The identification and deletion of the polyketide synthase-nonribosomal peptide synthase gene responsible for the production of the phytotoxic triticone A/B in the wheat fungal pathogen Pyrenophora tritici-repentis.
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ABSTRACT:
SUBMITTER: Rawlinson C
PROVIDER: S-EPMC6915911 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) causes tan (syn. yellow) spot of wheat and accounts for significant yield losses worldwide. Understanding the molecular mechanisms of this economically important crop disease is crucial to counteract the yield and quality losses of wheat globally. Substantial progress has been made to comprehend the race structure of this phytopathogen based on its production of necrotrophic effectors and genomic resources of Ptr. However, one limitation for studying Ptr in a laboratory environment is the difficulty to isolate high spore numbers from vegetative growth with mycelial contamination common. These limitations reduce the experimental tractability of Ptr.ResultsHere, we optimized a multitude of parameters and report a sporulation method for Ptr that yields robust, high quality and pure spores. Our methodology encompasses simple and reproducible plugging and harvesting techniques, resulting in spore yields up to 1500 fold more than the current sporulation methods and was tested on multiple isolates and races of Ptr as well as an additional seven modern Australian Ptr isolates. Moreover, this method also increased purity and spore harvest numbers for two closely related fungal pathogens (Pyrenophora teres f. maculata and f. teres) that cause net blotch diseases in barley (Hordeum vulgare), highlighting the usability of this optimized sporulation protocol for the wider research community.ConclusionsLarge-scale spore infection and virulence assays are essential for the screening of wheat and barley cultivars and combined with the genetic mapping of these populations allows pinpointing and exploiting sources of host genetic resistance. We anticipate that improvements in spore numbers and purity will further advance research to increase our understanding of the pathogenicity mechanisms of these important fungal pathogens.
| S-EPMC8136220 | biostudies-literature
Project description:Pyrenophora tritici-repentis, the causal agent of tan spot disease of wheat, mediates disease by the production of host-selective toxins (HST). The known toxins are recognized in an 'inverse' gene-for-gene manner, where each is perceived by the product of a unique locus in the host and recognition leads to disease susceptibility. Given the importance of HSTs in disease development, we would predict that the loss of any of these major pathogenicity factors would result in reduced virulence and disease development. However, after either deletion of the gene encoding the HST ToxA or, reciprocally, heterologous expression of ToxA in a race that does not normally produce the toxin followed by inoculation of ToxA-sensitive and insensitive wheat cultivars, we demonstrate that ToxA symptom development can be epistatic to other HST-induced symptoms. ToxA epistasis on certain ToxA-sensitive wheat cultivars leads to genotype-specific increases in total leaf area affected by disease. These data indicate a complex interplay between host responses to HSTs in some genotypes and underscore the challenge of identifying additional HSTs whose activity may be masked by other toxins. Also, through mycelial staining, we acquire preliminary evidence that ToxA may provide additional benefits to fungal growth in planta in the absence of its cognate recognition partner in the host.
| S-EPMC4386829 | biostudies-literature
Project description:BACKGROUND:Pyrenophora tritici-repentis (Ptr) is a necrotrophic fungal pathogen that causes the major wheat disease, tan spot. We set out to provide essential genomics-based resources in order to better understand the pathogenicity mechanisms of this important pathogen. RESULTS:Here, we present eight new Ptr isolate genomes, assembled and annotated; representing races 1, 2 and 5, and a new race. We report a high quality Ptr reference genome, sequenced by PacBio technology with Illumina paired-end data support and optical mapping. An estimated 98% of the genome coverage was mapped to 10 chromosomal groups, using a two-enzyme hybrid approach. The final reference genome was 40.9 Mb and contained a total of 13,797 annotated genes, supported by transcriptomic and proteogenomics data sets. CONCLUSIONS:Whole genome comparative analysis revealed major chromosomal segmental rearrangements and fusions, highlighting intraspecific genome plasticity in this species. Furthermore, the Ptr race classification was not supported at the whole genome level, as phylogenetic analysis did not cluster the ToxA producing isolates. This expansion of available Ptr genomics resources will directly facilitate research aimed at controlling tan spot disease.
