Rice (Oryza sativa) plants used in this study include the susceptible accession LTH, the resistant accession IRBL9-W, the japonica accession Taipei 309 (TP309) and its transgenic lines overexpressing miR169a, and the indica accession Kasalath and its transgenic lines overexpressing mimicry of miR169a (MIM169a). All rice plants were grown in a growth room maintained at 26°C and 70% relative humidity with a 14/10-h day/night regime. Nicotiana benthamiana plants were planted at 22°C with a 16/8 h light/dark photoperiod in a growth room and used for agro-infiltration experiments.

To generate miR169a overexpressing transgenic plants, its genomic sequence containing 421 bp upstream and 500 bp downstream sequences was amplified from Nipponbare (NPB) DNA with primers OsmiR169a-F and OsmiR169a-R (Supplementary Table S2), and the amplified fragment was cloned into KpnI-SalI sites of the binary vector 35S-pCAMBIA1300, resulting in the overexpression construct p35S:miR169a. To make artificial target mimicry construct, we exploited the Arabidopsis gene IPS1 (INDUCED BY PHOSPHATE STARVATION1) that contains sequences complement to miR399 with a mismatched loop at the expected miRNA cleavage site (Franco-Zorrilla et al., 2007). Artificial target mimicry sequences of miR169a were inserted into the IPS1 to replace the miR399 target site with primers miR169-IPS1-F, miR169-IPS1-R, miR169mimic-F, and miR169mimic-R (Supplementary Table S2) as described previously (Franco-Zorrilla et al., 2007), and cloned into KpnI-SpeI sites of binary vector 35S-pCAMBIA1300, resulting in the overexpression construct p35S:MIM169a. Construct p35S:miR169a was transformed into TP309 and construct p35S:MIM169a was transformed into Kasalath via Agrobacterium tumefaciens (strain GV3101)-mediated transformation and the transgenic plants were screened as previously described (Li et al., 2014). To express the YFP-tagged 3′-UTRs of target genes, the 3′-UTRs of Os03g29760 (NF-YA2), Os03g44540 (NF-YA3), and Os03g48970 (NF-YA4) containing the target site of miR169 were amplified from Nipponbare (NPB) cDNAs using gene-specific primers (Supplementary Table S2). The isolated fragments were then fused to the C-terminus of YFP and inserted into KpnI-SpeI sites of binary vector 35S-pCAMBIA1300. Agrobacterium strain GV3101 was used for agroinfection assay in N. benthamiana (Huang et al., 2014).

Three M. oryzae strains, Y34, Guy11 and eGFP-tagged Zhong8-10-14 (GZ8), were used in this study. M. oryzae strains were cultured in complete medium at 28°C with 12-h/12-h light/dark cycles for sporulation. After 2 weeks, spores were collected and the inoculum concentration was adjusted to 5 × 10⁵ spores mL^–1 for spray inoculation on three-leaf-stage plants (Qu et al., 2006). T3 seedlings of miR169a transgenic plants were used for spray inoculation. Disease phenotypes on the leaf two were recorded at 5 days post inoculation (dpi). The blast disease lesions were examined and classified into six types (0–5 type; 0–2 type is classified as resistant phenotype, and 3–5 type is classified as susceptible phenotype) based on the sporulation rate on the lesions in infected rice leaves following a previous report (Bonman et al., 1986). In brief, 0 = no disease lesions observed; 1 = small pinpoint-like disease lesions between two small vascular bundles; 2 = lesions with diameter 0.5–1 mm and develop over the two small vascular bundles but do not reach the big vascular bundles; 3 = disease lesions with diameter about 1–3 mm and develop between the two big vascular bundles; 4 = disease lesions with diameter about 3–4 mm and develop over the two big vascular bundles; 5 = disease lesions with diameter over 4 mm and develop over the main vein. For evaluation of the transgenic lines expressing the target mimicry of miR169a, 8-week old T0 plants were inoculated following the drop inoculation method (Park et al., 2012), and the negative transgenic plants were used as control. In brief, leaves from 8-week-old plants were slightly wounded with a mouse ear punch, and 5 μL of spore suspension (5 × 10⁵ spores mL^–1) was added to the wound. Lesions were measured at 5 dpi. Relative fungal mass was calculated using the DNA level of M. oryzae Pot2 against the rice genomic ubiquitin DNA level by qPCR (Park et al., 2012). To observe the infection process of M. oryzae, the eGFP-tagged M. oryzae strain GZ8 was inoculated on 5-cm-long leaf sheaths as described (Kankanala et al., 2007). The inoculated epidermal layer was excised and analyzed by Laser Scanning Confocal Microscopy (Nikon A1) at 12 and 36 hpi, respectively.

