australia] is an important disease in Australia and in many parts of the world. North American isolates of the pathogen have been shown to produce combinations of three host-specific toxins, ToxA, ToxB and ToxC. Each toxin interacts with a host sensitivity locus, respectively Tsn1, Tsc2 and Tsc1. The virulence of an isolate is partially correlated with the presence of these toxins and resistance in the host is associated with absence of the sensitivity loci. Breeding for resistance to tan spot can, therefore, be aided by knowledge of the prevalence of the toxin-encoding genes in local pathogen populations. Two of the toxins, A and B, are encoded by known genes and molecular tests for the genes have been developed. We screened a diverse collection of 119 tan spot isolates collected between 1984 and 2008 and from all affected regions of Australia (Queensland, New South Wales, Victoria and Western Australia). In all cases, the gene for ToxA was present and the gene for ToxB was absent. The implications for resistance breeding and epidemiology of the disease are discussed. We also define a diagnostic molecular marker for P. tritici-repentis.

Genetics of wheat–Pyrenophora tritici-repentis interactions

Tan spot, caused by an ascomycete fungus Pyrenophora tritici-repentis, is one of the most devastating foliar diseases of wheat. This fungus induces two distinct symptoms, tan necrosis and extensive chlorosis, on susceptible wheat cultivars. Besides causing average yield losses of 5–10%, tan spot also causes significant losses in grain quality by grain shriveling, red smudge, and black point. Conservation agriculture in combination with wheat monoculture involving cultivation of susceptible cultivars has resulted in frequent onset of tan spot epidemics worldwide. Development of new resistant wheat cultivars, in conjunction with crop rotation, will provide an effective, economical, and environmentally safe means of controlling tan spot. Presently, eight races of P. tritici-repentis have been identified worldwide based on the ability to induce necrosis and chlorosis symptoms on a set of differential wheat cultivars. P. tritici-repentis is a homothallic fungus having both sexual and asexual reproduction resulting in high genetic diversity worldwide. Both quantitative and qualitative mode of inheritance for resistance to tan spot of wheat has been reported. The tan spot fungus produces multiple host-specific toxins and host resistance is highly correlated to insensitivity to toxins. Genetic studies have further confirmed that wheat–P. tritici-repentis follows the toxin model of gene-for-gene hypothesis although other mechanism of host–pathogen interaction may exist and exploitation of all resistance phenomenon is to be adopted to develop durable resistant cultivars.

Population Genetics of Fungal and Oomycete Effectors Involved in Gene-for-Gene Interactions

Antagonistic coevolution between plants and pathogens has generated a broad array of attack and defense mechanisms. In the classical avirulence (Avr) gene-for-gene model, the pathogen gene evolves to escape host recognition while the host resistance (R) gene evolves to track the evolving pathogen elicitor. In the case of host-specific toxins (HST), the evolutionary arms race may be inverted, with the gene encoding the pathogen toxin evolving to maintain recognition of the host sensitivity target while the host sensitivity gene evolves to escape binding with the toxin. Pathogen effector genes, including those encoding Avr elicitors and HST, often show elevated levels of polymorphism reflecting the coevolutionary arms race between host and pathogen. However, selection can also eliminate variation in the coevolved gene and its neighboring regions when advantageous alleles are swept to fixation. The distribution and diversity of corresponding host genes will have a major impact on the distribution and diversity of effectors in the pathogen population. Population genetic analyses including both hosts and their pathogens provide an essential tool to understand the diversity and dynamics of effector genes. Here, we summarize current knowledge about the population genetics of fungal and oomycete effector genes, focusing on recent studies that have used both spatial and temporal collections to assess the diversity and distribution of alleles and to monitor changes in allele frequencies over time. These studies illustrate that effector genes exhibit a significant degree of diversity at both small and large sampling scales, suggesting that local selection plays an important role in their evolution. They also illustrate that Avr elicitors and HST may be recognizing the same R genes in plants, leading to evolutionary outcomes that differ for necrotrophs and biotrophs while affecting the evolution of the corresponding R genes. Under this scenario, the optimal number of R genes in a plant genome may be determined by the relative abundance of necrotrophic and biotrophic pathogens in the plant's environment.

