Osa-miR162a balances rice yield and resistance to Magnaporthe oryzae

doi:10.21203/rs.2.22473/v1

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Osa-miR162a balances rice yield and resistance to Magnaporthe oryzae

https://doi.org/10.21203/rs.2.22473/v1

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published 10 Jun, 2020

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MicroRNAs (miRNAs) play essential roles in rice immunity against Magnaporthe oryzae, the causative agent of rice blast disease. Osa-miR162a targets Dicer-like 1 (DCL1) genes, which play vital roles in miRNA biogenesis and act as negative regulators in rice immunity. Here we demonstrate that Osa-miR162a improves rice immunity against M. oryzae and balances the trade-off between rice yield and resistance. Overexpression of Osa-miR162a compromises rice susceptibility to M. oryzae accompanying enhanced induction of defense-related genes and accumulation of hydrogen peroxide (H₂O₂). In contrast, blocking miR162 by overexpressing a target mimic of miR162 enhances susceptibility to blast fungus associating with compromised induction of defense-related gene expression and H₂O₂accumulation. Moreover, the transgenic lines overexpressing Osa-miR162a display decreased seed setting rate resulting in reduced yield per plant, whereas blocking miR162 leads to an increased number of grains per panicle, resulting in increased yield per plant. Altered accumulation of miR162 had limited impact on the expression of OsDCL1. Together, our results indicate that Osa-miR162a improves rice blast resistance and plays a role in the balance of trade-off between resistance and yield.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

miR162

OsDCL1

blast disease resistance

yield traits

hydrogen peroxide

defense-related gene

Plants mount a multi-layered immune system in fighting against the invasion of pathogens, and small RNAs are essential regulators in this process (Katiyar-Agarwal and Jin 2010; Weiberg et al. 2014). miRNA is a subset of non-coding small RNAs of 20–24 nucleotides (nt). miRNAs play vital roles in the regulation of gene expression either by chromatin methyl modification, mRNA cleavage, or translational inhibition (Yu et al. 2017). In plants, miRNAs are transcribed from miRNA genes and subsequently cleaved by the endoribonucleases Dicer-like proteins (DCLs) and processed into approximately 20–24 nt miRNAs (Kurihara and Watanabe 2004). Finally, the mature miRNAs are recruited into ARGONAUTE (AGO) proteins to form miRNA-Induced Silence Complex (miRISC), the critical component required for suppression of target genes (Vaucheret et al. 2004). Obviously, DCLs act as core components during miRNA biogenesis. In turn, the expression of DCL1 is regulated by miR162 (Wu et al. 2009; Zhou et al. 2010a).

miR162 is a conserved miRNA family involved in multiple abiotic stress responses in plants. For example, miR162b was induced by ABA treatment to improve adaptation to drought stress through suppressing OsTRE1 (Trehalase precursor 1; Tian et al. 2015). In maize, miR162 was responsive to salt stress with enhanced accumulation at 0.5 hours post-treatment of salt, whereas decreased at five and 24 hours post-treatment (Ding et al. 2009). In switchgrass (Panicum virgatum), the accumulation of miR162 was significantly changed under drought stress (Sun et al. 2012). In cotton (Gossypium hirsutum L.), miR162 was responsive to salinity (Salih et al. 2018). Besides, miR162 also participates in biotic stress responses. For example, virus suppressors, such as Tombusvirus p19 and Cucumovirus 2b, had a binding preference for miR162 to counteract host antiviral RNA silencing, a plant immune response during viral infection initiation (Pertermann et al. 2018).