For examining H₂O₂ accumulation in infected rice leaves, we followed the procedure published previously by Xiao et al. (2003). DAB and trypan blue were used to stain H₂O₂ and fungal structures, respectively. Images were acquired with a microscope (Zeiss imager A2).

Three-leaf-stage plants were inoculated with M. oryzae by spraying spore suspensions at a concentration of 5 × 10⁵ spores mL^–1, and samples were collected at 0, 6, 12, 24, and 48 hpi. TP309 is used as the control of miR169a transgenic lines. For examining the target gene expression in transgenic lines, negative transgenic lines were used as the control of MIM169a transgenic lines. Total RNA was extracted from collected samples using TRIzol reagent (Invitrogen) and was reverse transcribed to cDNA using the SuperScript first-strand synthesis system primed by oligo dT (Invitrogen). To test the expression of miRNAs, NCode miRNA first-strand cDNA module (Invitrogen) was selected to elongate and reverse transcribe miRNA following the manufacturer’s instructions. Quantitative RT-PCR was performed using isoform-specific primers (Supplementary Table S2) and SYBR Green mix (TaKaRa). snRNA U6 served as internal reference for the detection of miRNAs in quantitative RT-PCR. The rice ubiquitin (UBQ) gene was selected as an internal reference for data normalization. The data were determined by a one-way ANOVA followed by post hoc Tukey HSD analysis with significant differences (P < 0.01).

Agrobacterium strain GV3101 harboring the respective expression constructs in the binary vector pCAMBIA1300 was incubated at 28°C overnight in LB media containing rifampin (50 μg/mL), kanamycin (50 μg/mL), and carbenicillin (50 μg/mL) at a 250 r/min shaking table. The bacteria were collected at 3000 rpm for 5 min and resuspended in an MMA buffer (10 mM MES, 10 mM MgCl₂, 100 μM AS), respectively. The Agrobacteria harboring the expression constructs were infiltrated into leaves of N. benthamiana for transient expression assay. Leaves were examined between 36 and 80 hpi for image acquisition using a Nikon A1 Confocal Laser Scanning Microscope (Nikon Instruments, Inc., Chengdu, China) as previously described (Huang et al., 2014). Western blotting analyses were performed following a previous protocol (Chen et al., 2008). In brief, 15 micrograms of total protein were electrophoresed on 10% SDS-PAGE gel, and the protein blot was reacted with anti-GFP sera (BBI Life Science) to detect and determine YFP accumulation.