Coordinate control of oxidative stress tolerance, vegetative growth, and fungal pathogenicity via the AP1 pathway in the rough lemon pathotype of Alternaria alternata

Successful pathogenesis by the necrotrophic pathogen Alternaria alternata in citrus has been thought to be dependent on the production of host-specific toxins. This study determined whether the fungal pathogen can modulate reactive oxygen species (ROS) in the host plant by cloning and characterizing an RLAP1 gene in the rough lemon pathotype of A. alternata, which encodes a polypeptide that resembles many YAP1-like transcription activators of yeasts and fungi. Expression of the RLAP1 gene in a wild-type strain was responsive to many ROS-generating compounds. Mutants disrupted at the RLAP1 locus were hypersensitive to H 2O 2 and ROS-generating oxidants, showed reduced vegetative growth, were less effective in detoxifying H 2O 2, were non-pathogenic to rough lemon, and yet were unaltered in conidial formation or toxin production. When inoculated through wound sites, the mutants failed to incite any lesions. However, co-inoculation of the mutants with an NADPH oxidase inhibitor apocynin or diphenylene iodonium slightly restored fungal pathogenicity; introduction of the RLAP1 gene to a null mutant fully restored all phenotypes to wild type. Upon activation by H 2O 2, RLAP1::sGFP fusion protein is localized to the nucleus. Overall, the results reveal the essential role of effective detoxification via an RLAP1-mediated pathway in oxidative stress response and pathogenicity in A. alternata. It is proposed that the function of RLAP1 in A. alternata is to protect the pathogen from the toxicity of ROS generated by the citrus host.

A proteomic evaluation of Pyrenophora tritici‐repentis, causal agent of tan spot of wheat, reveals major differences between virulent and avirulent isolates

Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host-specific toxins, including Ptr ToxB, a chlorosis-inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici-repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome-level comparison by 2-DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up-regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes alpha-mannosidase and exo-beta-1,3-glucanase, heat-shock and BiP proteins, and various metabolic enzymes. These proteome-level differences suggest a reduced general pathogenic ability in race 4 of P. tritici-repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of Alternaria alternata

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity.

The jasmonate signaling pathway in tomato regulates susceptibility to a toxin-dependent necrotrophic pathogen

The plant hormone, jasmonic acid (JA), is known to have a critical role in both resistance and susceptibility against bacterial and fungal pathogen attack. However, little is known about the involvement of JA in the interactions between plants and toxigenic necrotrophic fungal pathogens. Using the tomato pathotype of Alternaria alternata (Aa) and its AAL-toxin/tomato interaction as a model system, we demonstrate a possible role for JA in susceptibility of plants against pathogens, which utilize host-specific toxins as virulence effectors. Disease development and in planta growth of the tomato pathotype of Aa were decreased in the def1 mutant, defective in biosynthesis of JA, compared with the wild-type (WT) cultivar. Exogenous methyl jasmonate (MeJA) application restored pathogen disease symptoms to the def1 mutant and led to increased disease in the WT. On the other hand, necrotic cell death was similarly induced by AAL-toxin both on def1 and WT, and MeJA application to the tomatoes did not affect the degree of cell death by the toxin. These results indicate that the JA-dependent signaling pathway is not involved in host basal defense responses against the tomato pathotype of Aa, but rather might affect pathogen acceptability via a toxin-independent manner. Data further suggest that JA has a promotional effect on susceptibility of tomato to toxigenic and necrotrophic pathogens, such that pathogens might utilize the JA signaling pathway for successful infection.

Alternaria Toxins—Potential Selective Agents in Biotechnology Approaches for Plant Improvement

ABSTRACTIncreasing sustainable agricultural production has always been related with cultivation of crops that are better able to tolerate biotic stresses (pests, diseases and weeds) and abiotic stresses (drought, salinity, and temperature stress). During last decades breeding programs apply different biotechnology approaches to improve the performance and productivity of crops, incl. increased resistance to plant diseases caused by toxin produced fungal pathogens. Fungi from genus Alternaria produce a group of mycotoxins as alternariols, altertoxins, tenuasonic acid etc. Host specific toxins of Alternaria spp. plant pathogens play an important role in pathogenesis and they could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Toxin compounds' effect on development of necrotic lesions on tobacco and tomato leaves was observed. The correlation between plant susceptibility to the pathogen and sensitivity to the toxin substances is discussed in the light of possibilities for application in the crop breeding programmes.

