Exploration of wheat and pathogen transcriptomes during tan spot infection
BMC Research Notes volume 11, Article number: 907 (2018)
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.
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.
The necrotrophic fungal pathogen Pyrenophora tritici-repentis causes tan spot disease of wheat (Triticum aestivum). Tan spot is an economically significant leaf disease, which has a major impact on the wheat industry worldwide. Here, we present exploratory RNA sequence data sets with the following aims: (1) to investigate in planta gene expression of both the host and pathogen during wheat tan spot infection by P. tritici-repentis, (2) to investigate in vitro P. tritici-repentis gene expression during vegetative and sporulating growth stages, and (3) to provide RNA sequencing for bioinformatics support of gene predictions in P. tritici-repentis  and wheat.
In total, six RNA libraries were Illumina HiSeq sequenced to yield 24 and 25 million read pairs respectively for 3 and 4 days post-infection with P. tritici-repentis, 28 and 23 million read pairs respectively for 3 and 4 days post-inoculation of control wheat, and 23 and 26 million read pairs for 7-day old vegetative fungal mycelia and 9-day old sporulating mycelia respectively (Data file 1) (Table 1). The time points were chosen to maximise the appearance of early disease symptoms in planta and capture a latent growth and sporulating growth phase in vitro.
To determine host gene expression during P. tritici-repentis infection, datasets from infected and non-infected leaf samples were individually aligned to the Chinese Spring wheat genome (IWGS V1.0) . Over half of the reads for each dataset mapped to the wheat genome (Data file 1). A total of 33,449 genes (24%) of the 137,056 high-confidence wheat reference genes were detected in both the control and infected groups (Data file 2).
For P. tritici-repentis expression during host infection, datasets from 3 and 4 days post-infection were also individually aligned to the P. tritici-repentis genome of isolate M4 . Only 0.4–0.6% of the sample reads mapped to the genome (Data file 1). A total of 9101 and 9824 transcripts were detected at 3 and 4 days post-infection respectively (Data file 3).
To profile P. tritici-repentis genes expressed at different mycelia growth stages, the in vitro vegetative and sporulating datasets were individually aligned to the M4 genome  with approximately half of the reads in concordant alignment (Data file 1). A total of 10,933 M4 transcripts were expressed in vitro and of these 8548 transcripts were found expressed in both vegetative and sporulating mycelia (Data file 4).
Plant and fungal material
The fully extended leaves of the 2-week-old susceptible wheat (Triticum aestivum) variety Machete were inoculated with the P. tritici-repentis race 1 M4 isolate or a mock control solution . Infected and control leaves were collected at 3 and 4 days post-inoculation (DPI). In vitro M4 samples of vegetative mycelia and sporulating mycelia grown on V8PDA agar  were harvested at 7 days and 9 days respectively. All samples were snap frozen in liquid nitrogen immediately after harvest, and stored at − 80 °C prior to RNA extraction.
RNA extraction and sequencing
RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific, USA), further purified using Zymo-Spin columns (Zymo Research, USA) as per the manufacturer’s guidelines prior to LiCl precipitation. RNA samples were pooled from 3 biological replicates. Isolated RNA was ribo-depleted and sequenced as un-stranded, 100 bp pair-end (PE) reads on an Illumina HiSeq2000 machine. A total of 30.6 Gb of raw sequence of 6 libraries was obtained. Further method details can be found in Supplementary file 1.
Reads were quality checked with FASTQC  and trimmed using TrimmomaticPE V0.32 . The trimmed reads were aligned to the P. tritici-repentis M4 reference genome (NCBI GenBank accession NQIK00000000.1)  and wheat Chinese Spring genome IWGS V1.0  using Bowtie2/TopHat2 version 2.0.9 [6, 7]. Expression analysis was conducted with the Cufflinks package guided by the reference genes for M4 and high confidence genes in wheat .
The data sets generated were pooled from three biological RNA samples and therefore have no replicates for differential expression studies. The downloadable sequence data is stored raw and requires quality filtering before use.
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PM conducted the bioinformatics analysis and wrote the draft manuscript. PTS and CM conducted the molecular analysis. CM and PTS contributed to reviewing and editing this manuscript. CM led the project conceptualization. All authors agree to the publication policies of BMC Data Notes. All authors read and approved the final manuscript.
We thank the Australian Government National Collaborative Research Infrastructure Strategy (NCRIS) for providing access to Pawsey Supercomputing under a National Computational Merit Allocation Scheme (NCMAS), Nectar Research/Pawsey Nimbus Cloud resources and the Australian Wheat Pathogens Consortium, BioPlatforms Australia (BPA).
The authors declare that they have no competing interests.
Availability of data materials
The datasets generated during and/or analysed during the current study are available in the figshare repository . All RNA-seq read data is available through European Nucleotide Archive (ENA) under study primary accession PRJEB29046 or secondary accession ERP111314. The RNA-seq read data can also be accessed from . Direct links to data sets are: Sample M1, 7 day in vitro vegetative mycelia (184.108.40.20650); Sample M2, 9 day in vitro sporulating mycelia (220.127.116.1151); Sample P1, 3 DPI P. tritici-repentis infection in planta (18.104.22.16855); Sample P2, 4 DPI P. tritici-repentis infection in planta (22.214.171.12457); Sample C1, 3 days post-control inoculation in planta (126.96.36.19954); Sample C2, 4 days post-control inoculation in planta (188.8.131.5256).
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Ethics approval and consent to participate
The RNA sequencing was generously supported through BioPlatforms Australia and funded by the Australian Government National Collaborative Research Infrastructure Strategy and Education Investment Fund Super Science Initiative. The analysis, data interpretation and writing of the manuscript were generously funded through the Grains and Research Development Corporation (project code CUR0023).
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Moolhuijzen, P., See, P.T. & Moffat, C.S. Exploration of wheat and pathogen transcriptomes during tan spot infection. BMC Res Notes 11, 907 (2018). https://doi.org/10.1186/s13104-018-3993-2