To address the question of how different miR169 isoforms are differentially responsive to M. oryzae infection, we examined the accumulation of the five most abundant miR169 isoforms in the susceptible accession LTH and the monogenic resistant accession IRBL9-W that contains the resistance (R) gene Pi-9 (Tsumematsu et al., 2000) and confers high resistance to M. oryzae (Khanna et al., 2015). LTH is widely used as a susceptible reference in blast inoculation experiments for it is susceptible to all M. oryzae strains ever tested (Lin et al., 2001). We first confirmed the disease phenotypes by inoculating the virulent strain Guy11 on three-leaf-stage seedlings. While large disease lesions were observed in LTH, small lesions were occasionally found in IRBL9-W (Figure 1A), indicating susceptibility and resistance, respectively. We next performed a time-course assay to examine the accumulation of miR169 isoforms in LTH and IRBL9-W by miRNA quantitative RT-PCR. According to the small RNA deep sequencing data in a previous report (Li et al., 2014), the accumulation of miR169a, miR169b/c, miR169f.1/g, miR169h/i/j/k/l/m, and miR169n/o is mostly abundant and thus was examined in this study, whereas, the accumulation of miR169d, miR169e, miR169p, and miR169q is rarely or not detected (below 50 reads or no reads per one million total reads) and thus not included in this study. Our data confirmed that the abundance of different miR169 isoforms was quite different, with the least abundant being for miR169f/g and miR169n/o, the most abundance being for miR169b/c, and the middle being for miR169a and miR169h/i/j/k/l/m (Figures 1B,C). Moreover, the accumulation pattern of miR169b/c was quite similar to the sum accumulation pattern of miR169 (Figure 1D). It is intriguing that the accumulation of all the tested miR169 isoforms was increased in LTH upon M. oryzae infection, whereas, the accumulation of miR169b/c was quite stable in IRBL9-W, although the accumulation of the other miR169 isoforms was also increased at certain time points (Figures 1B,C). However, the high abundance of miR169b/c might mask the fluctuation of the other isoforms so that its pattern is similar to the sum of miR169 (Figure 1D). Particularly, the total accumulation of all miR169 isoforms was highly up-regulated upon M. oryzae inoculation in LTH, but significantly down-regulated at 12 hpi, up-regulated at 24 hpi and back to the background level at 48 hpi in IRBL9-W (Figure 1D), which was consistent with the total reads in LTH and IRBLKm-Ts (in Supplementary Table S1 of Li et al., 2014). These data imply that miR169 may integratively act as a negative regulator in rice immunity against M. oryzae.

All miR169 isoforms target the same eight NF-YA genes (Wu et al., 2009; Li Y. F. et al., 2010; Zhou et al., 2010) because of their high sequence identity (Supplementary Figure S1). Thus, it is feasible to investigate the integrative role of all miR169 isoforms in rice immunity against M. oryzae by over expressing one of the miR169 isoforms, although the authentic miR169-NF-YA regulation module could be much more complicated due to different miR169 isoform has different expression pattern and is differentially responsive to different stimuli (Zhao et al., 2007, 2009). Following this scenario, we constructed rice transgenic lines overexpressing miR169a within the TP309 background. Two independent transgenic lines with high miR169a accumulation were identified and used for phenotypic analyses (Figure 2A). Three-leaf-stage seedlings were separately spray-inoculated with three M. oryzae strains with different virulence. While Y34 is an incompatible strain to TP309, Guy11 and the enhanced GFP-tagged Zhong8-10-14 (GZ8) are virulent strains. Disease phenotypes were recorded at 5 dpi. All the three strains formed more and larger disease lesions in the transgenic lines than those in the control plants (Figures 2B–D), indicating enhanced susceptibility by overexpression of miR169a. Then, the composition of lesion types was quantitatively analyzed. While the Y34-infected lesions in the control plants were mostly scored as types 1–2 (resistant phenotype), the lesions in the transgenic lines overexpressing miR169a were scored as types 3–5 (susceptible phenotype, Figure 2B), indicating that overexpression of miR169a converted the partial resistance into fully susceptibility to Y34. Moreover, the susceptible lesion types (3–5) of GZ8 and Guy11 in the control plants were all exacerbated in the transgenic lines overexpressing miR169a. The percentage of susceptible lesion types (types 3–4) of GZ8 was increased from 50% in the control plants to 80% in the transgenic lines (Figure 2C). Guy11-infected susceptible lesion types accounting for more than 80% in the control plants were exacerbated into 100% in the transgenic lines overexpressing miR169a, in which type 5 was increased from 10% in the control plants to more than 50% in the transgenic lines overexpressing miR169a (Figure 2D). These data demonstrate that overexpressing miR169a makes the susceptibility of TP309 to these strains into hyper-susceptibility.