| S-EPMC5913888 | biostudies-literature
Project description:OBJECTIVES:Tan spot is a yield-reducing disease that affects wheat and is caused by the fungus Pyrenophora tritici-repentis (Ptr). Eight races of Ptr have been identified based upon production of the effectors Ptr ToxA, Ptr ToxB, and Ptr ToxC. Wheat cultivars have also been characterized by their resistance and susceptibility to races of Ptr and sensitivity to the effectors. The objective of this research was to assess differences in gene expression between Ptr resistant and susceptible wheat cultivars when either inoculated with Ptr race 2 spores or directly infiltrated with Ptr ToxA. DATA DESCRIPTION:A greenhouse experiment was used to assess wheat-Ptr interaction. Wheat seedlings were grown for two weeks prior to the experiment under greenhouse conditions. Four treatments were used: (1) spray-inoculation with a suspension of Ptr spores (3000 spores/mL) (2) spray inoculation with water as a control (3) needleless syringe injection with Ptr ToxA, and (4) needleless syringe injection with water as a control. Plants were transferred to a humidity chamber and leaf sample were taken at 0, 8, and 16 h. After RNA extraction and sequencing, 48 RNA datasets are reported. This data will be useful in understanding how resistant wheat responds to Ptr compared to susceptible wheat.
| S-EPMC6670118 | biostudies-literature
Project description:Bread wheat (Triticum aestivum L.) is one of the world's most important crop species. The development of new varieties resistant to multiple pathogens is an ongoing task in wheat breeding, especially in times of increasing demand for sustainable agricultural practices. Despite this, little is known about the relations between various fungal disease resistances at the genetic level, and the possible consequences for wheat breeding strategies. As a first step to fill this gap, we analyzed the genetic relations of resistance to the three fungal diseases - powdery mildew (PM), septoria tritici blotch (STB), and tan spot (TS) - using a winter wheat multiparent advanced generation intercross population. Six, seven, and nine QTL for resistance to PM, STB, and TS, respectively, were genetically mapped. Additionally, 15 QTL were identified for the three agro-morphological traits plant height, ear emergence time, and leaf angle distribution. Our results suggest that resistance to STB and TS on chromosome 2B is conferred by the same genetic region. Furthermore, we identified two genetic regions on chromosome 1AS and 7AL, which are associated with all three diseases, but not always in a synchronal manner. Based on our results, we conclude that parallel marker-assisted breeding for resistance to the fungal diseases PM, STB, and TS appears feasible. Knowledge of the genetic co-localization of alleles with contrasting effects for different diseases, such as on chromosome 7AL, allows the trade-offs of selection of these regions to be better understood, and ultimately determined at the genic level.
| S-EPMC6505172 | biostudies-literature
Project description:Key messageGenetic mapping of sensitivity to the Pyrenophora tritici-repentis effector ToxB allowed development of a diagnostic genetic marker, and investigation of wheat pedigrees allowed transmission of sensitive alleles to be tracked. Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, is a major disease of wheat (Triticum aestivum). Secretion of the P. tritici-repentis effector ToxB is thought to play a part in mediating infection, causing chlorosis of plant tissue. Here, genetic analysis using an association mapping panel (n = 480) and a multiparent advanced generation intercross (MAGIC) population (n founders = 8, n progeny = 643) genotyped with a 90,000 feature single nucleotide polymorphism (SNP) array found ToxB sensitivity to be highly heritable (h2 ≥ 0.9), controlled predominantly by the Tsc2 locus on chromosome 2B. Genetic mapping of Tsc2 delineated a 1921-kb interval containing 104 genes in the reference genome of ToxB-insensitive variety 'Chinese Spring'. This allowed development of a co-dominant genetic marker for Tsc2 allelic state, diagnostic for ToxB sensitivity in the association mapping panel. Phenotypic and genotypic analysis in a panel of wheat varieties post-dated the association mapping panel further supported the diagnostic nature of the marker. Combining ToxB phenotype and genotypic data with wheat pedigree datasets allowed historic sources of ToxB sensitivity to be tracked, finding the variety 'Maris Dove' to likely be the historic source of sensitive Tsc2 alleles in the wheat germplasm surveyed. Exploration of the Tsc2 region gene space in the ToxB-sensitive line 'Synthetic W7984' identified candidate genes for future investigation. Additionally, a minor ToxB sensitivity QTL was identified on chromosome 2A. The resources presented here will be of immediate use for marker-assisted selection for ToxB insensitivity and the development of germplasm with additional genetic recombination within the Tsc2 region.
