The first genome assembly of fungal pathogen Pyrenophora tritici-repentis race 1 isolate using Oxford Nanopore MinION sequencing

Objectives: The assembly of fungal genomes using short-reads is challenged by long repetitive and low GC regions. However, long-read sequencing technologies, such as PacBio and Oxford Nanopore, are able to overcome many problematic regions, thereby providing an opportunity to improve fragmented genome assemblies derived from short reads only. Here, a necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) isolate 134 (Ptr134), which causes tan spot disease on wheat, was sequenced on a MinION using Oxford Nanopore Technologies (ONT), to improve on a previous Illumina short-read genome assembly and provide a more complete genome resource for pan-genomic analyses of Ptr. Results: The genome of Ptr134 sequenced on a MinION using ONT was assembled into 28 contiguous sequences with a total length of 40.79 Mb and GC content of 50.81%. The long-read assembly provided 6.79 Mb of new sequence and 2846 extra annotated protein coding genes as compared to the previous short-read assembly. This improved genome sequence represents near complete chromosomes, an important resource for large scale and pan genomic comparative analyses

A new PacBio genome sequence of an Australian Pyrenophora tritici-repentis race 1 isolate

Objectives: The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of tan spot a major disease of wheat. We have generated a new genome resource for an Australian Ptr race 1 isolate V1 to support comparative 'omics analyses. In particular, the V1 PacBio Biosciences long-read sequence assembly was generated to confirm the stability of large-scale genome rearrangements of the Australian race 1 isolate M4 when compared to the North American race 1 isolate Pt-1C-BFP. Results: Over 1.3 million reads were sequenced by PacBio Sequel small-molecule real-time sequencing (SRMT) cell to yield 11.4 Gb for the genome assembly of V1 (285X coverage), with median and maximum read lengths of 8959 bp and 72,292 bp respectively. The V1 genome was assembled into 33 contiguous sequences with a of total length 40.4 Mb and GC content of 50.44%. A total of 14,050 protein coding genes were predicted and annotated for V1. Of these 11,519 genes were orthologous to both Pt-1C-BFP and M4. Whole genome alignment of the Australian long-read assemblies (V1 to M4) confirmed previously identified large-scale genome rearrangements between M4 and Pt-1C-BFP and presented small scale variations, which included a sequence break within a race-specific region for ToxA, a well-known necrotrophic effector gene

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

© 2018 Moolhuijzen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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

Retrieving fin-de-siècle women poets: the transformative myths, fragments and voices of Webster, Blind and Levy

The critical recuperation of late nineteenth-century women poets, most still waiting in the margins of the literary canon, has owed significantly to the renovated interest and study of the poetical works of Augusta Webster, Mathilde Blind and Amy Levy (1860-90) by the postmodern reader. One of the reasons for this ‘salvage’ may be that they represent and embody the profound and extraordinary changes encompassing the British fin-de-siècle, in which the transition from the Victorians to the Moderns implied the transformation or reconfiguration of certain myths or (hi)stories and the critical re-use or ‘recycling’ of major literary forms. If, for Webster and Blind, involvement in radical politics (namely, feminism and socialism) certainly implied a stance as outsiders, Blind and Levy were even more set apart by their foreignness, with Levy’s different religion and sexuality increasing the distance even further. With recourse to close reading and cultural critique, this paper will analyse how these three women poets re-use fragments (‘verbal ruins’) of national and international history, as well as classic myth, in order to question and transform the images and representations of man and woman in their respective connections with the world. It will demonstrate that while Webster’s poetry (Dramatic Studies of 1866 and Portraits of 1870) is firmly grounded on social demands and the exploration and dramatization of the nature of female experience, Blind’s epic and dramatic verse (The Ascent of Man of 1889 and Dramas in Miniature of 1891) creates new myths of human destiny, reclaiming the Poet’s role as the singer of the age’s scientific deeds, and Levy’s lyrics (Xantippe of 1881 and A Minor Poet of 1884) signal the New Woman poet’s role as victim of the pressures of emancipation. With the support of critics as Isobel Armstrong, Helen Groth and Angela Leighton, the paper will furthermore discuss the way in which these poets explore the selves that women inherit and create and the languages that re-define them, often through the expansive, public forms of dramatic and narrative verse; through these hybrid and fragmentary forms, Webster, Blind and Levy literally give voice to unspeakable feelings and situations, in which the anomalous and marginal are made central.info:eu-repo/semantics/acceptedVersio

Correction to: PacBio genome sequencing reveals new insights into the genomic organisation of the multi-copy ToxB gene of the wheat fungal pathogen Pyrenophora tritici-repentis

