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

Abstract

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.

Figure 1. Size distribution and 5′ nucleotide preferences of sRNAs mapped to transposons, RxLR and CRN effector gene subsets in P. infestans isolates R0 and 3928A.

Abundance of each size class of sRNAs based on nucleotide (nt) length in: A. Transposons B. RxLRs and C. CRNs. The relative frequency of each 5′ terminal nucleotide of sRNAs aligned to: D. Transposons E. RxLRs and F. CRNs.

Figure 2. Northern hybridizations detecting P. infestans antisense sRNAs derived from transposons, RxLR and CRN effector genes in R0 and 3928A isolates.

A. sRNAs hybridizing to Copia3-LTR, PITG_22969 (CRN), Crypton6, PiAvrblb1 (RxLR) were detected. Loading controls (U4 spliceosomal RNA) are shown below each autoradiograph. B. Determination of 5′ terminal modifications to sRNAs. From left, terminator exonuclease (TE) and tobacco acid pyrophosphatase (TAP) treatment of 21 and 32 nt sRNAs from Copia 3-LTR and Crypton6, respectively in isolate R0. C. ß-elimination assay for 3′ modifications to sRNAs, determined for 21 nt sRNAs for Copia3-LTR in isolate R0. Lane +β is after treatment with periodate, showing a 1 nt downward shift in size compared to the untreated sample (−β).

Figure 3. Relative transcript abundance (qRT-PCR) of RxLR and CRN effector genes at different infection time points in P. infestans isolates R0 and 3928A.

A. PiAvr3a, B. PiAvr1, C. PiAvrblb1, D. PiAvrblb2, E. PITG_06308, F. PiAvr3b, G. PiAvr4, H. PITG_23226, I. PITG_14783, J. PITG_22969. A–I encode RxLR effectors; J encodes a CRN effector. The transcript profiles are shown at 24, 48 and 72 h post-inoculation of potato cultivar Bintje (no known resistance genes) relative to the mRNA level in cultured non-sporulating mycelium (M). In each graph, the light grey bar represents R0, and the dark bar represents 3928A. All calculations and statistical analyses were carried out as described in . Error bars represent confidence intervals calculated using three technical replicates for each sample within the qRT-PCR assay. The abundance of mRNA for each gene is shown as a proportion of the actin A (PiactA) transcript on the y-axis of each graph. Amplifications repeated on independent occasions with different starting RNA and cDNA samples resulted in similar transcript accumulation profiles for all genes tested.

Figure 4. DCL-dependent generation of 21 nt sRNAs, and AGO involvement in 32 nt sRNA generation.

A. Copia3-LTR 21 nt sRNAs in PiDcl1 (D1t7, D1t8) and PiRnh5 (D2t1) silenced lines. An upward mobility shift to approximately 24/25 nt was observed for the most silenced PiDcl1 line, D1t8. B. PITG_22969 (CRN) 21 nt sRNAs are abolished upon silencing of PiDcl1 (D1t7, D1t8) or PiRnh5 (D2t1). C. Crypton6 32 nt sRNAs in PiDcl1 (D1t7, D1t8) and PiRnh5 (D2t1) silenced lines. Silencing of these genes had no effect on the accumulation of 32 nt sRNAs. D. PiAvrblb1 32 nt sRNAs in PiDcl1 (D1t7, D1t8) and PiRnh5 (D2t1) silenced lines. Silencing of these genes had no effect on the accumulation of 32 nt sRNAs. E and F. PiAvrblb1 32 nt sRNAs in PiAgo1, 4, or 5 (A1b, A4a, A5c) silenced lines. The accumulation of 32 nt sRNAs is strongly reduced in PiAgo4 or 5 lines, while over-accumulation occurs for PiAgo1. Silenced lines used were PiDcl1 (Dicer-like; D1t8, t7), PiRnh5 (Dicer-like helicase; D2t1), PiAgo1 (Argonaute 1; A1b) PiAgo4 (Argonaute 4; A4a), and PiAgo5 (Argonaute 5; A5c). Loading controls (U4 spliceosomal RNA) are shown below each autoradiograph.

Figure 5. Proposed model for sRNA pathways in P. infestans.

A. siRNA pathway. Transcription of repeat regions or transposons, heterochromatic regions or natural antisense transcripts and exogenous dsRNAs will lead to formation of long precursor dsRNAs. PiDcl1 processes these dsRNA into predominantly 21 nt siRNAs, in some instances in association with PiRnh5 (Dicer-like helicase). The 21 nt siRNAs generated by PiDcl1 associate with one of the PiAgo proteins, leading to degradation of target mRNAs. B. Long siRNA (lsiRNA) pathway. lsiRNAs are generated by PiAgo4 and 5 from transposons, coding genes, or overlapping regions of antisense transcription, or dsRNA generated by RNA polymerases. This may be a PiDcl1 independent pathway. Both pathways could be linked via one of the AGOs to heterochromatin formation aided by histone deacetylases and chromodomain proteins. RdR could further amplify the silencing.

Figure 6. Secondary structure predictions of six miRNA candidates in P. infestans.

Precursor miRNA sequences were folded with the RNAfold program. The miRNA sequences are shown in red.