To explain why overexpression of miR169a leads to enhanced susceptibility, we examined typical defense responses, including expression levels of defense-related genes, production of hydrogen peroxide (H₂O₂) and microscopic pathogenesis of M. oryzae. First, three-leaf-stage seedlings were inoculated with M. oryzae strains Guy11. Then, the expression levels of three defense-related genes were examined by quantitative RT-PCR, including the two defense marker genes, Oryza sativa pathogenesis-related protein10b (OsPR10b) (Yamaguchi et al., 2013) and Os04g10010 (Schwessinger et al., 2015), and OsNAC4, an earlier induced basal defense-related gene (Park et al., 2012). The three genes were significantly induced in the wild type at 6 hpi of M. oryzae (Figures 3A–C), in which OsNAC4 was peaked at 12 hpi, whereas OsPR10b and Os04g10010 were further increased along with the time points. On the contrary, the expression levels of these genes were much lower and the induced time was delayed in the transgenic lines, although their expressions were also induced (Figures 3A–C). Next, we examined the production of H₂O₂ upon inoculation of M. oryzae. Consistent with the reduced expression of defense-related genes, H₂O₂ was hardly detected in the infected sheath cells of transgenic lines at 24 hpi, while easily observed in the control plants at the same time point (Figure 3D). These data indicate that overexpression of miR169a compromises M. oryzae-related defense responses in rice plants.

In addition, we compared the infection process of M. oryzae strain GZ8 on leaf sheath of the wild type and the transgenic lines by using Laser Scanning Confocal Microscopy. At 12 hpi, more than half of the inoculated spores germinated and formed appressoria on the sheath from the transgenic lines overexpressing miR169a, which was in contrast to no or seldom appressoria formation on the sheath from the control plants (Figures 4A,B). At 36 hpi, more than 70% spores formed the invasive hyphae in the transgenic lines, which was in contrast to about 30% invasive hyphae formed in the control plants (Figures 4A,B). These results indicate that overexpressing miR169 can compromise rice defense responses so as to facilitate M. oryzae invasion at the early infection stage.

All of the known target genes of miR169a encode NF-YA (Supplementary Table S1) (Wu et al., 2009; Li Y. F. et al., 2010; Zhou et al., 2010). As the role of miRNAs is to suppress target gene expression, we speculated that the expression of the target genes should be reduced in the lines overexpressing miR169a. To this end, we examined the mRNA levels of target genes in the lines overexpressing miR169a by quantitative RT-PCR. As anticipated, the transcription levels of six target genes, including Os02g53620 (NF-YA11), Os03g07880 (NF-YA1), Os03g44540 (NF-YA3), Os03g48970 (NF-YA4), Os07g41720 (NF-YA6) and Os12g42400 (NF-YA10), were repressed significantly in the transgenic lines; particularly, the expression of Os03g44540 (NF-YA3) and Os12g42400 (NF-YA10) was down-regulated to less than 20% of that in the control plants (Figure 5A). However, the transcription level of Os03g29760 (NF-YA2) was significantly increased in one line and did not change in the other line (Figure 5A), and the simplest explanation is that miR169a may repress its translation or that Os03g29760 (NF-YA2) is feedback regulated by its products. Then, we established an YFP-based reporter system to examine whether miR169a inhibits translation of Os03g29760. To this end, we made a construct expressing the Yellow Fluorescent protein (YFP) with the 3′-UTR of Os03g29760 as the terminator (YFP-3′-UTR_Os03g29760) because the target sites of miR169a on all of the eight NF-YA genes are located in the 3′-UTR (Supplementary Figure S2). Next, YFP-3′-UTR_Os03g29760 was separately expressed or co-expressed with miR169a in N. benthamiana and the protein levels were compared by both Western blotting analysis and examining the intensity of YFP. The results showed that when expressed alone, YFP-3′-UTR_Os03g29760 was highly accumulated (Figure 5B) and expressed in both the cytoplasm and the nucleus (Figure 5C). However, its expression level was obviously reduced when miR169a was co-expressed, particularly at higher OD value of the infiltrated Agrobacteria. By contrast, the co-expression of YFP that did not contain the 3′-UTR of Os03g29760 and miR169a did not affect the protein accumulation of YFP (Figures 5B,C). To further confirm the translation inhibition by miR169 on 3′-UTR of Os03g29760, we constructed the target mimicry of miR169a, MIM169a, which could act as a sponge to trap miR169a because of the insertion of three nucleotides between positions 10 and 11, and as a result, the cleavage on the formed double strand RNA was inhibited because of the loop formation at the mismatched nucleotides (Franco-Zorrilla et al., 2007). When YFP-3′-UTR_Os03g29760, miR169a and MIM169a were co-expressed, the protein accumulation of YFP-3′-UTR_Os03g29760 was obviously increased with the addition of MIM169a, indicating that the suppression on YFP-3′-UTR_Os03g29760 by miR169a was recovered by MIM169a (Figures 5B,D). These results suggest that miR169a may authentically repress the expression of Os03g29760 at translational level.

