The 25-26 nt small RNAs in Phytophthora parasitica are associated with efficient silencing of homologous endogenous genes

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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.

Figures

  • FIGURE 1 | The basic characteristic of small RNAs in P. parasitica. (A) The annotation of small RNAs (sRNAs). (B) The length distribution of sRNAs at stages of vegetative mycelial growth and plant infection (24 hpi). The rRNA- and mitochondrial RNA-derived sRNAs were filtered out. (C) The first base preference of sRNAs. (D) The 3′ 2-nt overhang enrichment analysis. Only the sRNAs derived from 25 to 26 nt sRNA clusters and detected in all three mycelial samples were used for overhang enrichment analysis.
  • FIGURE 2 | The P. parasitica sRNAs are typically derived from both strands with specific length and base preference. (A) The ratios of antisense and sense sRNAs in sRNA clusters. Each point represents a sRNA cluster. (B,C) The length distribution (B) and the first base preference (C) of the strand-specific and non-strand-specific clusters. The strand-specific clusters (II) were defined as the clusters where the ratios of antisense sRNAs were more than 0.9 or less than 0.1. The others were non-strand-specific clusters (I). (D) Accumulation of sense and antisense sRNAs in 25–26 nt sRNA-associated gene loci. (E,F) The length distribution (E) and the first base preference (F) of sense and antisense sRNAs derived from 25 to 26 nt sRNA-associated genes. (G) Accumulation of 21 nt sRNAs derived from the sense and antisense strands of the 21 nt sRNA-associated gene loci. (H,I) The length distribution (H) and the first base preference (I) of 21 nt sRNAs derived from the sense and antisense strands of the 21 nt sRNA-associated gene loci. Only the 30 genes with the most abundant 21 nt antisense sRNAs were used in (G–I). All sRNA reads showed in this figure were normalized based on the mapped times of sRNA reads. Type 1 genes, the 25–26 nt sRNA-associated gene loci. Type 2 genes, the 21 nt sRNA-associated gene loci.
  • FIGURE 3 | The 25–26 nt sRNAs are associated with efficient gene silencing in P. parasitica. (A) Gene expression levels of sRNA-associated genes compared with other genes. (B) Gene expression levels of sRNA-associated genes with different anti-sRNA accumulation levels in P. parasitica and P. infestans. Genes were grouped based on the accumulation levels of anti-sRNAs, which were represented by the normalized anti-sRNA reads and labeled in x axis. The gene numbers and the p-values of Wilcoxon rank sum test were marked in black and blue, respectively. (C) The number of genes without orthologs in P. infestans in sRNA-associated genes. (D) The distribution of sRNAs in the upstream and downstream of the 25–26 nt sRNA-associated genes. The gene body, upstream and downstream regions were divided into 100 bins, respectively, and the sRNA RPKM value of each bin was calculated. (E) The distribution of sRNAs in exon and intron regions of sRNA-associated genes. The exon and intron regions were divided into 100 bins, respectively, and the sRNA RPKM value of each bin was calculated. The genes without introns were filtered out to reduce interference. Type 1 genes, the 25–26 nt sRNA-associated gene loci. Type 2 genes, the 21 nt sRNA-associated gene loci.
  • FIGURE 4 | The levels of gene expression and the accumulation of the 25–26 nt antisense sRNAs in P. parasitica. (A) Heatmap visualization of sRNA and gene expression levels of all P. parasitica genes. (B) Distribution of sRNAs in the Supercontig 7000000185249100 (also named Supercontig_2.1 in the published genome sequence version 2). The values of repeat time, gene FPKM and sRNA RPKM were adjusted and represented by bar length. The values higher than one were adjusted to the sum of its logarithm and one. To calculate repeat time, the genome sequences were split into adjacent 100 bp bins.
  • FIGURE 5 | The 25–26 nt sRNAs are typically derived from gene-sparse and repeat-rich regions in P. parasitica. (A) The 25–26 nt sRNA accumulation levels in the intergenic regions of different sizes. The intergenic regions were grouped based on the length and labeled in x axis. (B) The length distribution of the 5′ and 3′ flanking intergenic regions of 25–26 nt sRNA-associated genes (Type 1). (C) The 25–26 nt sRNA accumulation levels in the 100 bp blocks with different repeat times. (D) The 25–26 nt sRNA accumulation levels of the 100 bp blocks with different distance from the nearest 25–26 nt sRNA-generating repeat blocks. The position of the 25–26 nt sRNA-generating repeat blocks were labeled as “0”. (E) Distances between the 25–26 nt sRNA-associated genes and the upstream and downstream repeats. Up, the distance between the gene and the nearest upstream repeat bin. Down, the distance between the gene and the nearest downstream repeat bin. Up&Down, the distance between the gene and the nearest repeat bin. The genomic sequences were split into adjacent 100 bp blocks in (C–E). The repeat time and sRNA RPKM value of each bin was calculated as described in Materials and Methods.

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Jia, J., Lu, W., Zhong, C., Zhou, R., Xu, J., Liu, W., … Shan, W. (2017). The 25-26 nt small RNAs in Phytophthora parasitica are associated with efficient silencing of homologous endogenous genes. Frontiers in Microbiology, 8(MAY). https://doi.org/10.3389/fmicb.2017.00773

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