In Arabidopsis and rice, miR162 was reported to suppress the expression of DCL1, which plays essential roles in miRNA biogenesis and metabolism (Tijsterman and Plasterk 2004; Xie et al. 2004; Zhou et al. 2010b). In Arabidopsis, the loss-of-function mutants of AtDCL1 showed multiple altered phenotypes associating with the reduction of miRNA levels (Park et al. 2002). Besides, AtDCL1 was required for miR393-mediated anti-bacterial defenses (Navarro et al. 2006). In rice, strong loss of function of OsDCL1 led to developmental arrest at the seedling stage, whereas weak loss of function of OsDCL1 resulted in pleiotropic developmental defects, including dark green, dwarfism, and different leaves and root phenotypes in comparison with control (Liu et al. 2005). Besides the roles in rice development, OsDCL1 also regulates rice resistance against pathogens. For example, the silence of DCL1 enhanced resistance to the virulent Magnaporthe oryzae strains with improved defense responses, including hydrogen peroxide accumulation and cell death at the infected sites (Zhang et al. 2015). In contrast, activation of OsDCL1a enhances susceptibility to fungal pathogen Fusarium fujikuroi and M. oryzae accompanying with compromised pathogen-inducible defense responses and impacts miRNA network (Salvador-Guirao et al. 2019). These results indicate that OsDCL1 regulates rice growth and immunity against M. oryzae. Although miR162 suppresses the expression of OsDCL1 (Zhou et al. 2010b), the effect of Osa-miR162a on rice blast resistance and yield is unclear. It is also unknown whether there are other target genes of miR162 involved in these processes.

In rice, the miR162 family has two isoforms, namely Osa-miR162a and Osa-miR162b. Previously, we found that Osa-miR162a was responsive to M. oryzae in both susceptible and resistance accessions (Li et al. 2014). To elucidate the roles of miR162 in rice growth and blast disease resistance, we constructed transgenic lines overexpressing miR162a and a target mimic of miR162, respectively. We examined the blast disease phenotypes and yield traits in these transgenic lines. Our data demonstrate that overexpression of miR162a led to enhanced resistance but penalized yield, whereas blocking miR162 via a target mimic resulted in suppressed resistance but improved yield. Besides, altered accumulation of miR162 showed limited impact on OsDCL1 expression. Overall, our results indicate that Osa-miR162a positively regulates rice resistance agianst M. oryzae and the trade-off between resistance and yield via OsDCL1.

miR162a is up-regulated in both susceptible and resistant accessions by M. oryzae

To explore the involvement of Osa-miR162a in rice blast resistance, we examined the amounts of Osa-miR162a in a highly susceptible accession Lijiangxin Tuan Heigu (LTH) and a resistant accession IRBLKm-Ts. LTH is sensitive to over 1,300 regional isolates of M. oryzae worldwide and often used as a susceptible reference in blast disease assay (Lin et al. 2001). IRBLKm-Ts carries a resistance (R) locus Pi-km and exhibits resistance to M. oryzae isolates carrying Avr-Pikm (Tsumematsu et al. 2000). We identified the disease phenotype of LTH and IRBLkm-Ts by spray-inoculation of M. oryzae strain Guy11. LTH showed susceptible phenotype, whereas IRBLkm-Ts showed resistance (Fig. 1a). We then examined the expression of miR162a in both accessions. LTH displayed unchanged or decreased miR162a abundance following the inoculation of Guy11 in comparison with mock samples. In contrast, IRBLKm-Ts showed reduced accumulation at 12 and 48 hpi but increased at 24 hpi (Fig. 1b), indicating that miR162a was involved in rice-M. oryzae interaction and possibly played positive roles in rice blast disease resistance.

Overexpression of Osa-miR162a enhances rice resistance to M. oryzae

To explore the roles of miR162a in rice blast resistance, we constructed transgenic lines overexpressing Osa-miR162a (OX162). We got over 20 transgenic lines displaying increased miR162 abundance in comparison with wild type (WT) control. We selected two lines showing moderate accumulation of miR162 and producing seeds, namely OX162-11 and OX162-24, for subsequent study (Fig. 2a). We then examined blast disease phenotype of these lines following the punch-inoculation of virulent strains, namely GFP-tagged Zhong 8-10-14 (GZ8) and 97-27-2. OX162 lines displayed compromised susceptibility with smaller disease lesions than control (Fig. 2b). Consistently, the fungal biomass in OX162 was significantly less than that in control (Fig. 2c). Then we examined the disease phenotype of OX162 by spray-inoculation of GZ8 and got the results similar to that by punch-inoculation (Additional file 1: Figure S1). Moreover, we also identified the resistance of OX162 against four M. oryzae strains isolated by punch-inoculation. Similarly, OX162 displayed smaller disease lesions than control (Additional file 2: Figure S2). These results indicate that miR162a improves rice blast resistance.

