Osa-miR162a is differentially responsive to M. oryzae in susceptible and resistant accessions
To explore the involvement of Osa-miR162a in rice blast resistance, we examined the amounts of Osa-miR162a in a highly susceptible accession Lijiang xin 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 with serious disease lesions, whereas IRBLkm-Ts showed resistant phenotype with few small lesions (Fig. 1a). LTH displayed unchanged or decreased Osa-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 Osa-miR162a was involved in rice-M. oryzae interaction.
Overexpression of Osa-miR162a enhances rice resistance to M. oryzae
To explore the roles of Osa-miR162a in rice blast resistance, we constructed transgenic lines overexpressing Osa-miR162a (OX162) under Nipponbare (NPB) background. We got over 20 transgenic lines displaying increased amounts of mature Osa-miR162a in comparison with NPB control. We selected two lines showing moderate accumulation of Osa-miR162a and producing seeds, namely OX162-11 and OX162-24, for subsequent study (Fig. 2a). We then examined the blast disease phenotype of these lines following the punch-inoculation of two virulent strains, namely GZ8 and 97-27-2. GZ8 was an enhanced Green Fluorescence Protein (GFP)-tagged strain Zhong 8-10-14, which was derived from the paddy yard in China, and 97-27-2 was a strain derived from the paddy yard in Sichuan Province, China. OX162 lines displayed enhanced resistance 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 from paddy yard in Sichuan Province, China. Similarly, OX162 displayed smaller disease lesions than control (Additional file 2: Figure S2). These results indicate that overexpression of Osa-miR162a enhances rice blast resistance.
To understand how OX162 lines suppress fungal growth, we observed the infection process of GZ8 in leaf sheath. At 24 hpi, more than 55 % of spores formed appressoria, and quite a few of which formed invasive hyphae in sheath cells of WT. However, in OX162, less than 50 % of spores developed appressoria, and no invasive hyphae were observed (Fig. 2d). At 36 hpi, more than 50 % of the spores started to infect the neighbor cells near the local infected cell of WT, whereas less than 20 % of the spores invaded in the neighbor cells of OX162 (Fig. 2d). The quantification assay confirmed that overexpression of Osa-miR162a delayed the infection progress of blast fungus (Fig. 2e).
To understand why overexpression of Osa-miR162a delayed the infection of M. oryzae, we examined immune responses, such as H2O2 accumulation and induction of defense-related genes in OX162 and control. OX162 showed more H2O2 accumulation in the leaf cells around the infected sites than NPB following 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 in OX162 and NPB. 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 induction of KS4 was enhanced to higher levels in OX162 than in NPB control at 12, 24 and 48 hpi, and NAC4 was enhanced to higher levels in OX162 than in NPB control at 12 hpi and subsequently decreased at 24 hpi, then enhanced at 48 hpi again (Fig. 3b). Similarly, the expression of PR1a and PR10b was significantly induced to higher levels at 12 and 48 hpi than that in NPB control (Fig. 3b). These data indicate that overexpression of Osa-miR162a enhanced the immune responses triggered by M. oryzae.
Blocking Osa-miR162a leads to increased blast disease susceptibility
To further confirm the roles of Osa-miR162a in rice immunity again blast fungus, we constructed the transgenic lines overexpressing a target mimic of Osa-miR162a (MIM162). MIM162 contained a sequence reversely complementary to Osa-miR162a with three nucleotides insertion between 10 to 11 nucleotide sites. Therefore, the target mimic acted as a sponge to absorb Osa-miR162 and block its binding to target genes (Additional file 3: Figure S3). We then examined the amount of Osa-miR162a in MIM162. MIM162 displayed significantly less accumulation of Osa-miR162a than NPB control (Fig. 4a). Next, we conducted a disease assay on MIM162. As expected, MIM162 showed enhanced susceptibility to M. oryzae with larger disease lesions following punch- or spray-inoculation (Fig. 4b and c, and Additional file 1: Figure S1). We also examined the disease phenotypes of MIM162 against four M. oryzae strains derived from the paddy yards by punch-inoculation. Similarly, 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. At 24 hpi, more than 70 % of spores formed appressoria or invasive hyphae in MIM162, in comparison with less than 60 % of that in NPB control (Fig. 4d and e). At 36 hpi, more than 75 % of spores invaded into the neighbor cells in MIM162, in comparison with less than 60% of that in NPB (Fig. 4d and e). These results indicate that blocking Osa-miR162 facilitates the invasion of M. oryzae.
