Abstract
MicroRNAs (miRNAs) are a group of small non-coding endogenous RNAs. In plants, miRNAs play vital functions in regulating growth, development, and stress response. However, the role of miRNAs in Arabidopsis-Phytophthora capsici (P. capsici) model pathosystem is poorly understood. Here, we used a high-throughput sequencing approach to identify pathogen-responsive miRNAs using 15 small RNA (sRNA) libraries prepared from Arabidopsis thaliana leaves collected at 0, 3, 6, 12, and 24 h post-inoculation with P. capsici. A total of 293 known miRNAs and 6 potential novel sRNAs (miRNAs or siRNAs) were identified, of which 33 miRNAs were differentially expressed at four different infection stages. To verify the reliability of the sRNA-seq results, we investigated the expression of five sRNAs upregulated throughout the four infection stages and their potential target genes using northern blot analysis and/or stem-loop quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the potential target genes of the differentially expressed miRNAs, both conserved and novel, were enriched in pathways such as starch and sugar metabolism, spliceosome, and plant-pathogen interaction, indicating that the splicing machinery and pathogenesis-related (PR) proteins play important roles in the response to P. capsici infection. Taken together, these results provide novel insights into the molecular mechanisms of pathogenesis by P. capsici. Additionally, these results will serve as a strong foundation for further in-depth analysis of miRNAs involved in the resistance to Phytophthora species in other crops.
Highlights
MicroRNAs are a class of endogenous non-coding single-stranded small (21–22 nt) RNAs commonly found in eukaryotes (Reinhart et al, 2002)
Arabidopsis ecotype Col-0 was used in this study because it is highly susceptible to P. capsici isolate LT263
To identify miRNAs most likely involved in Arabidopsis immunity against P. capsici, we performed a thorough evaluation of the infection process of P. capsici isolate LT263 in Arabidopsis Col-0 leaves; Col-0 was selected for this experiment because it is highly susceptible to P. capsici (Wang et al, 2013)
Summary
MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded small (21–22 nt) RNAs commonly found in eukaryotes (Reinhart et al, 2002). Endogenous MIR genes are transcribed to form the primary transcript, pre-miRNA, which is gradually processed in the nucleus in an ATP-dependent manner to first form a pre-miRNA with a hairpin structure and form an miRNA:miRNA∗ double-stranded complex after methylation (Liu et al, 2018). The first layer of immunity is based on the perception of pathogen-associated molecular patterns (PAMPs) and is known as PAMP-triggered immunity (PTI), which prevents a large number of potential pathogenic microbes from invasion (Jones and Dangl, 2006). Recent studies demonstrate that small RNAs (sRNAs) are involved in both PTI and ETI in plants (Padmanabhan et al, 2009)
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