To understand how OX162 lines suppress fungal growth, we observed the infection process of GZ8 in the leaf sheath. At 24 hpi, more than 60% of spores formed invasive hyphae in sheath cells of WT. However, in OX162, we only observed many appressoria (Fig. 2d). At 36 hpi, the hyphae started to infect the neighbor cells near the local infected cell of WT but were limited in the local infected cells of OX162 (Fig. 2d). The quantification assay confirmed that overexpression of miR162 delayed the infection progress (Fig. 2e).

To understand why overexpression of miR162 delayed the infection of M. oryzae, we examined immune responses, such as hydrogen peroxide (H₂O₂) accumulation and expression of resistance-related genes in OX162 in comparison with control. OX162 showed greatly more H₂O₂ than WT upon GZ8 inoculation (Fig. 3a). Four defense-related genes, namely ENT-KA RENE SYNTHASE 4 (KS4), NAC DOMAIN-CONTAINING PROTEIN 4 (NAC4), PATHOGENESIS-RELATE GENG 1A (PR1a), and PR10b, were examined by RT-qPCR. KS4 is an early-induced basal defense-related gene (Park et al. 2012). NAC4 is involved in plant cell death and highly expressed in a lesion mimic mutant spl11 at the lesion forming period (Yin et al. 2000). PR1a and PR10b are pathogenesis-related genes (Yamaguchi et al. 2013). The expression of KS4 and NAC4 was induced to higher levels in OX162 than in control at 12 hpi (Fig. 3b and c). Similarly, the expression of PR1a and PR10b was significantly induced to higher levels at 12 and 48 hpi than that in control (Fig. 3d and e). These data indicate that overexpression of miR162a improves the immune responses triggered by M. oryzae.

Blocking miR162a leads to increased blast disease susceptibility

To confirm the roles of miR162a in rice immunity again blast fungus, we constructed the transgenic lines overexpressing a target mimic of miR162a (MIM162). MIM162 contained a sequence reversely complementary to miR162a with three nucleotides insertion between 10 to 11 nucleotide. Therefore, the target mimic acted as a sponge to absorb miR162 and block its binding to target genes (Additional file 3: Figure S3). We then examined miR162 abundance and OsDCL1a mRNA amounts in MIM162. MIM162 displayed significantly less accumulation of miR162 than control (Fig. 4a). Next, we conducted a disease assay on MIM162. As expected, MIM162 showed enhanced susceptibility to M. oryzae with larger disease lesions and supported more fungal growth than control by punch- and spray-inoculation, respectively (Fig. 4b and c, and Additional file 1: Figure S1). We also examined the disease phenotypes of MIM162 against four M. oryzae strains by punch-inoculation. MIM162 displayed larger disease lesions and supported more fungal growth than control (Additional file 2: Figure S2).

Moreover, the infection process of GZ8 in MIM162 was faster than that in control. In WT plants, a few of spores formed invasive hyphae in sheath cells at 24 hpi and started to infect the neighbor cells at 36 hpi. In contrast, in MIM162, many hyphae had invaded into neighbor cells at 24 hpi, and more than 60% hyphae invaded more cells at 36 hpi than that in WT (Fig. 4d and e). These results indicate that blocking Osa-miR162a facilitates the M. oryzae invasion.

Blocking miR162 also led to compromised immune responses. WT plant showed visible H₂O₂ accumulation in leaves and cells where M. oryzae appressorium located, whereas MIM162 displayed little H₂O₂ amounts at the invaded sites (Fig. 5a). The induction of defense-related genes, KS4, NAC4, PR1a, and PR10b, was lower in MIM162 than that in WT. These data indicate that blocking miR162 compromises immune responses against M. oryzae (Fig. 5b-d).

miR162a regulates rice yield traits

Besides blast disease resistance, we found that miR162a also regulated agronomic traits. Both OX162 and MIM162 exhibited comparable phenotypes with WT, including gross morphology, panicle number per plant, and panicle length (Fig. 6a and Table 1). However, OX162 showed narrower seeds, resulting in lower seed weight than control, and significantly lower seed setting rate, leading to lower grain number than control (Fig. 6b and Table 1). Substantially, OX162 displayed a lower yield per plant (Table 1). In contrast, MIM162 exhibited similar seed size as control (Fig. 6b), slightly lower seed weight (Table 1), lower seed setting rate, but more grains per plant in comparison with control (Fig. 6c and Table 1). As a result, MIM162 showed higher yield per plant than control (Table 1). These results indicate that miR162a is involved in the regulation of rice yield traits and negatively regulates rice yield, and miR162 acts as a regulator in the trade-off between rice blast disease resistance and yield.