We also examined the defense responses in MIM162. NPB plant showed visible H2O2 accumulation at the infected sites of leaves and cells following GZ8 infection, whereas MIM162 displayed little H2O2 amounts at the invasive sites (Fig. 5a). Consistently, the induction of defense-related genes, KS4, NAC4, PR1a, and PR10b, was lower in MIM162 than that in NPB (Fig. 5b). These data indicate that blocking Osa-miR162 compromises immune responses induced by M. oryzae.
Osa-miR162a regulates rice yield traits
We found that Osa-miR162a also controlled rice 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 slight 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 Osa-miR162a compromises yield, whereas blocking Osa-miR162a enhances yield.
OsDCL1 is involved in Osa-miR162-regulated rice blast resistance
Rice Osa-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 Osa-miR162 and the two genes in 11 independent OX162 lines and 11 independent MIM162 lines, respectively. All the 22 detected lines displayed normal gross morphology and produced seeds. Seven of the 11 tested OX162 lines showed significantly higher Osa-miR162 accumulation (Fig. 7a). Conversely, six of the seven lines displayed significant decrease, and the one line showed slight decrease of OsDCL1 mRNA levels (Fig. 7a). Besides, among the 11 tested MIM162 lines, nine lines displayed significantly lower Osa-miR162 abundance in comparison with NPB control (Fig. 7b). Conversely, five of the nine lines displayed significant increase of OsDCL1 mRNA amounts, and three of the nine lines showed slight increase (Fig. 7b). These results indicated the regulation of Osa-miR162a on OsDCL1.
Intriguingly, the mRNA levels of OsDCL1 decreased mildly in all the seven lines overexpressing Osa-miR162 (Fig. 7a). Even in the four OX162 lines showing over 40-fold amounts of Osa-miR162a, the mRNA levels of OsDCL1 only decreased less than 50 % of that of control (Fig. 7a), indicating that OsDCL1 is critical for rice normal development, and Osa-miR162 had a limited impact on the expression of OsDCL1 in these lines. However, the mRNA levels of OsDUF292 were markedly higher in both OX162 and MIM162 (Additional file3：Figure S3b), suggesting Osa-miR162 may not directly target OsDUF292.
To 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 M. oryzae (Fig. 7c), and were reversely consistent with the amounts of Osa-miR162a in LTH following inoculation (Fig. 1b), indicating OsDCL1 was involved in rice resistance to blast fungus and down-regulated by Osa-miR162a. In contrast, The mRNA amounts of OsDCL1 were decreased in IRBLKm-Ts and Yahui2115 (Fig. 7c), further suggesting the involvement of OsDCL1 in rice resistance to M. oryzae. We then examined the roles of OsDCL1 in rice blast resistance by inoculation of GZ8 on an OsDCL1 RNA inference line (DCL1i, (Liu et al. 2005)) showing significantly lower OsDCL1 mRNA amounts than control (Fig. 7d). DCL1i line exhibited enhanced resistance to GZ8 with smaller disease lesions and supported less fungal growth (Fig. 7e and f), which was similar to the disease phenotype of OX162, and was consistent with the previous report that down-regulation of OsDCL1 leads to enhanced resistance to blast fungus (Zhang et al. 2015). Together, these results indicate that OsDCL1 possibly participates in Osa-miR162-regulated rice resistance against blast fungus.