Prevalence of ToxA-sensitive alleles of the wheat gene Tsn1 in Australian and Chinese wheat cultivars

A recent survey of worldwide isolates of Stagonospora nodorum showed that all Australian isolates expressed the host-specific toxin ToxA (Stukenbrock and McDonald 2007). In contrast, very few Chinese isolates did. All the Australian Pyrenophora tritici-repentis isolates that were tested expressed ToxA. We therefore postulated that the wheat gene that confers sensitivity to ToxA, Tsn1, would vary in prevalence in wheat cultivars in use in the two countries. Contrary to expectation, 10 out of 21 Chinese cultivars responded to ToxA as did 26 out of 46 Australian cultivars. The result suggests that ToxA has not had a determining effect on the survival of wheat cultivars in either country. They also suggest that despite the widespread use of Tsn1 markers in Australia, sensitive alleles are still commonplace. The removal of sensitive alleles from breeders’ lines could be readily achieved and could significantly affect the resistance of wheat to both diseases.

An approach to the biosynthesis of the polyketide toxins excreted by the phytophagen fungus Botrytis cinerea

Botrytis cinerea is a well known pathogen of a number of commercial crops and produces many structurally diverse metabolites. There is no evidence for the production of host-specific toxins by this fungus, which is in accordance with the broad host range of this pathogen. Botrytis produces two series of phytotoxic metabolites: a family of characteristic sesquiterpene metabolites which contain the basic botryane skeleton, principally botrydial and dihydrobotrydial and two polyketide lactones types, botcinins and botrylactone [1,2,3]. The higher production of polyketide toxins from a botrydial sesquiterpene cyclase nock out mutant strain, led us to reinvestigate the structure and biosynthesis of these polyketide compounds.

At Death's Door: Alternaria Pathogenicity Mechanisms

The fungal genus Alternaria is comprised of many saprophytic and endophytic species, but is most well known as containing many notoriously destructive plant pathogens. There are over 4,000 Alternaria/host associations recorded in the USDA Fungal Host Index ranking the genus 10th among nearly 2,000 fungal genera based on the total number of host records. While few Alternaria species appear to have a sexual stage to their life cycles, the majority lack sexuality altogether. Many pathogenic species of Alternaria are prolific toxin producers, which facilitates their necrotrophic lifestyle. Necrotrophs must kill host cells prior to colonization, and thus these toxins are secreted to facilitate host cell death often by triggering genetically programmed apoptotic pathways or by directly causing cell damage resulting in necrosis. While many species of Alternaria produce toxins with rather broad host ranges, a closely-related group of agronomically important Alternaria species produce selective toxins with a very narrow range often to the cultivar level. Genes that code for and direct the biosynthesis of these host-specific toxins for the Alternaria alternata sensu lato lineages are often contained on small, mostly conditionally dispensable, chromosomes. Besides the role of toxins in Alternaria pathogenesis, relatively few genes and/or gene products have been identified that contribute to or are required for pathogenicity. Recently, the completion of the A. brassicicola genome sequencing project has facilitated the examination of a substantial subset of genes for their role in pathogenicity. In this review, we will highlight the role of toxins in Alternaria pathogenesis and the use of A. brassicicola as a model representative for basic virulence studies for the genus as a whole. The current status of these research efforts will be discussed.

