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.     

The Stagonospora nodorum-wheat pathosystem involves multiple proteinaceous host-selective toxins and corresponding host sensitivity genes that interact in an inverse gene-for-gene manner

We recently showed that the wheat pathogen Stagonospora nodorum produces proteinaceous host-selective toxins (HSTs). These toxins include SnTox1 as well as SnToxA, a HST first identified from Pyrenophora tritici-repentis that was implicated in a very recent horizontal gene transfer event from S. nodorum to P. tritici-repentis. Compelling evidence implicating SnToxA and SnTox1 in disease development has been obtained. Here, we report the partial purification and characterization of a third HST designated SnTox2, as well as the genetic characterization of the corresponding host-sensitivity gene. SnTox2 was protease sensitive and is estimated between 7 and 10 kDa in size. Sensitivity to SnTox2 was conferred by a single dominant gene designated Snn2, which mapped to the short arm of wheat chromosome 2D. Genetic analysis of reaction to conidial inoculations in a segregating wheat population indicated that both the Snn2-SnTox2 and the Tsn1-SnToxA interactions were involved in disease development, and together they accounted for the majority of the phenotypic variation. Therefore, S. nodorum produces multiple toxins that rely on specific interactions with host gene products to cause disease. The identification of multiple HST-host gene interactions important for disease development and the availability of the S. nodorum whole genome sequence indicate the potential for this pathosystem to serve as a toxin-based, inverse gene-for-gene model.     

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

Tumor‐targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)‐targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC‐10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co‐expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile. Biotechnol. Bioeng. 2016;113: 2698–2711. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.