Abstract
Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host–pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25–26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25–26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25–26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25–26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.
Highlights
Small RNAs are typically processed by endoribonuclease III Dicer or Dicer-like protein from double-stranded RNA or single-stranded RNA precursor capable of forming stable stem-loop structure, followed by loading into Argonaute (AGO), the effector of RNA-induced silencing complex (RISC) and RNAinduced transcriptional silencing complex (RITS) (Ghildiyal and Zamore, 2009)
The most famous one is microRNA (Ameres and Zamore, 2013; Alptekin et al, 2016a). miRNA is well known in animals and plants, but typically absent from other species, few miRNAlike genes have been reported in fungi and oomycetes (Lee et al, 2010; Fahlgren et al, 2013)
For P. parasitica sRNA-associated genes, the gene upstream and downstream regions, and intron regions generated sRNAs at a similar level to the exon regions (Figures 3D,E and Supplementary Figure S6), and 25–26 nt sRNA clusters typically overlapped with more than one gene locus (Figure 4B). These results showed that the 25–26 nt sRNAs were unlikely the products of mRNA-derived double-stranded RNA (dsRNA), and might mediate gene silencing at the transcriptional level
Summary
As important non-coding RNA regulators, small RNAs (sRNAs) have been widely found in eukaryotes and shown to play key roles in developmental regulation, transposon suppression, response to environment, host–pathogen interaction and other diverse biological processes (Katiyar-Agarwal and Jin, 2010; Axtell, 2013; Lucas et al, 2014; Budak et al, 2015; Alptekin et al, 2016b). Small RNAs are typically processed by endoribonuclease III Dicer or Dicer-like protein from double-stranded RNA (dsRNA) or single-stranded RNA precursor capable of forming stable stem-loop structure, followed by loading into Argonaute (AGO), the effector of RNA-induced silencing complex (RISC) and RNAinduced transcriptional silencing complex (RITS) (Ghildiyal and Zamore, 2009). They regulate corresponding target gene expression in a sequence-specific manner. We found that the 25–26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in oomycetes
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