miR162 regulates rice blast resistance by suppressing OsDCL1

Rice miR162 was reported to target LOC_Os03g02970 (Zhou et al. 2010b) and was predicted to target LOC_Os03g15230 (http://plantgrn.noble.org/psRNATarget/). LOC_Os03g02970 encodes OsDCL1a, and LOC_Os03g15230 encodes an expressed protein OsDUF292 (domains of unknown function protein 292; Additional file3：Figure S3b). We examined the expression of miR162 and the two genes in OX162 and MIM162. We tested 11 independent OX162 lines and 11 independent MIM162 lines, respectively. Eight of the 11 tested OX162 lines showed significantly higher miR162 accumulation (Additional file3：Figure S3c). Conversely, the mRNA amounts of OsDCL1a decreased in the eight OX162 lines (Fig. 7a). Besides, among the 11 tested MIM162 lines, ten lines displayed lower miR162 abundance (Additional file3：Figure S3d). Conversely, the mRNA amounts of OsDCL1a increased in eight of the 11 MIM162 lines, except MIM162-22 and MIM162-23 showing unchanged OsDCL1a accumulation (Fig. 7b). These results confirmed the regulation of miR162a on OsDCL1. Intriguingly, the mRNA levels of OsDCL1a decreased slightly in all the eight lines overexpressing miR162 (Fig. 7a). Even in the four OX162 lines showing over 40-fold amounts of miR162a (Additional file3：Figure S3c), the mRNA levels of OsDCL1a only decreased less than 40% of that of control, indicating that the down-regulation of OsDCL1a is limited by increasing the abundance of miR162. However, the mRNA levels of OsDUF292 were markedly higher in both OX162 and MIM162 (Additional file3：Figure S3e and f), suggesting miR162 may not directly target OsDUF292.

In a previous report, the silencing of OsDCL1 led to enhanced rice resistance to blast fungus and increased H₂O₂ accumulation and cell death at the infection sites, suggesting OsDCL1 negatively regulates rice blast disease resistance (Zhang et al. 2015). In this study, we confirm the role of OsDCL1 in rice blast resistance by inoculation of GZ8 on an OsDCL1 RNA inference line (DCL1i, Liu, 2005 #1062). DCL1i showed significantly lower OsDCL1 mRNA amounts than control (Fig. 7c). Consistent with the previous report, DCL1i exhibited compromised susceptibility to GZ8 with smaller disease lesions and supported less fungal growth (Fig. 7d and e). These data imply that miR162 positively regulates rice blast disease resistance via suppressing the expression of OsDCL1. To further explore the roles of OsDCL1 in rice resistance, we examined the expression of OsDCL1 in susceptible accession LTH and resistance accessions IRBLKm-Ts and Yahui2115 upon M. oryzae infection, respectively. Yahui2115 is an elite hybrid restorer line carrying several blast resistance genes and widely used in breeding programs (Shi et al. 2015). The mRNA amounts of OsDCL1 increased in LTH at 24 and 48 hpi of GZ8, whereas decreased or unchanged in IRBLKm-Ts and Yahui2115 (Fig. 7f-h), indicating that down-regulation of OsDCL1 contributed to resistance in the two accessions. These results indicate that miR162a positively regulates rice immunity against blast fungus by suppressing the expression of OsDCL1.