Emergence of Tan Spot Disease Caused by Toxigenic Pyrenophora tritici-repentis in Australia Is Not Associated with Increased Deployment of Toxin-Sensitive Cultivars

The wheat disease tan (or yellow leaf) spot, caused by Pyrenophora tritici-repentis, was first described in the period 1934 to 1941 in Canada, India, and the United States. It was first noted in Australia in 1953 and only became a serious disease in the 1970s. The emergence of this disease has recently been linked to the acquisition by P. tritici-repentis of the ToxA gene from the wheat leaf and glume blotch pathogen, Stagonospora nodorum. ToxA encodes a host-specific toxin that interacts with the product of the wheat gene Tsn1. Interaction of ToxA with the dominant allele of Tsn1 causes host necrosis. P. tritici-repentis races lacking ToxA give minor indistinct lesions on wheat lines, whereas wheat lines expressing the recessive tsn1 are significantly less susceptible to the disease. Although the emergence and spread of tan spot had been attributed to the adoption of minimum tillage practices, we wished to test the alternative idea that the planting of Tsn1 wheat lines may have contributed to the establishment of the pathogen in Australia. To do this, wheat cultivars released in Australia from 1911 to 1986 were tested for their sensitivity to ToxA. Prior to 1941, 16% of wheat cultivars were ToxA-insensitive and hence, all other factors being equal, would be more resistant to the disease. Surprisingly, only one of the cultivars released since 1940 was ToxA insensitive, and the area planted to ToxA-insensitive cultivars varied from 0 to a maximum of only 14% in New South Wales. Thus, the majority of the cultivars were ToxA-sensitive both before and during the period of emergence and spread of the disease. We therefore conclude that the spread of P. tritici-repentis in Australia cannot be causally linked to the deployment of ToxA-sensitive cultivars.

Host-specific toxins: effectors of necrotrophic pathogenicity

Host-specific toxins (HSTs) are defined as pathogen effectors that induce toxicity and promote disease only in the host species and only in genotypes of that host expressing a specific and often dominant susceptibility gene. They are a feature of a small but well-studied group of fungal plant pathogens. Classical HST pathogens include species of Cochliobolus, Alternaria and Pyrenophora. Recent studies have shown that Stagonospora nodorum produces at least four separate HSTs that interact with four of the many quantitative resistance loci found in the host, wheat. Rationalization of fungal phylogenetics has placed these pathogens in the Pleosporales order of the class Dothideomycetes. It is possible that all HST pathogens lie in this order. Strong evidence of the recent lateral gene transfer of the ToxA gene from S. nodorum to Pyrenophora tritici-repentis has been obtained. Hallmarks of lateral gene transfer are present for all the studied HST genes although definitive proof is lacking. We therefore suggest that the Pleosporales pathogens may have a conserved propensity to acquire HST genes by lateral transfer.

Recent Fungal Diseases of Crop Plants: Is Lateral Gene Transfer a Common Theme?

A cursory glance at old textbooks of plant pathology reveals that the diseases which are the current scourge of agriculture in many parts of the world are a different set from those that were prominent 50 or 100 years ago. Why have these new diseases arisen? The traditional explanations subscribe to the "nature abhors a vacuum" principle-that control of one disease creates the condition for the emergence of a replacement-but does little to explain why the new pathogen succeeds. The emergence of a new disease requires a series of conditions and steps, including the enhanced fecundity of the new pathogen, enhanced survival from season to season, and spread around the world. Recently, evidence was obtained that wheat tan spot emerged through a lateral gene transfer event some time prior to 1941. Although there have been sporadic and persistent reports of lateral gene transfer between and into fungal plant pathogens, most examples have been dismissed through incomplete evidence. The completion of whole genome sequences of an increasing number of fungal pathogens no longer allows such proposed cases of lateral gene transfer to be dismissed so easily. How frequent are lateral gene transfers involving fungal plant pathogens, and can this process explain the emergence of many of the new diseases of the recent past? Many of the apparently new diseases are dependant on the expression of host-specific toxins. These are enigmatic molecules whose action requires the presence of plant genes with products that specifically encode sensitivity to the toxin and susceptibility to the disease. It is also notable that many new diseases belong to the fungal taxon dothideomycetes. This review explores the coincidence of new diseases, interspecific gene transfer, host-specific toxins, and the dothideomycete class.

Production of plants resistant to Alternaria carthami via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamustinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 105 spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min−1 mg−1 protein) from organogenesis and 47% (0.23 μmol min−1 mg−1 protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg−1 protein) and 19.5% (145 U mg−1 protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.

