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. 2017 Apr 2;19(3):607–614. doi: 10.1111/mpp.12546

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae

Xionghui Zhong 1,†, Jiuxia Yang 1,†, Yanlong Shi 1, Xuli Wang 1,✉, Guo‐Liang Wang 1,2,✉
PMCID: PMC6638105  PMID: 28220688

Summary

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.

Keywords: 26S proteasome pathway, DnaJ protein, E3 ligases, Magnaporthe oryzae, Oryza sativa, Pyricularia oryzae, rice blast

Introduction

Plant diseases are major constraints in crop production worldwide. To subvert pathogen attacks, plants have evolved a two‐branched innate immune system: microbial‐ or pathogen‐associated molecular pattern (MAMP or PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI). In the first branch, PAMPs are perceived by pattern recognition receptors (PRRs) on plant cell membranes, resulting in PTI to limit pathogen ingress (Jones and Dangl, 2006). This recognition triggers rapid and transient production of reactive oxygen species (ROS) in plant tissues and confers basal resistance to pathogen infection (Segonzac et al., 2011). As a result of co‐evolution, some pathogens can counteract PTI through the secretion of effectors into the host cell. However, these effectors can be recognized by cognate resistance (R) proteins of plants, leading to the activation of ETI responses. ETI often results in a hypersensitive reaction (HR) and confers high levels of resistance to pathogens (Coll et al., 2011; Heath, 2000).

Heat shock protein 40 (HSP40), as an important component of the heat shock protein machinery, plays a vital role in a variety of abiotic and biotic stress responses (Salasmunoz et al., 2016; Xia et al., 2014), growth and development processes (Shen et al., 2011). HSP40 protein is also termed DnaJ or J protein because it contains a conserved ∼70‐amino‐acid‐long J domain, which has a central tripeptide of histidine (His), proline (Pro) and aspartic acid (Asp) (the HPD motif). DnaJs are categorized into four types on the basis of the presence of specific conserved domains. The prototypical type I DnaJ proteins contain four structural domains: a J domain in the N‐terminus, followed by a glycine/phenylalanine (G/F)‐rich region, a zinc‐finger domain containing four repeats of the motif CXXCXGXG (X represents any amino acid) and a less conserved C‐terminal peptide‐binding domain (CTD) (Shi et al., 2005). Type II DnaJ proteins lack the zinc‐finger domain of type I (Walsh et al., 2004). Type III DnaJ proteins only contain the J domain, which could be present in any part of the protein sequence (Knox et al., 2011). In addition, ‘J‐like proteins’ have been designated as Type IV J proteins because they lack the central HPD motif (Kampinga and Craig, 2010).

Many DnaJ proteins have been reported from a variety of organisms, and a growing body of evidence indicates that DnaJ proteins also play important roles in plant immunity. For example, the association between the capsid protein (CP) and host DnaJ‐like proteins (HSP40) is essential for Potato virus Y infection of tobacco (Hofius et al., 2007). Similarly, the movement protein (MP) of Tomato spotted wilt virus mediates recruitment of host DnaJ‐like proteins for viral intercellular transportation (Soellick et al., 2000). The tobacco type I DnaJ protein NbMIP1 functions as a co‐chaperone during virus infection and plant immunity (Du et al., 2013). In addition, the soybean type III DnaJ protein GmHSP40 participates in HR‐like cell death and disease resistance in soybean (Liu and Whitham, 2013). Another example is the tomato chloroplast‐localized DnaJ gene LeCDJ2 which functions in drought tolerance and resistance to Pseudomonas solanacearum in transgenic tobacco (Wang et al., 2014).

Rice (Oryza sativa), one of the most important staple foods, is attacked by many pathogens, causing significant yield losses and threatening global food security. The control of these diseases depends on an understanding of rice resistance mechanisms. The rice genome includes 104 DnaJ genes (Sarkar et al., 2013), but only a few of these genes have been functionally characterized. OsDjA7/8, the first characterized DnaJ gene, is involved in DNA replication and repair by interaction with the proliferating cell nuclear antigen (PCNA) gene (Yamamoto et al., 2005). OsDjA7/8 also functions in chloroplast development in rice (Zhu et al., 2015). Some functions of the DnaJ protein RNB8 (NM_001060020) have also been characterized; it facilitates cell‐to‐cell movement of virus by interacting with Pc4 (a putative MP of Rice stripe virus) (Lu et al., 2009). However, the function of the rice DnaJs in response to fungal pathogens has not yet been reported. In this study, we used reverse genetic analysis to demonstrate the role of the OsDjA6 gene in resistance to Magnaporthe oryzae, a fungal pathogen that causes rice blast disease and threatens rice production worldwide (Dean et al., 2005). The expression of OsDjA6 in rice is significantly induced after M. oryzae infection. OsDjA6 RNA interference (RNAi) plants exhibit an increased ROS burst and higher resistance to M. oryzae. We also revealed that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. Our results reveal that OsDjA6 negatively regulates rice innate immunity, probably via the 26S proteasome pathway.

