Evolution of the Toxb Gene in Pyrenophora tritici-repentis and Related Species.

Pyrenophora tritici-repentis (tan spot) is a destructive foliar pathogen of wheat with global impact. This ascomycete fungus possesses a highly plastic open pangenome shaped by the gain and loss of effector genes. This study investigated the allelic variations in the chlorosis-encoding gene ToxB across 422 isolates representing all identified pathotypes and worldwide origins. To gain better insights into ToxB evolution, we examined its presence and variability in other Pyrenophora spp. A ToxB haplotype network was constructed, revealing the evolutionary relationships of this gene (20 haplotypes) across four Pyrenophora species. Notably, toxb, the homolog of ToxB, was detected for the first time in the barley pathogen Pyrenophora teres. The ToxB/toxb genes display evidence of selection that is characterized by loss of function, duplication, and diverse mutations. Within the ToxB/toxb open reading frame, 72 mutations were identified, including 14 synonymous, 55 nonsynonymous, and 3 indel mutations. Remarkably, a, ∼5.6-kb Copia-like retrotransposon, named Copia-1_Ptr, was found inserted in the toxb gene of a race 3 isolate. This insert disrupted the ToxB gene's function, a first case of effector gene disruption by a transposable element in P. tritici-repentis. Additionally, a microsatellite with 25 nucleotide repeats (0 to 10) in the upstream region of ToxB suggested a potential mechanism influencing ToxB expression and regulation. Exploring ToxB-like protein distribution in other ascomycetes revealed the presence of ToxB-like proteins in 19 additional species, including the Leotiomycetes class for the first time. The presence/absence pattern of ToxB-like proteins defied species relatedness compared with a phylogenetic tree, suggesting a past horizontal gene transfer event during the evolution of the ToxB gene. [Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB.

BACKGROUND: In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements. RESULTS: A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr's adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr's effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb 'Starship' transposon (dubbed 'Horizon') with a clearly defined target site and target site duplications. 'Horizon' was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative 'Starship' (dubbed 'Icarus') in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and 'Icarus' were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as 'one-compartment' based on calculated gene distances and evolutionary rates. CONCLUSIONS: These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

Diversity of Fusarium spp. Associated with Wheat Node and Grain in Representative Sites Across the Western Canadian Prairies.

Fusarium head blight (FHB) and Fusarium crown and root rot (FCRR) are major wheat diseases. Populations of FHB and FCRR pathogens are highly dynamic, and shifts in these populations in different regions is reported. Analyzing fungal populations associated with wheat node and grain tissues collected from different regions can provide useful information and predict diseases that might affect subsequent crops and effective disease management practices. In this study, wheat node and grain samples were collected from four representative sites across the western Canadian prairies in the 2018 growing season to characterize the major Fusarium spp. and other mycobiota associated with wheat in these regions. In total, 994 fungal isolates were recovered, and based on culture and molecular diagnostic methods, three genera constituted over 90% of all fungal isolates, namely Alternaria (39.6%), Fusarium (27.8%), and Parastagonospora (23.9%). A quantitative PCR (qPCR) diagnostic toolkit was developed to quantify the most frequently isolated Fusarium spp. in infected wheat tissues: Fusarium avenaceum, F. culmorum, F. graminearum, and F. poae. This qPCR specificity was validated in silico, in vitro, and in planta and proved specific to the target species. The qPCR results showed that F. graminearum was not detected frequently from wheat node and grain samples collected from four locations in this study. F. poae was the most abundant Fusarium species in grain samples in all tested locations. However, in node samples, F. culmorum (Beaverlodge and Scott) and F. avenaceum (Lacombe and Lethbridge) were the most abundant species. Trichothecene genotyping showed that the 3ADON is the most dominant trichothecene genotype (68%), followed by type-A trichothecenes (29.5%), whereas the 15ADON trichothecene genotype was least dominant (2.5%) and the NIV genotype was not detected. Moreover, a total of 129 translation elongation factor 1-alpha (TEF1α) sequences from nine Fusarium spp. were compared at the haplotype level to evaluate genetic variability and distribution. F. avenaceum and F. poae exhibited higher diversity as reflected by higher number of haplotypes present in these two species compared with the rest.

The Changing Virulence of Stripe Rust in Canada from 1984 to 2017.