To figure out whether miR169 suppresses the expression of the other target genes at protein levels, we also tested the expression of two target genes by using the reporter system. As anticipated, the YFP intensity of YFP-3′-UTR_Os03g44540 and YFP-3′-UTR_Os03g48970 were both reduced by co-expression of miR169a, whereas when MIM169a was co-expressed, the suppression on YFP-3′-UTR_Os03g44540 and YFP-3′-UTR_Os03g48970 by miR169a was recovered (Supplementary Figure S3). These results are consistent with the transcriptional level of the two target genes, and demonstrate that miR169a can indeed repress the expression of the two target genes at protein levels.

Unfortunately, we did not detect the expression of Os07g06470 (NF-YA5) by qRT-PCR and failed to amplify this gene to make a transient expression construct. It is unclear whether and how the expression of Os07g06470 (NF-YA5) is regulated by miR169a.

To further test the role of miR169a in rice immunity against the blast fungus, we constructed the target mimicry of miR169a, MIM169a, and obtained transgenic lines expressing MIM169a. Because overexpressing miR169a significantly repressed the expression of its target genes and led to enhanced susceptibility to M. oryzae, expressing MIM169a is predicted to up-regulate these target genes and result in enhanced resistance. Therefore, we first examined the expression of the target genes in the transgenic lines. As anticipated, the expression of all the detected target genes were significantly up-regulated in the two transgenic lines harboring the MIM169a construct (Figure 6A). Particularly, the expression of Os12g42400 (NF-YA10) was up-regulated about sixfold in the transgenic line #12 and 2-fold in the transgenic line #13, whereas, the mRNA levels of all the other six target genes were generally up-regulated from 2- to 4-fold in the two independent MIM169a transgenic lines compared with that in the control plants. Next, we tested the resistance of the transgenic lines to M. oryzae strains GZ8. Indeed, the lesions on the leaves from transgenic lines were obviously smaller than that on the leaves from the control plants, and the fungal biomass on the transgenic lines were also decreased significantly than that on the control plants (Figure 6B), indicating that the up-regulation of miR169’s target genes alleviated rice susceptibility to M. oryzae. Taken together, these results confirmed that miR169 natively regulates rice immunity, whereas, its target genes, or some of its target genes, positively regulate rice immunity against M. oryzae.

Now that all the detected seven target genes can be down-regulated by miR169 at either transcriptional or translational level, and up-regulated by the miR169’s target mimicry, it is questionable whether all of them similarly or equally regulate rice immunity against M. oryzae. To address this question, we performed a time course examination of their expression in LTH and IRBL9-W upon M. oryzae inoculation. According to the dynamic expression pattern, we divided the target genes into three groups. The first group contained one gene, Os02g53620 (NF-YA11), its transcription level was decreased significantly upon M. oryzae infection in both LTH and IRBL9-W (Figure 7A). The second group included three genes, Os03g44540 (NF-YA3), Os03g48970 (NF-YA4), and Os12g42400 (NF-YA10), their expression levels were decreased significantly and rapidly in LTH, but decreased slightly and slowly in IRBL9-W with Os03g44540 (NF-YA3) and Os03g48970 (NF-YA4) being transiently increased at 12 hpi and then decreased at the later time points of M. oryzae inoculation (Figure 7B). The third group included Os03g07880 (NF-YA1), Os03g29760 (NF-YA2) and Os07g41720 (NF-YA6), their transcription levels were kept stable or decreased in LTH, but increased significantly at one or all examined time points in IRBL9-W (Figure 7C). Therefore, different target genes of miR169 are differently responsive to M. oryzae infection in the resistant and susceptible accessions and thus may act differently in rice immunity against M. oryzae. The three genes, Os03g07880 (NF-YA1), Os03g29760 (NF-YA2), and Os07g41720 (NF-YA6) most likely contribute to the resistant phenotype of IRBL9-W.