Results

RNA expression of OsDjA6 is increased on M. oryzae infection

To assess whether DnaJ genes contribute to the defence response to M. oryzae, we analysed the expression data of the whole family of genes in the rice massively parallel signature sequencing (MPSS) database (Nakano et al., 2006). The expression of 29 DnaJ genes was up‐regulated at 6, 12 and 24 h in compatible and incompatible interactions. We generated RNAi lines for six DnaJ genes and evaluated their resistance to M. oryzae. Among them, OsDjA6 RNAi lines displayed significant enhanced resistance compared with wild‐type plants to M. oryzae. OsDjA6 was induced in the resistance and susceptible reaction. The expression of OsDjA6 was validated by quantitative real‐time polymerase chain reaction (qRT‐PCR) using cDNA samples of the rice cultivar Nipponbare that had been infected with the compatible M. oryzae isolate RO1‐1, the incompatible isolate C9240 or 0.05% Tween‐20; the RNA samples were obtained at 0, 12, 24, 48, 72, 96 and 120 h after inoculation. In accordance with the MPSS results, the expression level of OsDjA6 began to increase at 12 h and peaked at 96 h in the compatible reaction; however, it showed no significant change in the incompatible reaction or in the 0.05% Tween‐20 control (Fig. 1). These results indicate that OsDjA6 can be beneficial to M. oryzae infection.

Figure 1.

Figure 1

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Expression analysis of OsDjA6 in Nipponbare after Magnaporthe oryzae inoculation. Quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis of the time course of OsDjA6 RNA expression after plants had been inoculated with isolate RO1‐1, C9240 or 0.05% Tween‐20. Each bar represents mean ± standard deviation (SD) (n = 3).

Subcellular localization of OsDjA6 in rice protoplasts

To investigate the subcellular localization of OsDjA6, we fused the green fluorescent protein (GFP) gene to the N‐terminus of the OsDjA6 coding region. The derived construct was transiently expressed in rice protoplasts by the polyethylene glycol (PEG) method (Zhang et al., 2011). Similar to the GFP protein, OsDjA6‐GFP was detected in both the nucleus and cytoplasm of the transfected rice protoplasts (Fig. 2).

Figure 2.

Figure 2

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Subcellular localization of OsDjA6 in rice protoplasts. PYBA1155 and OsDjA6‐PYBA1155 plasmids were transformed into rice protoplasts with the polyethylene glycol (PEG) method, and the transformed protoplasts were observed under a confocal microscope after 24 h. Bar, 20 μm.

Knockdown of OsDjA6 enhances resistance to M. oryzae

To explore the biological function of OsDjA6 in rice immunity, we generated the OsDjA6 RNAi construct, which was designed to target the nucleotide sequence 622–918 of OsDjA6. The construct was inserted into the japonica cultivar TG394 via Agrobacterium tumefaciens‐mediated transformation. We obtained 42 independent T0 lines and selected three homozygous T3 lines (RNAi‐4, RNAi‐9, RNAi‐18) for molecular and phenotypic analyses, in which OsDjA6 expression was significantly reduced (Fig. 3A). OsDjA6 RNAi plants were morphologically similar to wild‐type TG394 plants. When the 3‐week‐old plants were infected with compatible M. oryzae isolate RO1‐1, many more disease lesions were observed in TG394 plants than in RNAi plants at 7 days after spray inoculation (Fig. 3C). To confirm the results obtained from spray inoculation, we performed a punch inoculation and measured the basal resistance levels of the transgenic plants (Takahashi et al., 1999). After punch inoculation, we found that the disease lesion area were larger in wild‐type plants than in RNAi plants (Fig. 3B, D). We quantified sporulation on the infected leaves. The number of M. oryzae spores generated in infected leaves was about two‐fold greater in TG394 plants than in OsDjA6 RNAi plants (Fig. 3E). We also quantified the fungal biomass in the inoculated leaves using DNA‐based qPCR analysis. The relative fungal biomass (MgPot2/OsUBQ) was about three‐fold higher in wild‐type TG394 leaves than in OsDjA6 RNAi leaves (Fig. 3F). In conclusion, these data demonstrate that knockdown of OsDjA6 in rice plants increases resistance to M. oryzae. Thus, OsDjA6 is a negative regulator of rice basal resistance.