MiRNAs play important roles in controlling plant developmental progress and responses to biotic or abiotic stresses (de Lima et al. 2012; Seo et al. 2013). In recent years, rice-M. oryzae interaction has become a model in the study of plant-fungi interaction (Liu and Wang 2016). More than 70 miRNAs were responsive to M. oryzae or its elicitors and eleven of which have been identified as regulators in rice-M. oryzae interaction (Li et al. 2019b; Zhou et al. 2019). For example, seven miRNAs were identified as negative regulators in rice immunity against M. oryzae by disease assays on overexpressing or silencing transgenic lines, namely miR164a, miR169a, miR167d, miR319, miR396, miR444, and miR1873 (Li et al. 2017; Xiao et al. 2017; Xiao ZY 2017; Chandran et al. 2018; Wang et al. 2018; Zhang et al. 2018a; Zhang et al. 2018b; Zhao et al. 2019; Zhou et al. 2019). In contrast, four miRNAs were characterized as positive regulators, namely miR160a, miR166k-miR166h, miR398b, and miR7695 (Campo et al. 2013; Li et al. 2014; Salvador-Guirao et al. 2018; Li et al. 2019a). Here we characterized Osa-miR162a as a positive regulator in rice immunity against M. oryzae. First, the expression of Osa-miR162a was unchanged or decreased in susceptible accession LTH but up-regulated in resistance accession IRBLKm-Ts upon M. oryzae infection (Fig. 1). Second, overexpression of Osa-miR162a improved rice resistance against M. oryzae with higher induction of defense responses (Fig. 2 and 3). In contrast, blocking Osa-miR162a by expressing a target mimic compromised rice resistance associating with the suppression of defense responses (Fig. 4 and 5).

Besides the regulation of blast disease resistance, miR162 also regulates rice yield and yield traits. The trade-off between yield and disease resistance is a common phenomenon in crop production. For example, the mutation in OsSPL28 (SQUAMOSA promoter-binding protein-like transcription factors 28) showed enhanced resistance to M. oryzae but led to a reduction in rice yield (Qiao et al. 2010). miRNAs may act as the crucial fine-tuning regulators to balance rice blast resistance and yield by regulating the expression of their target genes. For example, miR396 fine-tunes rice blast disease resistance and yield by controlling the expression of Growth Regulating Factor (GRF) transcription factor genes. The transgenic lines overexpressing miR396 showed enhanced susceptibility to M. oryzae, whereas the transgenic lines blocking miR396 exhibited compromised rice susceptibility but increased yield (Chandran et al. 2019). In this study, we demonstrated that Osa-miR162a balanced the trade-off between rice blast resistance and yield. Overexpression of Osa-miR162a improved rice blast disease resistance but compromised yield. In contrast, blocking of miR162 facilitated the infection of M. oryzae but enhanced yield, indicating that the accurate accumulation of miR162a is required for the development of yield traits and manipulation of immune responses.

miR162a may affect the whole miRNA network by target DCL1. OX162 displayed blast resistance similar to OsDCL1 RNAi lines: higher blast disease resistance (Fig. 7) with increased induction of H₂O₂ and cell death at infected sites (Liu et al. 2005; Zhang et al. 2015). In the weak OsDCL1-knocking down mutant, more than ten miRNAs were identified as OsDCL1-regulated miRNAs for their differential expression in OsDCL1 RNAi lines and control (Liu et al. 2005; Zhang et al. 2015). For example, the accumulation of eight miRNAs was abolished or greatly reduced in a weak loss of function of DCL1-knocking down transformants, including miR156, miR159, miR166, miR167, miR168, miR168-3p, miR396, and miR528 (Liu et al. 2005). Besides, in another OsDCL1 RNAi lines, the abundance of 12 miRNAs was decreased significantly, including miR156, miR166, miR167, miR168, and miR396 (Zhang et al. 2015). Intriguingly, among these miRNAs, four miRNAs were characterized as regulators in rice-M. oryzae interaction, namely miR166, miR167, miR169, and miR396; moreover, four miRNAs were involved in rice development and growth, namely miR156, miR166, miR167 and miR396 (Tang and Chu 2017), suggesting that miR162a might regulate rice development and rice-blast fungus interaction by controlling these miRNAs via OsDCL1.

OsDCL1 is required for rice development. OsDCL1 RNAi lines with heavy loss of function showed developmental arrest at the seedling stage, including shoot and root abnormalities at an early developing stage, wilting of leaves and early senescence at a later stage, and eventually died without seed production (Liu et al. 2005). The lines with weak loss of function also displayed pleiotropic developmental defects but with seed production (Liu et al. 2005). In this study, we found that the OX162 lines with seed production all showed slightly decreased expression of OsDCL1. In the OX162 lines showing over 40-fold amounts of miR162a (Additional file3：Figure S3c), the expression of OsDCL1a decreased less than 40% of WT (Fig. 7a), whereas, in the MIM162 lines showing great decreasing of miR162 (Additional file3：Figure S3d), the expression of OsDCL1 was increased less than four-fold of that of WT (Fig. 7b). These results indicate that the regulation of Osa-miR162a on OsDCL1 is limited, and certain amount of OsDCL1 is required for rice development and growth, because too lower expression of OsDCL1a is possibly fatal. We selected two OX162 lines showing an appropriate 10% to 20% decrease of DCL1 amounts and two MIM162 lines displaying less than a 2-fold increase of OsDCL1a mRNA levels in comparison with that of control for this study. The two OX162 also showed slight defects in yield traits but enhanced blast disease resistance, suggesting that we could control the abundance of Osa-miR162a to manipulate resistance without significant loss of yield.