| S-EPMC7021774 | biostudies-literature
Project description:Tan Spot (TS), causal agent Pyrenophora tritici-repentis (Ptr), is a major threat to wheat production due to the lack of resistant cultivars. In our previous work, we identified MAGIC population parental lines exhibiting TS resistance and susceptibility, namely Robigus and Hereward, respectively. To understand the mechanisms underlying these phenotypes, we performed RNA-seq analysis of leaves before and during Ptr interaction. When comparing mock- and Ptr-inoculated samples, differentially expressed genes (DEGs) were identified with DESeq2, leading to the targeting of 15193 DEGs. Functional annotation showed the pathways enzyme classification, solute transport, RNA biosynthesis, protein modification and homeostasis represented 49.5% of DEGs in Robigus. Cellular metabolism pathways were induced, as well as vesicle trafficking, actin polymerization and cellulose. The upregulation of these cell wall related genes along with microscopic data suggested that barrier defence is a major feature of TS resistance in Robigus. Conversely, photosynthesis was the top fifth pathway in Hereward, totalling 389 repressed genes (12.63%). Photosynthesis collapse was linked to the activation of oligosaccharide metabolism and suppression of glycolysis, TCA cycle and amino acids degradation. This may reflect mobilization of host nutrients to Ptr. Our observations could inform wheat-breeding programmes targeting TS resistance.
2022-06-30 | GSE202986 | GEO
Project description:The necrotrophic fungal pathogen Pyrenophora tritici-repentis causes tan spot, a major disease of wheat, throughout the world. The proteinaceous effector ToxA is responsible for foliar necrosis on ToxA-sensitive wheat genotypes. The single copy ToxA gene was deleted from a wild-type race 1 P. tritici-repentis isolate via homologous recombination of a knockout construct. Expression of the ToxA transcript was found to be absent in transformants (toxa), as was ToxA protein production in fungal culture filtrates. Plant bioassays were conducted to test transformant pathogenicity. The toxa strains were unable to induce necrosis on ToxA-sensitive wheat genotypes. To our knowledge, this is the first demonstration of a targeted gene knockout in P. tritici-repentis. The ability to undertake gene deletions will facilitate the characterization of other pathogenicity effectors of this economically significant necrotroph.
| S-EPMC6638721 | biostudies-literature
Project description:Tan spot is a destructive foliar wheat disease worldwide and caused by the ascomycete fungus Pyrenophora tritici-repentis (Ptr); it has become more frequent in Tunisia over the last decade. In this study, the virulence of 73 single-spore isolates, collected from durum and bread wheat fields during 2017-2018 growing season, was evaluated on four differential wheat genotypes. This was followed by polymerase chain reaction tests with specific primers for the effector genes ToxA, ToxB, and toxb (ToxB-homolog). Sequence analysis to validate the identity of the amplified genes was followed, and ToxA amplicons from a subset of 22 isolates were analyzed to determine its haplotype identity. Ptr isolates from Tunisia were grouped in races 2, 4, 5, and 7, and 44% of the tested isolates did not fit under any known race, and were denoted here as atypical. These atypical isolates induced the same symptoms as race 7 isolates, extensive necrosis, and chlorosis on susceptible genotypes, but lacked the ToxA gene. ToxA is the only identified necrosis-inducing effector in Ptr, and was amplified in 51% of tested isolates, and shared identical sequence to previously identified haplotype (H15). ToxB and its homolog toxb were present in 97% and 93% of tested isolates, respectively. Ptr in Tunisia lacked Ptr ToxC activity, and none of the tested isolates induced the specific symptoms of that effector. Race 7 and the atypical isolates dominated the Tunisian Ptr population, while races 2, 4, and 5 were found at low percentages. In conclusion, ToxB and its homolog were the most dominant genes in Ptr from Tunisia, and the majority of the isolates induced necrosis and chlorosis on Ptr ToxA and Ptr ToxB susceptible wheat genotypes. However, only about half of that necrosis can be attributed to ToxA presence, this result necessitates further research to investigate the prevalence of additional necrotic effector(s). Terminology: in this paper, Pyrenophora tritici-repentis abbreviated as Ptr, the effectors are referred to by Ptr ToxA, Ptr ToxB and Ptr ToxC, and the genes coding for them are written in italic as ToxA, ToxB, and ToxC, respectively.
| S-EPMC6930679 | biostudies-literature
Project description:Water and ToxB-infiltrated leaves of sensitive cultivar Katepwa were collected at 0,3,9,14,24,48 hours post infiltration (hpi), total RNA was extracted and processed. Expression levels were obtained using Affymetrix GeneChip Wheat Genome Array
2012-09-01 | E-MTAB-963 | biostudies-arrayexpress