Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.     

Genetic Similarity among Isolates of Pyrenophora tritici-repentis, Causal Agent of Tan Spot of Wheat

Tan spot, a foliar disease of wheat, is caused by the fungus Pyrenophora tritici‐repentis. On susceptible wheat cultivars, P. tritici‐repentis induces two distinct symptoms: tan necrosis and extensive chlorosis. Presently isolates of P. tritici‐repentis are classified into 11 races based on their virulence on a set of wheat differential genotypes. In nature, this pathogen reproduces both sexually and asexually, but the extent of genetic variability in the P. tritici‐repentis population of western Canada is unknown. This study was conducted to assess the genetic variability among different isolates of P. tritici‐repentis and to determine if similarities among isolates are correlated with race classification or geographic origin of the isolates. Thirty‐three isolates of P. tritici‐repentis and one isolate each of P. teres f. sp. teres, P. teres f. sp. maculata, P. graminea, Helminthosporium sativum and an uncharacterized isolate were studied with 30 random amplified polymorphic DNA (RAPD) primers. Cluster analysis showed that all isolates had unique banding patterns and that clustering of isolates was independent of their race designation or geographic origin. Analysis of molecular variation (amova ) showed that 96.8% of variability occurred among isolates and among race variability accounted for only 3.2% of the total variability.     

Molecular mapping of resistance to <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> race 5 and sensitivity to Ptr ToxB in wheat

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an economically important foliar disease in the major wheat growing areas of the world. Multiple races of the pathogen have been characterized based on their ability to cause necrosis and/or chlorosis in differential wheat lines. Isolates of race 5 cause chlorosis only, and they produce a host-selective toxin designated Ptr ToxB that induces chlorosis when infiltrated into sensitive genotypes. The international Triticeae mapping initiative (ITMI) mapping population was used to identify genomic regions harboring QTLs for resistance to fungal inoculations of Ptr race 5 and to determine the chromosomal location of the gene conditioning sensitivity to Ptr ToxB. The toxin-insensitivity gene, which we are designating tsc2, mapped to the distal tip of the short arm of chromosome 2B. This gene was responsible for the effects of a major QTL associated with resistance to the race 5 fungus and accounted for 69% of the phenotypic variation. Additional minor QTLs were identified on the short arm of 2A, the long arm of 4A, and on the long arm of chromosome 2B. Together, the major QTL on 2BS identified by tsc2 and the QTL on 4AL explained 73% of the total phenotypic variation for resistance to Ptr race 5. The results of this research indicate that Ptr ToxB is a major virulence factor, and the markers closely linked to tsc2 and the 4A QTL should be useful for introgression of resistance into adapted germplasm.     

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 β-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 β-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.     

Genetics of tan spot resistance in wheat

Tan spot is a devastating foliar disease of wheat caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis. Much has been learned during the past two decades about the genetics of wheat–P. tritici-repentis interactions. Research has shown that the fungus produces at least three host-selective toxins (HSTs), known as Ptr ToxA, Ptr ToxB, and Ptr ToxC, that interact directly or indirectly with the products of the dominant host genes Tsn1, Tsc2, and Tsc1, respectively. The recent cloning and characterization of Tsn1 provided strong evidence that the pathogen utilizes HSTs to subvert host resistance mechanisms to cause disease. However, in addition to host–HST interactions, broad-spectrum, race non-specific resistance QTLs and recessively inherited qualitative ‘resistance’ genes have been identified. Molecular markers suitable for marker-assisted selection against HST sensitivity genes and for race non-specific resistance QTLs have been developed and used to generate adapted germplasm with good levels of tan spot resistance. Future research is needed to identify novel HSTs and corresponding host sensitivity genes, determine if the recessively inherited resistance genes are HST insensitivities, extend the current race classification system to account for new HSTs, and determine the molecular basis of race non-specific resistance QTLs and their relationships with host–HST interactions at the molecular level. Necrotrophic pathogens such as P. tritici-repentis are likely to become increasingly significant under a changing global climate making it imperative to further characterize the wheat–P. tritici-repentis pathosystem and develop tan spot resistant wheat varieties.     

RNA-mediated gene silencing of ToxB in Pyrenophora tritici-repentis

The fungus Pyrenophora tritici-repentis causes tan spot, a wheat leaf disease of worldwide importance. The pathogen produces three host-selective toxins, including Ptr ToxB, which causes chlorophyll degradation and foliar chlorosis on toxin-sensitive wheat genotypes. The ToxB gene, which codes for Ptr ToxB, was silenced in a wild-type race 5 isolate of the fungus thorough a sense- and antisense-mediated silencing mechanism. Toxin production by the silenced strains was evaluated in culture filtrates of the fungus via Western blotting analysis, and plant bioassays were conducted to test the virulence of the transformants in planta. The chlorosis-inducing ability of the silenced strains was correlated with the quantity of Ptr ToxB, and transformants in which toxin production was strongly decreased also caused very little disease on toxin-sensitive wheat genotypes. Cytological analysis of the infection process revealed that, in addition to a reduced capacity to induce chlorosis, the silenced strains with the greatest decrease in the levels of Ptr ToxB produced significantly fewer appressoria than the wild-type isolate, 12 and 24 h after inoculation onto wheat leaves. The results provide strong support for the suggestion that the amount of Ptr ToxB protein produced by fungal isolates plays a significant role in the quantitative variation in the virulence of P. tritici-repentis.     

