Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis

Abstract

Background: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots.

Results: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity.

Conclusions: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.

Fig. 1

The size distribution of sequenced small RNA reads. The size class distribution of redundant and non redundant small RNA sequences in the twelve tissue samples. The percentage of the different size classes (y-axis) and the different tissues, which includes seedling, root, leaf, stem and flower with biological replicates and BTI seedling and leaf cDNA libraries (x-axis) represented in N. benthamiana

Fig. 2

Conserved and other known miRNAs in N. benthamiana. Normalized read numbers of conserved and other known miRNAs across the tissues included in this study. Expression profiles are expressed in reads per million (RPM) genome matching reads. Heat map colours represents absolute normalized levels of miRNA expression ranging from less than 1 RPM (white) to more than 1000 RPM (red) as indicated in the colour key. We have identified 40 known or conserved miRNA families in the twelve sRNA libraries generated. All of the deeply conserved miRNA families (miR156/157, miR159/319, miR160, miR165/166, miR171, miR408, miR390/391 and miR395) were present in our data sets. The expression levels of different miRNA families were different and we also observed clear tissue-specific expressional changes within some miRNA families as it was expected

Fig. 3

Size distribution and starting nucleotide of the conserved and other known miRNAs. The relative abundance of different size categories (a), from 20 to 24 nucleotides is shown for the conserved and other known miRNAs presented in Fig. 2. The relative abundance of the 5′-nucleotide (b) is shown for the conserved and other known miRNAs presented in Fig. 2. In 31 miRNA families, 21 nt long miRNAs were the most significant size class. In 32 of the 40 conserved miRNA families, the majority of sequences started with uracyl at their 5′-end, although a portion of these have a different nucleotide composition at position 1 to a variable degree. Three miRNA families exhibited a preference for an adenine at position 1

Fig. 4

Expression analysis of selected conserved and other known miRNAs in different N. benthamiana tissues. Total RNA was extracted from different tissues including, seedling (Se), root (R), leaf (L), stem (St), flower (F) from N. benthamiana plants used in our laboratory and from plants from Boyce Thompson Institute (leaf^BTI - L^BTI, seedling^BTI - Se^BTI). The RNA was separated on PAGE and transferred to nylon membranes for Northern blot analysis of the miRNAs. Oligonucleotide probes were used to detect specific miRNAs, and an U6-specific probe was used to detect U6 RNA as a loading control for each membrane

Fig. 5

Novel miRNAs in N. benthamiana. Normalized read numbers of novel miRNAs across the tissues included in this study. Expression profiles are expressed in reads per million genome matching reads. Heat map colours represents absolute normalized levels of miRNA expression ranging from less than 1 RPM (white) to more than 1000 RPM (red) as indicated in the colour key

Fig. 6

Size distribution and starting nucleotide of the novel N. benthamiana specific miRNAs. The relative abundance of different size categories (a), from 20 to 24 nucleotides is shown for the novel miRNAs presented in Fig. 5. The relative abundance of the 5′-nucleotide (b) is shown for the novel miRNAs presented in Fig. 5

Fig. 7

Expression patterns of novel miRNAs found in N. benthamiana. Total RNA was extracted from different tissues including, seedling (Se), root (R), leaf (L), stem (St), flower (F) from N. benthamiana plants used in our laboratory and from plants from Boyce Thompson Institute (leaf^BTI - L^BTI, seedling^BTI - Se^BTI). The RNA was separated on PAGE and transferred to nylon membranes for Northern blot analysis of the novel miRNAs. Oligonucleotide probes were used to detect specific miRNAs, and an U6-specific probe was used to detect U6 RNA as a loading control for each membrane