Figure 3.

Figure 3

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OsDjA6 RNA interference (RNAi) plants are resistant to Magnaporthe oryzae. (A) Expression levels of the OsDjA6 gene in three T3 homozygous RNAi lines (RNAi‐4, RNAi‐9, RNAi‐18) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR). (B, C) Resistance of OsDjA6 RNAi plants to M. oryzae isolate RO1‐1 after punch and spray inoculation. (D) Lesion sizes of OsDjA6 RNAi plants compared with wild‐type TG394 plants after punch inoculation. (E) Number of spores produced on lesions of wild‐type TG394 and OsDjA6 RNAi plants after punch inoculation. (F) Quantification analysis of relative fungal biomass in wild‐type TG394 and OsDjA6 RNAi plants after punch inoculation. OsUBQ was used as an internal control. Each bar represents the mean ± standard deviation (SD) (n = 3). Asterisks indicate significant differences analysed by Student's t‐test (*P < 0.05, **P < 0.01, ***P < 0.0001). WT, wild‐type.

Silencing of OsDjA6 enhances PAMP‐triggered ROS production in rice leaves

To further investigate the function of OsDjA6 in the early molecular events of PTI, we measured ROS generation in OsDjA6 RNAi plants after flg22 and chitin treatments; these PAMP elicitors trigger OsFLS2‐ and OsCEBiP/OsCERK1‐mediated PTI signalling in rice (Kaku et al., 2006; Takai et al., 2008). Silencing of OsDjA6 led to a four‐fold higher ROS production relative to the wild‐type TG394 after flg22 treatment. The ROS level peaked at 14 min after flg22 treatment (Fig. 4A). Chitin‐induced ROS accumulation was two‐fold higher in OsDjA6 RNAi plants than in mock‐treated plants (Fig. 4B). These results indicate that silencing of OsDjA6 in rice enhances the flg22‐ and chitin‐induced ROS burst, further confirming the role of OsDjA6 in the suppression of the PTI response in rice.

Figure 4.

Figure 4

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Reactive oxygen species (ROS) production and expression of defence‐related genes in TG394 and OsDjA6 RNA interference (RNAi) plants. (A, B) ROS production in TG394 and OsDjA6 RNAi plants after flg22 and chitin treatments. Water treatment was used as control. (C–E) Expression analysis of defence‐related genes WRKY45 (C), NPR1 (D) and PR5 (E) in TG394 and OsDjA6 RNAi plants. Each bar represents the mean ± standard deviation (SD) (n = 3). Asterisks indicate significant differences analysed by Student's t‐test (*P < 0.05, **P < 0.01, ***P < 0.0001).

To test whether knockdown of OsDjA6 affected the expression of defence‐related genes in the salicylic acid (SA) pathway, we quantified the RNA levels of WRKY45, NPR1 and PR5 in OsDjA6 RNAi and TG394 plants. The RNA levels of these three genes were increased by two‐ to four‐fold in OsDjA6 RNAi lines relative to TG394 plants (Fig. 4C–E). Taken together, these results suggest that OsDjA6 acts as a negative regulator in the rice PTI response through the inhibition of ROS accumulation and the expression of defence‐related genes in the SA‐mediated pathway.

OsDjA6 interacts with the E3 ligase OsZFP1 in vitro and in vivo

To further assess the mechanism by which OsDjA6 suppresses rice resistance to M. oryzae, we identified OsDjA6‐associated proteins in rice using the yeast two‐hybrid screen. We fused the OsDjA6 coding region to the GAL4 DNA‐binding domain in the pBD‐Gal4 vector and used the construct as the bait for screening against a rice cDNA library fused to the yeast GAL4 activation domain in the pAD‐Gal4 plasmid (Vega‐Sanchez et al., 2008). Fourteen unique clones were obtained after sequencing over 200 transformants (Table 1). Interestingly, a plasmid harbouring an 1110‐bp rice cDNA that encodes a zinc finger C3H2C3‐type domain‐containing protein, designated as OsZFP1 (LOC_Os01g47740), appeared 36 times in the transformants. OsZFP1 belongs to the C3H2C3‐type RING protein family containing a single conserved RING domain with the sequence Cys‐X2‐Cys‐X14‐Cys‐X1‐His‐X2‐His‐X2‐Cys‐X10‐Cys‐X2‐Cys (X represents any amino acid) in its C‐terminal region. The 41‐amino‐acid RING domain is highly conserved among different plants.

