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

Abstract

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.

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

“…Surveys of Canadian wheat varieties found sensitivity to be present in around 30% of wheat varieties (Tran et al 2017: 24 of 100 cultivars. Lamari et al 2005a: 30 of 86 cultivars), while in a recent screen of 122 Australian varieties just 4% were ToxB sensitive (See et al 2019). Here, screening an AM panel of 480 varieties found ToxB sensitivity to be uncommon in the European germplasm screened, with just 7% possessing a sensitivity score ≥ 1.…”

“…ToxB protein was heterologously expressed via an Escherichia coli SHuffle strain and purified using immobilised metal affinity chromatography (IMAC) as described in See et al (2019). Briefly, the DNA sequence that encodes the mature ToxB protein (GenBank accession PZD27634) (Martinez et al 2001) was cloned into the pET21a(+) (Novagen) vector using the primers ToxB-sigP_F (5′-GGA ATT CCAT ATG AAC TGC GTC GCC AATAT-3′) and ToxB_R (5′-CCG CTC GAG ACA ACG TCC TCC ACT TTG CA-3′) that included the engineered NdeI and XhoI sites (underlined), respectively.…”

“…Briefly, the first leaf of seedlings at growth stage 12 (GS12, Zadoks et al 1974) was infiltrated with 50 µl of ToxB suspension at a concentration of 200 µg/mL, and the extent of infiltration along the leaf marked using a non-toxic pen. Phenotyping of ToxB sensitivity was carried out a week after infiltration and scored using a 0-5 scale, as described by See et al (2019). A score of 0 = no visible symptoms; 1 = slight chlorosis; 2 = full chlorosis; 3 = extensive chlorosis with/without slight necrosis; 4 = chlorosis with necrosis; 5 = full necrosis.…”

“…Sensitivity was scored on a 0 (no sensitivity) to 4 (highly necrotic) scale(See et al 2019), with five replicates per genotype. The relevant major resources associated with each variety are listed.…”

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

“…More recently, Ptr was found to interact specifically with barley, with the interaction mediated by the chlorosis-inducing necrotrophic effector Ptr ToxB (Aboukhaddour and Strelkov, 2016). While the symptoms induced by Ptr on barley were weaker than those on wheat, and a higher concentration of Ptr ToxB was needed to induce chlorosis on the barley (Aboukhaddour and Strelkov, 2016;See et al, 2019), the specificity between Ptr and barley was evident, since chlorosis developed on certain barley genotypes but not on others (Aboukhaddour and Strelkov, 2016). Furthermore, infiltration of Ptr ToxB by itself induced chlorosis on the same barley genotypes rated as susceptible to the producing fungal isolate, but not on genotypes rated as resistant.…”