Collectively, our results shows that Osa-miR162a improves rice resistance to M. oryzae and balances the trade-off between rice yield and resistance. Overexpression of Osa-miR162a leads to improved rice resistance to M. oryzae but compromised yield per plant slightly, whereas blocking miR162 by expressing a target mimic of miR162 results in susceptibility but increased yield per palnt. Further study reveals that miR162 had limited impact on the expression of OsDCL1. Silencing OsDCL1 leads to the similar resistance phenotype as that caused by overexpression Osa-miR162a. Thus, Osa-miR162a improves rice blast resistance via OsDCL1 and can be used to manipulate resistance without significant loss of yield.

Plant materials and growth conditions

Rice plants used in this study included susceptible accession Lijiangxin Tuan Heigu (LTH), resistance accessions IRBLkm-Ts and Yahui2115. Japonica accession NPB (Nipponbare) was used to construct transgenic lines overexpressing Osa-miR162a (OX162) or overexpressing a target mimic of Osa-miR162a (MIM162a), respectively. For transformation, fungal inoculation, and defense responses assay, rice plants were planted at a growth room at 26℃ and 70% relative humidity with 14/10-h of day/night light. For yield straits detection, rice plants were planted in paddy yard at a regular season in Wenjiang district, Sichuan Province, China.

Plasmid construction and genetic transformation

The transgenic plants were generated following previous protocols (Li et al. 2017). The sequence of the Osa-miR162a gene containing approximate 400 bp upstream and downstream sequences was amplified from NPB genomic DNA with primers Osa-miR162a-F and Osa-miR162a-R (Additional file 4: Table S1). Then the amplified fragment was cloned into KpnI-SalI sites of binary vector 35S-pCAMBIA1300 and got the overexpression construct p35S:miR169a. To construct the target mimicry of Osa-miR162a, target mimic sequences of Osa-miR162a were formed by annealing with primers MIM162a-F and MIM162a-R (Additional file 4: Table S1) and the annealing double-strand product was inserted into Arabidopsis IPS1 gene to substitute miR399 target site as described previously (Franco-Zorrilla et al. 2007; Li et al. 2017). The reconstructed IPS1 sequence was cloned into KpnI-SalI sites of the binary vector pCAMBIA1300, resulting in overexpressing construct p35S: MIM162. The vectors were transformed into NPB by Agrobacterium strain EHA105, and the positive transgenic lines were screened with Hygromycin B.

Pathogen infection and microscopy analysis

M. oryzae strains Guy11, 97-27-2, eGFP-tagged Zhong8-10-14(GZ8) were cultured in oatmeal/tomato media at 28℃ with 12-h/12-h light/dark cycles. Two weeks later, the hyphae were scratched, and the plates were continuously cultured at 28℃ with 24-h light for sporulation. After four days post-inoculation (dpi), spores were collected, and the inoculum concentration was adjusted to 5×10⁵spore ml^-1 for punch- or spray-inoculation. Three-leaf-stage seedlings were used for spray inoculation following previous reports (Qu et al. 2006). The disease symptom was observed at five dpi. The relative fungal biomass was measured using the DNA amounts of fungal Mopot2 against rice ubiquitin DNA amounts by reverse transcription-quantitative PCR (Li et al. 2017). GZ8 spores with concentration 2×10⁵ml^-1 were inoculated on the leaf sheaths to observe the infection process of M. oryzae as described previously (Kankanala et al. 2007). The epidermal layer was excised for observation at 24 and 36 hours post-inoculation (hpi). The fungal growth process was analyzed by Laser Scanning Confocal Microscopy (Nikon A1), and the infestation stage was analyzed as described previously (Li et al. 2014). 3, 3’-Diaminobenzidine (DAB) was used to stain H₂O₂ and fungal structures at 48 hpi (Xiao et al. 2003). Leaves were placed in DAB (1 mg /ml, Sigma, ALORICH, USA) and shook at room temperature for 12 h in a dark place, and then decolorized in 90% ethyl alcohol at 65°C for several times. H₂O₂ accumulation and fungal structures were observed with a microscope (Zeiss imager A2).