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, is an important wheat disease worldwide. In this study, the P. striiformis f. sp. tritici population in Canada, representing a time period from 1984 to 2017, was analyzed for virulence diversity and geographical distribution. Virulence of 140 P. striiformis f. sp. tritici isolates was evaluated on 17 near-isogenic wheat lines in the 'Avocet S' background, each containing a single resistance gene along with an 18th line 'Tyee'. Seedlings were inoculated with a urediniospore/talc mixture and infection types were evaluated on a scale of 0 to 9. In total, 89 races were identified with various combinations of defeated Yr genes. Clear changes in pathogen virulence have been observed through time that are confirmed by clustering algorithms. The results showed that the tested P. striiformis f. sp. tritici isolates remained avirulent on Yr1, Yr5, and Yr15, and have very low frequency of virulence on Yr76, but had high frequencies of virulence on Yr6, Yr7, Yr8, Yr9, Yr17, Yr43, Yr44, YrTr1, and YrExp2. P. striiformis f. sp. tritici virulence spiked on Yr7, Yr8, and Yr9 for the first time in 2000, and on Yr10 and Yr27 in 2010. Overall, the predominant races in Canada were very similar to those reported in the United States (PSTv-37, PSTv-41, and PSTv-52), which indicates long-distance migration of P. striiformis f. sp. tritici from the United States to Canada. Sixty-four races had unique virulence combinations that had not been previously reported in the United States, which suggested that evolution of virulence/avirulence for host resistance by mutation at local scale, is possible. Analysis of diversity between Canadian isolates and races from the United States since 2010 showed that the P. striiformis f. sp. tritici population in western Canada is similar to that in the western states of the United States, and that the population in eastern Canada is similar to the eastern and/or central regions of the United States, supporting the hypothesis that specific P. striiformis f. sp. tritici populations in North America travel through different wind trajectories.

Parastagonospora nodorum and Related Species in Western Canada: Genetic Variability and Effector Genes.

Parastagonospora nodorum is an important fungal pathogen that causes Septoria nodorum blotch (SNB) in wheat. This pathogen produces several necrotrophic effectors that act as virulence factors; three have been cloned, SnToxA, SnTox1, and SnTox3. In this study, P. nodorum and its sister species P. avenaria f. tritici (Pat1) were isolated from wheat node and grain samples collected from distanced sites in western Canada during 2018. The presence of effector genes and associated haplotypes were determined by PCR and sequence analysis. An internal transcribed spacer-restriction fragment length polymorphism test was developed to distinguish between leaf spotting pathogens (P. nodorum, Pat1, Pyrenophora tritici-repentis, and Bipolaris sorokiniana). P. nodorum was mainly recovered from wheat nodes and to a lesser extent from the grains, while Pat1 was exclusively isolated from grain samples. The effector genes were present in almost all P. nodorum isolates, with the ToxA haplotype 5 (H5) being most prevalent, while a novel ToxA haplotype (denoted here H21) is reported for the first time. In Pat1, only combinations of SnTox1 and SnTox3 genes were present. A ToxA haplotype network was also constructed to assess the evolutionary relationship among globally found haplotypes to date. Finally, cultivars representing wheat development in Canada for the last century were tested for sensitivity to Sn-effectors and to the presence of Tsn1, the ToxA sensitivity gene. Of tested cultivars, 32.9 and 56.9% were sensitive to SnTox1 and SnTox3, respectively, and Tsn1 was present in 59% of the cultivars. In conclusion, P. nodorum and Pat1 were prevalent wheat pathogens in Canada with a potential tissue-specific colonization capacity, while producing necrotrophic effectors to which wheat is sensitive.

Identification of a Locus Conferring Dominant Susceptibility to Pyrenophora tritici-repentis in Barley.

The fungus Pyrenophora tritici-repentis (Ptr) causes tan spot, a destructive foliar disease of wheat worldwide. The pathogen produces several necrotrophic effectors, which induce necrosis or chlorosis on susceptible wheat lines. Multiple races of Ptr have been identified, based on their ability to produce one or more of these effectors. Ptr has a wide host range of cereal and non-cereal grasses, but is known to cause damage only on wheat. Previously, we showed that Ptr can interact specifically with cultivated barley (Hordeum vulgare ssp. vulgare), and that the necrotrophic effector Ptr ToxB induces mild chlorosis in a highly selective manner when infiltrated into certain barley genotypes. In the present study, a barley doubled-haploid (DH) population was evaluated for reaction to Ptr race 5, a Ptr ToxB-producer. Then a comprehensive genetic map composed of 381 single nucleotide polymorphism (SNP) markers was used to map the locus conditioning this chlorosis. The F1 seedlings, and 92 DH lines derived from a cross between the resistant Japanese malting barley cultivar Haruna Nijo and the susceptible wild barley (H. vulgare ssp. spontaneum) OUH602 were inoculated with a conidial suspension of Ptr race 5 isolate at the two-leaf stage. The seedlings were monitored daily for symptoms and assessed for chlorosis development on the second leaf, 6 days after inoculation. All tested F1 seedlings exhibited chlorosis symptoms similar to the susceptible parent, and the DH lines segregated 1:1 for susceptible:resistant phenotypes, indicating the involvement of a single locus. Marker-trait linkage analysis based on interval mapping identified a single locus on the distal region of the short arm of chromosome 2H. We designate this locus Susceptibility to P. tritici-repentis1 (Spr1). The region encompassing this locus has 99 high confidence gene models, including membrane receptor-like kinases (RLKs), intracellular nucleotide-binding, leucine-rich repeat receptors (NLRs), and ankyrin-repeat proteins (ANKs). This shows the involvement of a dominant locus conferring susceptibility to Ptr in barley. Further work using high-resolution mapping and transgenic complementation will be required to identify the underlying gene.