Fragmentation of tRNA in Phytophthora infestans asexual life cycle stages and during host plant infection

Abstract

Background: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host.

Results: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line.

Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans.

Figure 1

Size distribution of sRNA reads mapping to tRNAs. Shown are total sRNA reads from the mycelium life cycle stage of the three sequenced P. infestans isolates. (A) R0 and 3928A. (B) 88069. The percentages of sense and antisense reads are displayed on the positive and negative y-axes, respectively.

Figure 2

The 5′ end nucleotide base identity of tRNA-mapping sRNAs in different life cycle stages. (A, C, E, G) Isolate R0. (B, D, F, H) Isolate 3928A. Most tRF size classes started with 5′ G. The 27 nt sRNAs most frequently had 5′ U, except for in germinating cysts.

Figure 3

Northern blot detection of sense sRNAs complementary to tRNA. Hybridizations detected sense tRFs from tRNA Ile_cluster0, tRNA Thr_cluster1 and tRNA Arg_cluster0 in (A) wild-type (wt) isolates 3928A and R0, and in (B) wt isolate 88069 and transformant lines silenced for PiDcl1, PiAgo1, PiAgo4, PiAgo5. Approximate sizes in nucleotides are indicated to the right of each blot. The membranes were re-probed for 5S rRNA to control for equal loading of sRNAs (bottom). The signals in (B) were quantified, and values relative to the wt and normalized to 5S rRNA, are shown below each blot.

Figure 4

Sequence read coverage at tRNA clusters. sRNA read counts mapped along tRNA Ile_cluster0, tRNA Thr_cluster1, tRNA Arg_cluster0 and Arg_cluster7 in isolate R0. The profile was very similar in isolate 3928A.

Figure 5

Putative tRNA structures and predicted cleavage sites. The predicted secondary structures of tRNA Ile_cluster0, tRNA Arg_cluster0, tRNA Thr_cluster1 and tRNA Arg_cluster7 are shown. The color code depicts base pair probabilities. Black arrows show 5′ cleavage sites determined by sRNA sequencing while cleavage sites suggested from Northern hybridizations are shown by red arrows for 5′ tRFs, internal (I) tRFs or 3′ tRFs. The lengths of 3′ tRFs are calculated excluding the 3′ CCA.