A novel Transposable element-derived microRNA participates in plant immunity to rice blast disease

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that direct post-transcriptional gene silencing in plant development and stress responses through cleavage or translational repression of target mRNAs. Here, we report the identification and functional characterization of a new member of the miR812 family in rice (named as miR812w) involved in disease resistance. miR812w is present in cultivated Oryza species, both japonica and indica subspecies, and wild rice species within the Oryza genus, but not in dicotyledonous species. miR812w is a 24nt-long that requires DCL3 for its biogenesis and is loaded into AGO4 proteins. Whereas overexpression of miR812w increased resistance to infection by the rice blast fungus Magnaporthe oryzae, CRISPR/Cas9-mediated MIR812w editing enhances disease susceptibility, supporting that miR812w plays a role in blast resistance. We show that miR812w derives from the Stowaway type of rice MITEs (Miniature Inverted-Repeat Transposable Elements). Moreover, miR812w directs DNA methylation in trans at target genes that have integrated a Stowaway MITE copy into their 3' or 5' untranslated region (ACO3, CIPK10, LRR genes), as well as in cis at the MIR812w locus. The target genes of miR812 were found to be hypo-methylated around the miR812 recognition site, their expression being up-regulated in transgene-free CRISPR/Cas9-edited miR812 plants. These findings further support that, in addition to post-transcriptional regulation of gene expression, miRNAs can exert their regulatory function at the transcriptional level. This relationship between miR812w and Stowaway MITEs integrated into multiple coding genes might eventually create a network for miR812w-mediated regulation of gene expression with implications in rice immunity.

Keywords: Oryza sativa; CRISPR; methylation; microRNA; plant immunity; transposable element.

Figure 1

Validation of the novel miRNA candidate under study as a novel miRNA from rice. The newly identified miRNA is a member of the rice miR812 family, which has been named as miR812w. (a) Structure of the miRNA precursor. The small RNAs identified in small RNA‐seq datasets (Baldrich et al., 2015) mapping to the 5′ and 3′ arm of this precursor (named as miR812w‐5p and miR812w‐3p) are 24‐nt in length and have 2‐nt overhangs at both 3′ ends. RPM, Reads per million. (b) Accumulation of miR812w precursor transcripts in leaves and roots of 21 days‐old rice plants (left panel) as determined by RT‐qPCR. Northern blot detection of miR812w‐5p and miR812w‐3p (right panel) in leaves of 21 days‐old rice plants. A representative blot out of three independent biological replicates is shown. (c) Northern blot analysis of osa‐miR812w in monocotyledonous and dicotyledonous species. (d) Northern blot analysis of osa‐miR812w in cultivated varieties of the genus Oryza (O. sativa, Asian rice; O. glaberrima, African rice) and wild species of the genus Oryza. The small RNA fraction obtained from 100 μg of total RNA was probed with a P³²‐labelled synthetic oligonucleotide complementary to the miR812w‐3p sequence (indicated in Table S4). RNA blots were also probed with the U6 probe for loading control (lower panel). Oligonucleotide sequences used as primers are listed Table S4. The complete list and geographical distribution of Oryza species analysed in this study is indicated at Table S1

Figure 2

Biogenesis of miR812w. (a) Northern blot analysis of miR812w in rice dcl1, dcl3 and dcl4 mutants. RNA blots were also probed with the U6 probe for loading control (lower panel). (b) Abundance of miR812w‐3p in dcl3a rice mutant lines. (c‐d) Total sRNA and AGO‐bound reads plotted over the miR812w precursor. The 5p and 3p arms are indicated. (b‐d) Small RNA data were obtained from published libraries from seedlings of O. sativa cv Nipponbare of dcl3a mutant lines (Wei et al., 2014) and immunoprecipitation with AGO proteins (Wu et al., 2009, 2010). Scale bar indicate small RNA read number normalized per one million reads (RPM)

Figure 3

CRISPR/Cas9‐induced mutations in the MIR812w gene and infection phenotype to M. oryzae. (a) Mutations generated by CRISPR/Cas9 editing are shown in the upper part of the miR812w precursor structure. Three miR812w alleles were obtained: Δ22nt, Δ3nt and +1nt. Dashes indicate deletions. In bold, single nucleotide insertion. The PAM motif (UGG) is next to miR812w‐5p. WT, Wild‐type. (b) Predicted secondary structure of the mutated miR812w precursor (∆22nt, ∆3nt, +1nt) using RNAfold. The length of the precursor (nt) and the minimum free energy of folding (MFE, kcal/mol) are indicated. The position of the CRISPR/Cas9 mutation is indicated by an arrow. (c) Accumulation of miR812w as determined by northern blot using a ³²P‐labelled oligonucleotide for detection of miR812w‐3p and miR812w‐5p (Table S4). The small RNA fraction was isolated from total RNA of leaves (WT, Δ22nt, +1nt plants). RNA blots were also probed with the U6 probe for loading control (lower panel). (d–e) Disease phenotype of mir812w‐∆22 and mir812w+1 rice plants. (d) Representative images of M. oryzae‐infected CRISPR/Cas9‐edited miR812w plants at 7dpi. (e) Quantification of blast lesions was carried out by image analysis (left panel). Relative quantification of fungal biomass was determined by qPCR using specific primers for M. oryzae 28S DNA (values are fungal DNA levels normalized against the rice Ubiquitin 1 gene (right panel). Primers are listed in Table S4. Data from one representative experiment of three independent experiments are presented as the mean ± SE (n = 15) (Student t‐test, *P < 0.05), which gave similar results