Following publication of the original article [1], it was reported that an update on the location of DW5 ToxB cluster relative to the M4 chromosome 10 fusion event was required. Consequently, the Abstract, Table 2, Fig. 7 and several relevant sections of the article have been updated. The changes are given in this Correction article with the updated text highlighted in bold face. Abstract A total of ten identical ToxB gene copies were identified and based on flanking sequence identity, nine loci appeared associated with chromosome 11 and a single copy with chromosome 5. Chromosome 11 multiple ToxB gene loci were separated by large sequence regions between 31 - 66 kb within larger segmental duplications in an alternating pattern related to loci strand, and flanked by transposable elements. Whole genome comparative analysis between Ptr races 1 and 5 A chromosome fusion between chromosome 10 and 11 (referred to as chromosome 10) in Australian isolate M4 resolved by optical mapping [2] was not observed for DW5, where DW5 contig 8 possessed both 5’ and 3’ telomere motifs (Table 2), which would represent a chromosome (telomere to telomere). Ptr ToxB multiloci analysis In this study, the ToxB loci were located on chromosome 5 and 11, which had assembly sizes of 3.36 and 2.18 Mb respectively, which are close to the previously estimated chromosome sizes by Martinez et. al, (2004). Of the ten ToxB loci, nine appeared to be associated with the smaller chromosome 11 located in the 3’ distal region. A Ptr chromosome noted for a chromosome fusion event for a race 1 isolate M4 [2]. The telomere to telomere support for eleven DW5 chromosomes is similar to the findings for another American race 1 isolate Ptr Pt-1C-BFP [3], unlike the 10 chromosome genome of Australian isolate M4 (1) (Fig. 7). (Table presented.). (Figure presented.). Ptr ToxB patterns of duplication In addition to the positioning of the ToxB duplication within the distal region of chromosome 11, ToxB loci were located equidistant downstream from dimer TnphaT transposases, a familiar gene found coupled to Ptr ToxA and within the horizontally transferred region, also found in Parastagonospora nodorum and Bipolaris sorokiniana [4, 5]

PacBio genome sequencing reveals new insights into the genomic organisation of the multi-copy ToxB gene of the wheat fungal pathogen Pyrenophora tritici-repentis

Background: Necrotrophic effector proteins secreted by fungal pathogens are important virulence factors that mediate the development of disease in wheat. Pyrenophora tritici-repentis (Ptr), the causal agent of wheat tan spot, has a race structure dependent on the combination of effectors. In Ptr, ToxA and ToxB are known proteinaceous effectors responsible for necrosis and chlorosis respectively. While Ptr ToxA is encoded by the single gene ToxA, ToxB has multiple loci in the Ptr genome, which is postulated to be directly related to the level of ToxB production and leaf chlorosis. Although previous analysis has indicated that the majority of the ToxB loci lie on a single chromosome, the exact number and chromosomal locations for all the ToxB loci have not been fully identified. Results: In this study, we have sequenced the genome of a race 5 ToxB-producing isolate (DW5), using PacBio long read technology, and found that ToxB duplications are nested in the complex subtelomeric chromosomal regions. A total of ten identical ToxB gene copies were identified and based on flanking sequence identity, nine loci appeared associated with chromosome 10 and a single copy with chromosome 5. Chromosome 10 multiple ToxB gene loci were separated by large sequence regions between 31 and 66 kb within larger segmental duplications in an alternating pattern related to loci strand, and flanked by transposable elements. Conclusion: This work provides for the first time the full accompaniment of ToxB loci and surrounding regions, and identifies the organization and distribution of ten ToxB loci to subtelomeric regions. To our knowledge, this is the first report of an interwoven strand-related duplication pattern event. This study further highlights the importance of resolving the highly complex distal chromosomal regions, that remain difficult to assemble, and can harbour important effectors and virulence factors

Exploration of wheat and pathogen transcriptomes during tan spot infection

Objectives: The fungus Pyrenophora tritici-repentis is the causal agent of tan spot, a major disease of wheat (Triticum aestivum). Here, we used RNA sequencing to generate transcriptional datasets for both the host and pathogen during infection and during in vitro pathogen growth stages. Data description: To capture gene expression during wheat infection with the P. tritici-repentis isolate M4, RNA datasets were generated for wheat inoculated with P. tritici-repentis (infection) and a mock (control) at 3 and 4 days post-infection, when scorable leaf disease symptoms manifest. The P. tritici-repentis isolate M4 was also RNA sequenced to capture gene expression in vitro at two different growth stages: 7-day old vegetative mycelia and 9-day old sporulating mycelia, to coincide with a latent growth stage and early sporulation respectively. In total, 6 RNA datasets are available to aid in the validation of predicted genes of P. tritici-repentis and wheat. The datasets generated offer an insight into the transcriptomic profile of the host-pathogen interaction and can be used to investigate the expression of a subset of transcripts or targeted genes prior to designing cost-intensive RNA sequencing experiments, that would be best further explored with replication and a time series analysis

On the accuracy of the PFA: analogies between Casimir and electrostatic forces

We present an overview of the validity of the Proximity Force Approximation (PFA) in the calculation of Casimir forces between perfect conductors for different geometries, with particular emphasis for the configuration of a cylinder in front of a plane. In all cases we compare the exact numerical results with those of PFA, and with asymptotic expansions that include the next to leading order corrections. We also discuss the similarities and differences between the results for Casimir and electrostatic forces.Comment: 17 pages, 5 figures, Proceedings of the meeting "60 years of Casimir effect", Brasilia, 200