Catenarin Production by Isolates of Pyrenophora tritici-repentis (Died.) Drechsler and its Antimicrobial Activity

A total of 88 Pyrenophora tritici‐repentis (Died.) Drechsler isolates from different hosts and localities were screened for catenarin production using thin layer chromatography. Catenarin was detected in 29% of the tested strains. The level of this compound ranged from 2 to 400 ppm and was especially high in case of fungus variants unable to biosynthesize melanins. The extracted pigment exhibited antibiotic properties against Gram‐positive bacteria (Aureobacterium liquefaciens, Arthrobacter globiformis, Bacillus brevis, B. circulans, B. subtilis and Curtobacterium plantarum). Catenarin also inhibited the growth of fungi accompanying P. tritici‐repentis during the saprophytic phase of development. The most sensitive species was Epicoccum nigrum, whose growth was inhibited up to 90%.     

Characterization of the ToxB gene from Pyrenophora tritici-repentis

The ToxB gene was cloned and characterized from a race 5 isolate of Pyrenophora tritici-repentis from North Dakota. ToxB contains a 261-bp open reading frame that encodes a 23 amino acid putative signal peptide and a 64 amino acid host-selective toxin, Ptr ToxB. Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a host-selective toxin.     

Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota

Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.     

Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota.

Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.     

Genome characterization of Pyrenophora tritici-repentis isolates reveals high plasticity and independent chromosomal location of ToxA and ToxB

The fungus Pyrenophora tritici - repentis (Died.) causes tan spot, an important leaf disease of wheat worldwide. Isolates of this pathogen have been collected and characterized into eight races on the basis of their ability to produce three different host-selective toxins. The karyotype of 47 isolates was determined by pulsed field gel electrophoresis. The collection originated from different parts of the world and included genotypes from all races. A single isolate was characterized for each of races 3, 4 and 6, whereas fourteen, five, nine, five and eleven isolates were karyotyped for races 1, 2, 5, 7 and 8, respectively. The survey showed that the chromosome number of P. tritici-repentis was highly variable, with some isolates having as few as eight chromosomes, but others having 11 or more. Similarly, the genome size ranged from 25.5 to 48.0 Mb, and individual chromosome sizes ranged from 1.3 to more than 5.7 Mb. Considerable variation was observed in karyotype patterns among the P. tritici-repentis isolates tested. A total of 29 different karyotypes was identified among the 47 isolates. These chromosome level variations were as variable for isolates within a race as for isolates across races. Southern blot analysis of the 47 isolates with ToxA and ToxB probes revealed that the toxin genes were always located on different chromosomes. Furthermore, with six chromosome-specific single-copy probes, the ToxA -carrying chromosome was shown to be homologous among the Ptr ToxA-producing isolates, with a related chromosome in the non-ToxA-producing isolates, suggesting that the chromosome on which ToxA generally resides is of an essential nature. Interestingly, a molecular rearrangement involving a translocation of ToxA to a different chromosome was identified in one isolate.     

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici‐repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12‐kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici‐repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non‐synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non‐ToxA isolate. This work demonstrates that proteins that confer host‐specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.     

Analysis of Genetic Diversity of Fusarium oxysporum f. sp. phaseoli Isolates, Pathogenic and Non-pathogenic to Common Bean (Phaseolus vulgaris L.)

Twenty isolates of Fusarium oxysporum from Brazil, pathogenic and non‐pathogenic to common bean, were analysed using random amplified polymorphic DNA (RAPDs) to study the genetic diversity. RAPD analysis using 23 oligonucleotides resulted in the amplification of 229 polymorphic and 7 monomorphic DNA fragments ranging from 234 to 2590 bp. High genetic variability was observed among the isolates, with the distances varying between 8% and 76% among pathogenic, 2% and 63% among the non‐pathogenic and 45% and 76% between pathogenic and non‐pathogenic isolates. The analysis of genetic distance data showed that the pathogenic isolates tended to group in one group and the non‐pathogenic in another. The genetic distance values of 30% among the pathogenic isolates in cluster A are compatible with the genetic distance values observed within the physiological races, but the distance values among the pathogenic isolates in clusters B and G are not compatible with the distance values observed within the race. Although our results are preliminary, it was not possible to exclude the existence of more than one race of this fungus in Brazil.     

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide‐binding leucine‐rich repeat protein (NLR)‐encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA‐Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.     

Genetic Diversity of Pyrenophora teres Isolates as Detected by AFLP Analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to analyse mainly 83 Czech isolates of Pyrenophora teres, P. graminea, P. tritici‐repentis and Helminthosporium sativum. Each species had distinct AFLP profiles. Using 19 primer combinations 948 polymorphic bands were detected. All main clusters in dendrogram correspond to the studied species. Even the two forms of P. teres–P. teres f. teres (PTT) and P. teres f. maculata (PTM) – formed different clusters. Genetic diversity, with regard to the locality and the year of the sample's collection, was analysed separately within the AFLP‐based dendrogram cluster of PTT and PTM. Unweighted pair‐group method (UPGMA) analysis of the 37 isolates of PTT and 30 isolates of PTM, using 469 polymorphic bands, showed that the variability seemed to have been influenced more by the year of sampling than by the geographic origin of the isolate. The presence of intermediate haplotypes with a relatively high number of shared markers between the two groups indicated that hybridization between the forms of P. teres could happen, but it is probably often overlapped by selection pressure or genetic drift.