Table 1.

Summary of OsDjA6‐interacting proteins identified by yeast two‐hybrid assays.

Gene locus Annotation
LOC_Os10g27050.1 OsPP2Ac‐4 – phosphatase 2A isoform 4 belonging to family 2, expressed
LOC_Os02g44360.1 Scarecrow transcription factor family protein, putative, expressed
LOC_Os05g03480.2 Acyl‐coenzyme A dehydrogenase, mitochondrial precursor, putative, expressed
LOC_Os06g05100.2 Transketolase, putative, expressed
LOC_Os03g15050.1 Phosphoenolpyruvate carboxykinase, putative, expressed
LOC_Os01g56580.1 CK1_CaseinKinase_1a.3 – CK1 includes the casein kinase 1 kinases, expressed
LOC_Os06g07350.1 RNA‐binding motif protein, putative, expressed
LOC_Os03g57340.1 Chaperone protein DnaJ, putative, expressed
LOC_Os10g01134.2 OsSCP46 – putative serine carboxypeptidase homologue, expressed
LOC_Os01g29430.2 Tetratricopeptide‐like helical, putative, expressed
LOC_Os02g32030.1 Elongation factor, putative, expressed
LOC_Os03g08010.1 Elongation factor Tu, putative, expressed
LOC_Os04g53620 Ubiquitin family protein, putative, expressed
LOC_Os01g47740.1 Zinc finger, C3HC4‐type domain‐containing protein, expressed
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Primers corresponding to the full‐length coding region were subsequently designed to amplify the gene from rice cDNA (Table S1, see Supporting Information). The open reading frame (ORF) of this gene consisted of a 1572‐bp sequence encoding 524 amino acids. We further confirmed the interaction between OsDjA6 and full‐length OsZFP1 in yeast (BD‐OsDjA6+AD‐OsZFP1) (Fig. 5A).

Figure 5.

Figure 5

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OsDjA6 interacts with OsZFP1 in vitro and in vivo. (A) Yeast two‐hybrid assays of OsDjA6 and OsZFP1. The transformants of Mav203 yeast were diluted 10, 100 and 1000 times, and grown on selective plates without leucine (Leu), tryptophan (Trp) or histidine (His) (SD‐LWH) and with 0 or 40 mm 3‐aminotriazole (3AT). (B) The cLUC‐OsZFP1 and cLUC‐OsZFP1 (H495Y) plasmids were transiently expressed in Nicotiana benthamiana using the agroinfiltration method. The infiltrated leaves were harvested at 48 h after infiltration. MG132 (50 μm) was infiltrated at 18 h before harvesting. (C) Co‐immunoprecipitation (Co‐IP) assay of OsDjA6 and OsZFP1 (H495Y) in N. benthamiana. The Co‐IP assay (IP) was carried out with anti‐haemagglutinin (anti‐HA) beads, and the proteins were analysed by western blot (IB) with anti‐HA and anti‐cLUC antibodies.

To determine whether this interaction occurs in planta, we conducted co‐immunoprecipitation (Co‐IP) experiments in Nicotiana benthamiana. In a preliminary experiment, OsZFP1 cDNA was cloned into a binary expression vector fused to the N‐terminal cLUC epitope tag (Chen et al., 2008). The cLUC‐OsZFP1 plasmid was expressed in N. benthamiana using the agroinfiltration method. Leaf tissues were collected 2 days after agroinfiltration to determine whether the OsZFP1 protein could be stably expressed. Intriguingly, OsZFP1 was totally degraded 2 days after agroinfiltration (Fig. 5B), which is similar to the case of the E3 ligase APIP6 (Park et al., 2012). Then, we generated a mutant protein OsZFP1 (H495Y) by replacing the His residue with the tyrosine (Tyr) residue in the RING domain at the amino acid position 495 of OsZFP1. cLUC‐OsZFP1 (H495Y) was efficiently expressed in agroinfiltrated leaves. To determine the interaction between OsZFP1 and OsDjA6 in vivo, we co‐expressed cLUC‐OsZFP1 (H495Y) plasmids with OsDjA6‐HA plasmids or empty vector (EV) in N. benthamiana leaves. cLUC‐OsZFP1 (H495Y) and OsDjA6‐HA proteins were detected in the total protein extract, confirming the expression of these proteins (Fig. 5C, left panel). cLUC‐OsZFP was co‐immunoprecipitated when the OsDjA6‐HA fusion protein was immunoprecipitated with anti‐haemagglutinin (anti‐HA) antibody coupled to beads, but not the HA protein alone (Fig. 5C, right panel). Taken together, these results show that OsDjA6 interacts with OsZFP1 in planta.

