Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection

Abstract

Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation.

Keywords: gma-miR2606b; gma-miR4416; miRNA; nodulation; root hair; soybean.

Figure 1

Size distribution of small RNAs identified from mock‐inoculated root hairs (Un_RH) and Bradyrhizobium japonicum‐infected root hairs (IN_RH) and stripped roots. Reads were normalized to transcripts per million. The size of small RNAs was plotted against frequency in each library. The majority of the tags were 21, 22 and 24 nucleotides in length.

Figure 2

Venn diagram of number of miRNAs expressed in stripped roots, Un_RH and In_RH. Normalized expression levels of miRNAs from stripped roots, Un_RH and In_RH libraries were compared. Among 86 miRNAs from three libraries, one, two and one miRNAs indentified only in stripped roots, In_RH and Un_RH libraries, respectively.

Figure 3

Volcano plot of differential miRNAs expression between libraries. (a) Differential miRNAs expression between root hairs (In_RH) and stripped roots. (b) Differential miRNAs expression between mock‐inoculated root hairs (Un_RH) and Bradyrhizobium japonicum‐infected root hairs (In_RH). Fold change and P values between libraries were calculated by edgeR method. P values were transformed into negative log10 and miRNAs fold change between libraries was transformed into log2. Negative log10 P values and log2 fold change are on the y‐ and x‐axes, respectively. miRNAs with more than fourfold change and P values <0.05 were shown in blue.

Figure 4

Validation of several rhizobium‐responsive miRNA candidates in root hairs by qRT‐PCR. The expression levels of 11 selected miRNAs were measured by qRT‐PCR (blue column) using cDNAs synthesized from RNA samples collected from In_RH or Un_RH tissues. Three replications were performed. The fold change of each miRNA was calculated by In_RH/Un_RH. The red bar indicates the fold change of each miRNA (In_RH/Un_RH) calculated from Illumina sequencing data.

Figure 5

Ecotopic expression of miR2606b and miR4416 in soybean hairy roots resulted in significant changes of nodule numbers. (a) Relative nodule number formed on soybean hairy roots expressing miR2606b and miR4416 as compared to roots transformed with the empty vector control. Asterisks indicate that the difference is statistically significant at P < 0.05 level. Error bar represents ±SE. (b) GFP‐expressing root nodules in transgenic roots (top) and thin section (bottom) of nodule showing the infected and uninfected cells.

Figure 6

Expression of miRNAs and the corresponding target genes in miRNA overexpression tissues. (a) qRT‐PCR analysis of gma‐miR2606 (in red bar) and its two targets (in white bar) in gma‐miR2606 overexpression tissues. (b) qRT‐PCR analysis of gma‐miR4416 (in red bar) and its six targets (in white bar) in gma‐miR4416 overexpression tissues. Three biological replicates were used. *indicates significant at the 0.05 probability level; ** indicates significant at the 0.01 probability level; *** indicates significant at the 0.005 probability level. Error bar represents ±SE.

Figure 7

Relative expression levels of GmRIP1 (Glyma11g29920) in water mock‐inoculated control (Un_RH) and Bradyrhizobium japonicum‐innoculated root hairs (In_RH). Three replications were performed. Bar represents ±SE. *** indicates significant at the 0.005 probability level. Error bar represents ±SE.