Fitotoxinlerin Bitki Hastalıklarındaki Rolü

Bacterial and fugal plant pathogens are known to produce toxic subtance in infectected plants, Which are named as phytotoxins. Phytotoxins are small molecules and have a variety of structures including sesquiterpenoids, sesterterpenoids, diketopiperazines, peptides, spirocyclic lactams, isocoumarins, polyketides, tetramic acids and perylenequinones. Phytotoxins may be classified as host specific toxins (HST) and non host specific toxins (NHST). Host specific toxins affect only plants of a genotype susceptible to the pathogens. On the other hand, non-host specific toxins affect a lot of plants. So far, Approximately 23 HST and 34 NHST have been determined. All of HSTs are produced by fungi, many of Which belonging to Alternaria ve Cochliobolus. NHSTs produce both bacteria and fungi. Phytotoxins cause a wide range of simtomps on host plants including wilt, chlorosis, blights, leaf spots, necrosis, water soaking and galls.

Dothideomycete–Plant Interactions Illuminated by Genome Sequencing and EST Analysis of the Wheat Pathogen Stagonospora nodorum

Stagonospora nodorum is a major necrotrophic fungal pathogen of wheat (Triticum aestivum) and a member of the Dothideomycetes, a large fungal taxon that includes many important plant pathogens affecting all major crop plant families. Here, we report the acquisition and initial analysis of a draft genome sequence for this fungus. The assembly comprises 37,164,227 bp of nuclear DNA contained in 107 scaffolds. The circular mitochondrial genome comprises 49,761 bp encoding 46 genes, including four that are intron encoded. The nuclear genome assembly contains 26 classes of repetitive DNA, comprising 4.5% of the genome. Some of the repeats show evidence of repeat-induced point mutations consistent with a frequent sexual cycle. ESTs and gene prediction models support a minimum of 10,762 nuclear genes. Extensive orthology was found between the polyketide synthase family in S. nodorum and Cochliobolus heterostrophus, suggesting an ancient origin and conserved functions for these genes. A striking feature of the gene catalog was the large number of genes predicted to encode secreted proteins; the majority has no meaningful similarity to any other known genes. It is likely that genes for host-specific toxins, in addition to ToxA, will be found among this group. ESTs obtained from axenic mycelium grown on oleate (chosen to mimic early infection) and late-stage lesions sporulating on wheat leaves were obtained. Statistical analysis shows that transcripts encoding proteins involved in protein synthesis and in the production of extracellular proteases, cellulases, and xylanases predominate in the infection library. This suggests that the fungus is dependant on the degradation of wheat macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing pycnidiospores.

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H2O2 generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H2O2 generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.

Heterologous expression and activity of Ptr ToxB from virulent and avirulent isolates of Pyrenophora tritici-repentis

The fungal pathogen Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat (Triticum aestivum). The fungus produces at least three host-specific toxins, including the chlorosis-inducing Ptr ToxB, a small protein encoded by the ToxB gene. A ToxB homolog is also found in race 4 isolates of P. tritici-repentis, which lack detectable toxin activity. It is not clear whether the absence of toxin activity in these isolates stems from differences in the ToxB gene sequence and (or) level of expression. To directly compare the chlorosis-inducing activity of different forms of Ptr ToxB, ToxB from a virulent race 5 isolate (Alg3-24) and a ToxB homolog from an avirulent race 4 isolate (90-2) were heterologously expressed as glutathione-S-transferase (GST)- and hexahistidine (His)-tagged fusion proteins in Escherichia coli. Infiltration of toxin solutions into the leaves of Ptr ToxB sensitive and insensitive wheat seedlings revealed that the overexpressed GST- and His-tagged toxin from Alg3-24 induced strong chlorosis symptoms in a genotype-specific manner, whereas the form of the toxin from isolate 90-2 induced only trace amounts of symptoms. This suggests that the absence of chlorosis-inducing activity in race 4 of P. tritici-repentis may be largely due to sequence differences in ToxB at the gene and protein level, regardless of the extent of gene expression.