A new ToxA haplotype in the wheat fungal pathogen Bipolaris sorokiniana.

The necrotrophic effector ToxA is a well-studied virulence factor produced by several fungal necrotrophs. Initially cloned from the wheat tan spot pathogen Pyrenophora tritici-repentis in 1996, ToxA was found almost a decade later in another fungal pathogen Parastagonospora nodorum and its sister species Parastagonospora pseudonodorum. In 2018, ToxA was detected in a third wheat fungal pathogenic species: Bipolaris sorokiniana that causes spot blotch disease. However, unlike the case with P. tritici-repentis and P. nodorum, the ToxA in B. sorokiniana has been investigated in recent years only. In this report, five Australian B. sorokiniana isolates were assessed for the presence of ToxA. Four isolates were found to contain ToxA. While one isolate harboured the previously reported ToxA haplotype sequence (ToxA19), three isolates contain a different haplotype, designated herein as ToxA25 that has a non-synonymous mutation resulting in an amino acid change of glycine to arginine at position 168. Both B. sorokiniana ToxA isoforms, when heterologously expressed in Escherichia coli, exhibited the classic ToxA-necrosis inducing activity on ToxA sensitive Tsn1 cultivars. Preliminary analysis of the B. sorokiniana isolates on Australian wheat cultivars showed that isolates with ToxA19, ToxA25 or ToxA-deficient displayed varying degrees of virulence, with the most aggressive isolates observed for those producing ToxA. Differences in spot blotch disease severity between Tsn1 and tsn1 cultivars were observed, however this was not limited to the ToxA-producing isolates. The overall results suggests that the virulence of the Australian B. sorokiniana isolates is diverse with the significance of ToxA-Tsn1 interactions depending on individual isolates.

A Race Profile of Tan Spot in Australia Reveals Race 2 Isolates Harboring ToxC1.

Tan spot disease is caused by Pyrenophora tritici-repentis (Ptr), one of the major necrotrophic fungal pathogens that affects wheat crops globally. Extensive research has shown that the necrotrophic fungal effectors ToxA, ToxB, and ToxC underlie the genetic interactions of Ptr race classification. ToxA and ToxB are both small proteins secreted during infection; however, the structure of ToxC remains unknown. In line with the recent discovery of the ToxC1 gene that is involved in ToxC production, a subset of 68 isolates collected from the Australian wheat cropping regions were assessed for the presence of all three effectors by pathotyping against four tan spot wheat differential lines and PCR amplification of ToxA, ToxB, and ToxC1. Based on the disease phenotypes, the 68 isolates were grouped into two races with 63 classified as race 1 and five as race 2. A representative selection of each race was tested against eight Australian commercial wheat cultivars and showed no distinction between the virulence levels. Sequencing of ToxA showed that both races had identical gene sequences of haplotype PtrA1. All the race 1 isolates possessed ToxC1 but three race 2 isolates also contained ToxC1 despite being unable to induce a spreading chlorotic symptom on the ToxC differential line. Quantitative trait loci mapping confirmed the absence of the ToxC-Tsc1 association in disease response caused by the ToxC1-containing race 2 isolate; however, ToxC1 expression was detected during plant infection. Altogether, these results suggest that there is a complex regulatory process involved in the production of ToxC within the Australian race 2 isolates.

Genetic analysis of wheat sensitivity to the ToxB fungal effector from Pyrenophora tritici-repentis, the causal agent of tan spot.

KEY MESSAGE: Genetic 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.

Heterologous Expression of the Pyrenophora tritici-repentis Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops.

Here, we evaluate the expression of the proteinaceous effectors ToxA and ToxB, produced by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, which confer tan spot disease susceptibility on wheat. These necrotrophic effectors were expressed in two heterologous systems: Escherichia coli and Pichia pastoris. The E. coli SHuffle system was demonstrated to be superior to P. pastoris in generating high-levels of recombinant proteins that were soluble and stable. In addition, protein extracts from P. pastoris induced non-specific chlorosis on wheat, postulated to be caused by co-purified glucanases secreted by the host. Up to 79.6 µg/ml of ToxB was obtained using the SHuffle system in the absence of the native signal peptide, whilst the ToxA yield was considerably lower at 3.2 µg/ml. Results indicated that a histidine tag at the ToxA C-terminus interfered with effector functionality. Heterologously expressed ToxA and ToxB were tested on a panel of Australian cereals, including 122 varieties of bread wheat, 16 durum, 20 triticale and 5 barley varieties, as well as common plant model species including tobacco and Arabidopsis thaliana. A varying degree of effector sensitivities was observed, with a higher ToxB sensitivity and prevalence in the durum and triticale varieties. ToxB-induced chlorosis was also detected on barley. The heterologous expression of effectors that are easily scalable, will facilitate effector-assisted selection of varieties in wheat breeding programs as well as the investigation of P. tritici-repentis effectors in host and non-host interactions.