Fig. 3

Noncoding RNAs are associated with TEs in B. hordei. A We mapped publicly available sRNA-seq datasets obtained from B. hordei-infected barley plants to the TE consensus database using bowtie [41] and identified candidate phasiRNAs using unitas [40]. The donut chart shows the number of consensus TEs to which phasiRNAs were linked (green portion of inner circle), the number of elements accounting for the different TE families (Table 1; second circle), and the number of consensus TEs whose expression peaks at specific time points (Fig. 2; outer circle). B Example of stacked phasiRNAs mapped to the consensus sequence of TE Ty3/mdg4-17 (6,054 bp in length). The TE self-propagation genes are indicated on top (green arrows); these genes encode RT (reverse transcriptase), RNase H, and DNA integrase. The scale below the black horizontal line indicates the TE length and position in bp. Mapped sRNAs are shown in the windows below the size scale for two examples: derived from infected leaf epidermal cells at 120 hpi [15] and derived from infected total leaf material at 24 hpi [39]. Grey blocks indicate single reads; the black boxes on top of each graph display predicted clusters of phasiRNAs. Colored blocks indicate read mismatches with the reference sequence: blue, C; red, T; orange, G; green, A. Data were visualized using Integrative Genomics Viewer v2.9.4 [42]. C The dot plot shows the time course expression patterns of selected consensus TEs using the RNA-seq dataset generated in this study. The y-axis shows the normalized expression expressed as transcripts per million (TPM); the x-axis indicates the respective time point of the asexual life cycle of B. hordei (Fig. 1A). The selected TE consensus elements are indicated on the top-left of each plot. D Example of an antisense lncRNA occurring in the consensus TE Ty1/Copia-63 (5,317 bp in length). The upper lane indicates annotated transcripts on the TE, where TE self-propagation genes are indicated in green (genes encode RT (reverse transcriptase), gag polyprotein, and DNA integrase) and three detected isoforms of associated lncRNAs in orange. The scale below the black horizontal line indicates the TE length and position in bp. The second panel indicates long reads obtained via ONT transcriptome sequencing at 144 hpi; colored lines indicate mismatches between read and reference sequence. The two lower panels show RNA-seq read mappings to this TE as colored lines. Green, stranded reads aligning to the sense strand; Orange, stranded reads aligning to the antisense strand. Grey lines indicate reads split due to predicted splicing events. Data were visualized using Integrative Genomics Viewer v2.9.4 [42]. E We amplified several TE antisense lncRNAs from B. hordei cDNA using sequence-specific primer pairs. The agarose gel shows PCR-amplified lncRNAs (indicated above each lane), arrows indicate the expected PCR products. Expected PCR amplicon sizes were: BLGHnc_000942-RA (Ty3/mdg4-23 antisense), 2,831 bp; BLGHnc_004556-RA (Ty1/Copia-23 antisense), 2,888 bp; BLGHnc_000243-RA (Ty3/mdg4-1 antisense), 1,150 bp; BLGHnc_003729-RB (Ty3/mdg4-9 antisense), 1,065 bp; BLGHnc_000866-RA (Ty3/mdg4-62 antisense), 2,187 bp; BLGHnc_03526-RA (Ty1/Copia-63 antisense), 1,419 bp; BLGHnc_003513-RB (intergenic), 922 bp; BLGHnc_004496-RA (intergenic), 2,128 bp. Bands corresponding to the expected product size were excised from the gel and their sequence identity confirmed by amplicon sequencing. NPC, no primer control. DNA Ladder, 1 kb plus (Invitrogen-Thermo Fisher, Waltham, MA, USA). F The genomic transcript models of BLGHnc_000942-RA, BLGHnc_004556-RA, BLGHnc_000243-RA, BLGHnc_003729-RB, BLGHnc_000866-RA, BLGHnc_03526-RA, BLGHnc_003513-RB, and BLGHnc_004496-RA. Orange blocks represent exons and grey lines spliced introns