RNA Extraction and Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Analysis

Total RNA was extracted from leaves using Trizol reagent (Invitrogen). The RNA quality and quantity were determined by a spectrophotometer (NanoDrop2000 Uvevis). First-strand cDNA was synthesized using the qPCR RT Master Mix with gDNA Remover (TOYOBO). Stem-loop pulse RT-qPCR was performed to analyze the amounts of miR162 with universal reverse primer and specific forward primer listed in Additional file 4: Table S1 following previous reports (Chen et al. 2005; Varkonyi-Gasic et al. 2007). U6 snRNA was used as an internal reference to normalize miRNA levels (Turner et al. 2013). qPCR was performed with specific primers for the detection of gene expression (Additional file 4: Table S1), and the rice ubiquitin (UBQ) gene was selected as an internal reference gene.

M. oryzae:Magnaporthe oryzae; miRNAs: MicroRNAs; DCL1: Dicer-like 1; H₂O₂: hydrogen peroxide; nt: nucleotides; AGO: ARGONAUTE; miRISC: miRNA-Induced Silence Complex; OsTRE1: trehalase precursor 1; LTH: Lijiangxin Tuan Heigu; R: resistance; OX162: transgenic lines overexpressing Osa-miR162a; GZ8: GFP-tagged Zhong 8-10-14; KS4: ENT-KA RENE SYNTHASE 4; NAC4: NAC DOMAIN-CONTAINING PROTEIN 4; PR1a: PATHOGENESIS-RELATE GENG 1A; MIM162: the transgenic lines overexpressing a target mimic of miR162a; OsDUF292 domains of unknown function protein; DCL1i: OsDCL1 RNA inference line; OsSPL28: SQUAMOSA promoter-binding protein-like transcription factors 28; GRF: Growth Regulating Factor.

Acknowledgements

We thank Dr. Cai-Lin Lei (Institute of Crop Science, Chinese Academy of Agricultural Sciences) for providing the monogenic resistant lines IRBLkm-Ts, Dr. Xiao-Feng Cao (Institute of Genetics and Development, Chinese Academy of Sciences) for providing DCL1 RNAi transgenic lines.

Authors’ contributions

YL and WW designed the research; XL, XM, HW, YZ, and XL conducted the experiments; TL, YZ, JZ, JZ, YH, MP, HF and JF analyzed the data; YLwrote the article; WW supervised and complemented the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31430072, 31672090 and U19A2033 to WW).

Availability of data and materials

The data sets supporting the conclusions of this article are included within the article and its addition files.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

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Table 1: the agronomic traits of OX162 and MIM162

Strain

Plant height（cm）

Panicle number

Panicle length（cm）

Setting

Rate

（%）

Grain

number

1000-grain weight

（g）

Yield

per plant

（g）

NPB

91.4±2.4^a

9.2±0.44^a

19.6±0.74^a

92.7±0.93^a

833±45^a

26.9±0.63^a

22.3±2.35^ab

OX-11

90.8±2.16^a

9.4±1.34^a

19.5±0.63^a

87.4±1.52^a

850±74^a

23.4±0.56^c

20.7±3.90^b

OX-24

89.6±2.88^a

10.6±1.5^a

19.6±0.7^a

60.7±7.06^a

839±65^a

22.8±0.38^c

18.3±2.76^b

MIM-1

94.8±4.02^a

9.4±0.89^a

19.8±0.89^a

90.5±3.14^a

987±36^a

25.9±0.38^ab

24.1±3.35^a

MIM-16

93.7±4.4^a

10.4±0.89^a

19.2±0.94^a

85.9±0.77^a

999±71^a

25.4±0.3^b

24.4±3.75^a

Notes: Data are shown as mean ± SD (n = 5). Means labeled with different letters indicate significant difference at 5% level via Tukey-Kramer test.

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Osa-miR162a balances rice yield and resistance to Magnaporthe oryzae

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