Figure 4

Resistance of rice plants overexpressing MIR812w (miR812w‐OE) to M. oryzae infection. Three independent miR812w‐OE lines and control plants (EV, AZ and WT) were assayed. WT, wild‐type; EV, transgenic empty vector; AZ, segregated azygous plants. (a) Accumulation of miR812w precursor transcripts (upper panel) and mature miR812w (lower panel) sequences in transgenic rice lines determined by RT‐qPCR and small RNA northern blot analysis, respectively. Lower panels show U6 small RNA (snRNA) as loading controls. (b–c) Disease phenotype of miR812w‐OE plants (d) Representative images of M. oryzae‐infected miR812w‐OE plants at 7dpi. Scale bar (1 cm). (e) Quantification of blast lesions and fungal biomass was carried out by image analysis and qPCR using specific primers for M. oryzae 28S DNA, respectively (as in Figure 3). Primers are listed in Table S4. Data from one representative experiment of three independent experiments are presented as the mean ± SE (n = 15) (Student t‐test, *P < 0.05), which gave similar results

Figure 5

MIR812w expression in wild‐type rice plants during M. oryzae infection, and H₂O₂ accumulation in miR812‐OE and CRISPR/Cas9‐edited plants. (a) The accumulation of miR812w precursor transcripts was determined by RT‐qPCR at the indicated times after inoculation with a suspension of M. oryzae spores (left panel) or treatment with M. oryzae elicitors (right panel). Data from one representative experiment of three independent experiments are presented as the mean ± SE (n = 3; each sample consisted of a pool of 10 individual leaves; *P < 0.05, ANOVA test). (b) DAB staining to visualize H₂O₂ accumulation in wild‐type, miR399‐OE and CRISPR/Cas9‐edited plants carrying the ∆22 deletion. miR812w‐OE plants inoculated with M. oryzae accumulated more H₂O₂ that did wild‐type plants, whereas little DAB staining could be observed in M. oryzae‐inoculated leaves of mir812w‐∆22 plants. (c) H₂O₂ accumulation in M. oryzae‐infected leaves was quantified on digital photographs using imageJ (https://imagej.nih.gov/ij/)

Figure 6

miR812w directs DNA methylation at DNA sequences (in trans) as well as at its own locus (in cis). (a) Analysis of DNA methylation at the miR812w target transcripts in wild‐type (WT) and miR812w‐Δ22 (‐Δ22nt) by bisulfite sequencing. The target genes examined were: ACO3 (Os02g53180, upper panel), CIPK10 (Os03g22050, medium panel) and LRR (Os06g04830, lower panel). Data was analysed using the Kismeth software (Gruntman et al., 2008). Numbers represent the percentage of methylation at each cytosine methylation context (CG, CHG and CHH). The first nucleotide of the target site is set as position +1. A 75nt surrounding the mir812w target site (shaded in yellow) is shown. Red colour indicates those cytosine contexts with lower methylation in miR812w‐Δ22 (‐Δ22nt) compared to wild‐type (WT) plants (in bold italics are indicated those with a decrease higher than 10%). Schematics representation for each target gene is shown (wide black boxes, exons; grey thin boxes, Untranslated regions (UTR); thin lines, introns). Purple boxes indicate the position and code ID for the Stowaway MITE inserted at the UTR of each target gene. (b) miR812w directs DNA methylation in cis at its own locus. DNA methylation status was analysed and represented as in (a). The 5′ nucleotide of mature miR812w‐5p (shaded in blue) and miR812w‐3p (shaded in pink) is set as position +1. The 22nt deletion next to miR812w‐5p is indicated in miR812w‐Δ22 rice plants with an orange square. Details on DNA methylation at the MIR812w precursor sequence can be found in Figure S10b. These experiments were carried out two times with similar results

Figure 7

Expression analysis of ACO3 (Os02g53180), CIPK10 (Os03g22050) and LRR (Os06g04830) in wild‐type and miR812w‐Δ22 rice plants by qRT‐PCR. The rice ubiquitin1 gene was used for normalization. Data from one representative experiment of three independent experiments are presented as the mean ± SE (n = 3, each biological replicate is a pool of 3 individual leaves) (Student t‐test, *P < 0.05). Primers are indicated in Table S4

Figure 8

Proposed model for the origin and mode of action of miR812w in rice. MITEs are non‐autonomous transposable elements (TEs) that derive from autonomous TEs and depend on their transposases for mobilization and integration elsewhere in the genome (i.e protein‐coding genes). Terminal inverted repeats (TIR, 10–15 bp) are indicated by black arrows. A proto‐MIR812w would be formed from a Stowaway MITE that further evolved to become a MIR812w precursor that is processed DCL3. During miR812w evolution, small RNAs might have been generated from the proto‐MIR812w. The functional strand of the miR812w‐5p/miR812w‐3p duplex (miR812w‐3p) would be loaded into the AGO4 clade protein to mediate DNA methylation at their target transcripts (in trans), as well as at its own locus (in cis). During evolution, MITEs from the Stowaway family would have integrated into the 3’ UTR region of protein‐coding genes, such as the ACO3, CIPK10 and LRR genes. The miR812w could then be derived from a Stowaway MITE, while the Stowaway MITE would serve as a source for the origin of potential target genes for miR812w. Adapted from Li et al., (2011)