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. 2018 Oct 31;13(10):e0206586.
doi: 10.1371/journal.pone.0206586. eCollection 2018.

Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element

Paula M Moolhuijzen  1 , Pao Theen See  1 , Richard P Oliver  1 , Caroline S Moffat  1
Affiliations

Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element

Paula M Moolhuijzen et al. PLoS One. .

Abstract

The ToxA effector is a major virulence gene of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot disease of wheat. ToxA and co-located genes are believed to be the result of a recent horizontally transferred highly conserved 14kb region a major pathogenic event for Ptr. Since this event, monitoring isolates for pathogenic changes has become important to help understand the underlying mechanisms in play. Here we examined ToxA in 100 Ptr isolates from Australia, Europe, North and South America and the Middle East, and uncovered in isolates from Denmark, Germany and New Zealand a new variation, a novel 166 bp insertion element (PtrHp1) which can form a perfectly matched 59 bp inverted repeat hairpin structure located downstream of the ToxA coding sequence in the 3' UTR exon. A wider examination revealed PtrHp1 elements to be distributed throughout the genome. Analysis of genomes from Australia and North America had 50-112 perfect copies that often overlap other genes. The hairpin element appears to be unique to Ptr and the lack of ancient origins in other species suggests that PtrHp1 emerged after Ptr speciation. Furthermore, the ToxA UTR insertion site is identical for different isolates, which suggests a single insertion event occurred after the ToxA horizontal transfer. In vitro and in planta-detached leaf assays found that the PtrHp1 element insertion had no effect on ToxA expression. However, variation in the expression of ToxA was detected between the Ptr isolates from different demographic locations, which appears to be unrelated to the presence of the element. We envision that this discovery may contribute towards future understanding of the possible role of hairpin elements in Ptr.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Gel electrophoresis showing variation of the ToxA gene amplicon sizes between Ptr isolates from various demographic locations.
Top gel shows, a larger than expected product size of approximately at 1 kb amplified in three isolates (EW4-4, SN001C and M14d). Isolates EW306-2-1 and M4 amplified the expected product sized of 832 bp, while ToxA was not detected in EW7m1. No template was used as a negative control. Bottom gel shows DNA amplification of a 490 bp region that is unique in Ptr genome [16] that was included as a positive control.
Fig 2
Fig 2. The ToxA insertion element PtrHp1 (166 bp) has a secondary hairpin structure.
Fig 3
Fig 3. P. tritici-repentis ToxA region nucleotide alignment.
(A) The sequence plot of EW4-4 (horizontal axis) and M4 (vertical axis) shows a deletion site in M4 (Contig1 5,732,328–5,732,329 bp) and an insertion (166bp) in EW4-4 between 1,484 and 1,649 bp inclusively. An EW4-4 inverted repeat downstream of the insertion site is visible at sequence positions 1,930 to 2,094bp. Under the dot plot an overview of the ToxA region (nucleotide multiple sequence alignment) shows the 3’UTR 166 bp insertion for isolates EW4-4, M14d and SN001C. Sequence alignment homology is shown (blue) and deletion (dotted line). *Asterisk indicates ToxA variants. (B) Ptr M4 ToxA and Parastagonospora nodorum Tp transposase (orthologue of SNOG16572) genes aligned to EW4-4 nucleotide region (4 kb) show the downstream inverse repeat (IR) position. ToxA coding sequence is CDS is shown in green and mRNA in blue.
Fig 4
Fig 4. Predicted secondary structure of the intergenic inverse repeat (IR).
The figure shows the predicted secondary structure of the intergenic inverse repeat found downstream of the ToxA PtrHp1 element insertion site.
Fig 5
Fig 5. Culture filtrate activity of Ptr isolates with or without the PtrHP1 element on the ToxA sensitive wheat variety Yitpi.
* Isolates with the PtrHp1 element; Δ Isolates without the ToxA locus. Photographs were taken 10 days post-infiltration.
Fig 6
Fig 6. In planta expression of ToxA during infection on Yitpi Tsn1 detached leaves assay.
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Grants and funding

This project was generously supported by the Grains Research and Development Corporation (GRDC) https://grdc.com.au/ and Curtin University https://www.curtin.edu.au/, project code CUR00023. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.