Integration of the Pokeweed miRNA and mRNA Transcriptomes Reveals Targeting of Jasmonic Acid-Responsive Genes

Abstract

The American pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of pokeweed treated with jasmonic acid (JA), a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

Prediction and Characterization of miRNA/Target Pairs in Non‐Model Plants Using RNA‐seq

“…3.trinity/Trinity.fasta Predicted miRNA sequences supplied from Neller, Klenov, Guzman, and Hudak (2018): 7.mirdeep.p/predicted_miRNAs.fa Reference file of known plant miRNAs: 7.mirdeep.p/known_miRNAs.fa The following programs will be run in the terminal through the command line.…”

“…Without this step, most reads would derive from rRNA due to its high relative abundance. The decision to use one strategy over the other depends Neller et al (2018). Our study included two conditions: an ethanol control treatment and jasmonic acid (JA) experimental treatment.…”

“…Use the provided file miRNA.counts.matrix, which contains counts of the 582 predicted miRNAs from Neller et al (2018).…”

“…Calculate the R 2 correlation value to quantify the strength of the linear relationship between the two methods. An example of such a plot comparing the fold change of eight JAresponsive miRNAs measured by RNA-seq and qRT-PCR is illustrated in Neller et al (2018).…”

“…To ensure that there is enough information for downstream analyses, we provide predicted_miRNAs.fa. It contains sequences of the 582 predicted miRNAs obtained by running miRDeep-P on the full datasets inNeller et al (2018).The provided reference file of known miRNAs was obtained by downloading all available mature miRNA sequences from the Plant Non-Coding RNA Database (http://structural biology.cau.edu.cn/PNRD/). 7.…”