Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans.

Phytophthora infestans is an oomycete that causes severe damage to potato, and is well known for its ability to evolve rapidly in order to overcome resistant potato varieties. An RNA silencing strategy was evaluated here to clarify if small interfering RNA homologous to selected genes in P. infestans could be targeted from the plant host to reduce the magnitude of the infection. As a proof-of-concept, a hairpin RNA (hp-RNA) construct using the GFP marker gene was designed and introduced in potato. At 72 hpi, a 55-fold reduction of the signal intensity of a corresponding GFP expressing P. infestans strain on leaf samples of transgenic plants, compared with wild-type potato, was detected. This suggests that an RNA interference construct in the potato host could be processed and target a transcript of the pathogen. Three genes important in the infection process of P. infestans, PiGPB1, PiCESA2, and PiPEC, together with PiGAPDH taking part in basic cell maintenance were subsequently tested using an analogous transgenic strategy. Out of these gene candidates, the hp-PiGPB1 targeting the G protein ß-subunit (PiGPB1) important for pathogenicity resulted in most restricted disease progress. Further, Illumina sequencing of inoculated transgenic potato leaves revealed sRNAs of 24/25 nt size homologous to the PiGPB1 gene in the transgenic plants indicating post-transcriptional silencing of the target gene. The work demonstrates that a host-induced gene-silencing approach is functional against P. infestans but is highly dependent on target gene for a successful outcome. This finding broadens the arsenal of control strategies to this important plant disease.

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

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.

Evidence for small RNAs homologous to effector-encoding genes and transposable elements in the oomycete Phytophthora infestans.

Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19-40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.