Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

Abstract

Background: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis.

Results: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species.

Conclusion: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.

Figure 1

Expression of miR168 and AGO1 in Arabidopsis plants treated with elicitors from F. oxysporum . (A) Stem-loop RT-qPCR analysis of miR168. RNAs were prepared from Arabidopsis plants treated with fungal elicitors for the indicated periods of time, and from control mock-inoculated plants (RNAs from samples harvested at 5, 30, 60 and 120 min were also used for microarray analysis). (B) qRT-PCR analysis of AGO1 using Ubiquitin10 (At5g65080) as the internal control. RNAs samples were the same as in (A). Results shown are from one of three independent experiments that gave similar results. Erro bars show the standard error. Asterisks indicate a significant difference between conditions (*, P ≤ 0.05 ; **; ≤0.01). c, control plants. e, elicitor-treated plants.

Figure 2

MiR168 activity in Arabidopsis plants revealed by GFP fluorescence patterns tissues of GFP -miR168* sensor plants. (A) Schematic representation of the miR168 sensor construct containing the GFP mRNA with a site complementary to miR168 (GFP-miR168*). (B) Plants constitutively expressing the GFP gene. Ten day-old plants were treated for 30 min with elicitors obtained from the fungus F. oxysporum. Water was used as mock control. Results obtained in elicitor-treated GFP-Arabidopsis plants are presented (similar patterns were observed in non treated GFP-Arabidopsis plants). GFP fluorescence images are shown. (C) Analysis of miR168 activity in control, non treated GFP-miR168* plants (sde1 background). Bright-field (left) and GFP fluorescence (right) images are shown. (D) miR168 activity in elicitor-treated GFP-miR168* Arabidopsis plants. Bright-field (left) and GFP fluorescence (right) images are shown. No GFP fluorescence was evident in roots of non treated plants due to miR168 guided silencing.

Figure 3

Expression of miR168 precursors and structural features of the MIR168 promoter. (A) qRT-PCR analysis of pre-miR168a (left panel) and pre-miR168b (right panel) expression in response to elicitor treatment. The relative expression level in comparison to the corresponding non treated controls is given for each time point (elicitor vs control non treated plants). Error bars represent the mean ± SD of two biological replicates and three technical replicates for each biological replicate (*, P ≤ 0.05 ; **; ≤0.01). All values were normalized against Ubiquitin. (B) Structural features of the MIR168a promoter from Arabidopsis. The location of known cis-acting elements is shown (for details on cis-elements, see Additional file 1: Table S2).

Figure 4

Functional analysis of the MIR168a promoter in transgenic Arabidopsis. (A) Schematic diagrame of the MIR168a promoter construct. (B) Arabidopsis plants constitutively expressing GFP. Ten day-old plants were treated with fungal elicitors for 30 min. Water was used as mock control. GFP fluorescence images are shown. (C) Control non treated pMIR168::GFP plants. Bright-field (left) and GFP fluorescence (middle and right) images are shown. (D) Elicitor-treated pMIR168::GFP plants Bright-field (left) and GFP fluorescence (middle and right) images are shown.

Figure 5

Genetic requirements for generation of the 24-nt hc-siRNA415. (A) Analysis of mutants impaired in small RNA biogenesis, dcl and rdr mutants. The same blot was successively hybridized, stripped, and re-hybridized to oligonucleotide probes corresponding to the complementary sequence of the indicated small RNAs. RNA blots were also probed with the U6 probe for loading control. (B) Analysis of ago4, nrpd2 (common to Pol IV and Pol V) and nrpe1 (Pol V) mutants. (C) Small RNA blot analysis of the hc-siRNA415 in different plant species.