Discussion

OsDjA6 negatively regulates rice resistance to M. oryzae

Many DnaJ proteins are involved in human diseases (Kakkar et al., 2012; Mitra et al., 2009). However, only a few DnaJs have been reported to play a role in plant immunity. The type III DanJ protein GmHSP40 in soybean, for example, plays a positive role in HR‐like cell death and disease resistance (Liu and Whitham, 2013). In another example, HSP40 is not only essential for host cell antiviral function, but also benefits virus replication (Knox et al., 2011). In addition, the tobacco type I DnaJ protein NbMIP1 is a specialized co‐chaperone with crucial roles during both Tobacco mosaic virus infection and plant innate immunity (Du et al., 2013). In this study, we have demonstrated that the chaperone DnaJ OsDjA6 participates in rice innate immunity from the following evidence. First, we found that OsDjA6 expression is highly induced in rice plants after infection with the compatible M. oryzae isolate RO1‐1 (Fig. 1), a finding that is consistent with the MPSS database (Nakano et al., 2006). This result suggests that M. oryzae might target OsDjA6 to inhibit the defence response during infection. Second, we performed spray and punch inoculations to investigate whether the reduction of OsDjA6 transcription in OsDjA6 RNAi plants affects basal resistance to M. oryzae. The results show that OsDjA6 RNAi plants are more resistant than wild‐type plants to M. oryzae (Fig. 3). In contrast, overexpression of OsDjA6 does not affect the resistance to M. oryzae compared with wild‐type plants (data not shown). Finally, the levels of ROS and defence‐related gene expression were constitutively up‐regulated in the OsDjA6 RNAi lines (Fig. 4). Taken together, these results indicate that OsDjA6 may negatively regulate rice resistance to M. oryzae. However, we cannot rule out the possibility that OsDjA6 may contribute to M. oryzae pathogenesis as a susceptibility gene in rice.

OsDjA6 participates in flg22‐ and chitin‐induced PTI in rice

flg22, a 22‐amino‐acid peptide, is from a conserved domain of bacterial flagellin (Meindl et al., 2000). Chitin, a long‐chain polymer of an N‐acetyl‐d‐glucosamine, is a typical component of the fungal cell wall (Reese et al., 2007). Both flg22 and chitin can trigger PTI in plants. To evaluate the role of OsDjA6 in PTI responses, we measured ROS production after flg22 and chitin treatments. Our results showed that OsDjA6 RNAi plants accumulate ROS to a higher level than wild‐type plants even without elicitor treatments, indicating that OsDjA6 functions as a suppressor of ROS accumulation in rice (Fig. 4).

Several studies have demonstrated that protein phosphatases negatively control the PTI response by modulating the state of phosphorylation of the PRR complex. For example, Arabidopsis protein phosphatase 2A (PP2A) negatively regulates plant immunity by controlling the phosphorylation state of the positive regulator BAK1 (Segonzac et al., 2014). Moreover, Arabidopsis protein phosphatase 2C (PP2C), a kinase‐associated protein phosphatase (KAPP), interacts with the FLS2 cytoplasmic domain in the yeast system, and its overexpression lines result in flg22 insensitivity (Gomezgomez et al., 2001). XB15, a PP2C, dephosphorylates XA21 in vitro, and negatively regulates cell death and XA21‐mediated immune responses in rice (Park et al., 2008). In our preliminary assays, we found that OsDjA6 interacts with OsPP2Ac‐4 in the yeast two‐hybrid screen (data not shown). Further analysis of the function of OsPP2Ac‐4 in rice innate immunity and of its relationship with OsDjA6 is required. In addition, the expression analysis shows that WRKY45, NPR1 and PR5 are highly up‐regulated in OsDjA6 RNAi plants. These results indicate that OsDjA6 might suppress the PTI response by modulating the SA‐mediated defence pathway.

OsDjA6 may bring substrate proteins to the E3 ligase OsZFP1 for degradation

Molecular chaperones are also known as protein folding and assembly factors. DnaJ proteins generally recruit and deliver client interacting targets to HSP70 (Summers et al., 2009). Several chaperone‐assisted degradation pathways have also been reported in mammalian cells, including CAP (chaperone‐assisted proteasomal degradation), CASA (chaperone‐assisted selective autophagy) and CMA (chaperone‐mediated autophagy) (Kettern et al., 2010). In our yeast two‐hybrid screens, we did not find any putative chaperone OsHSP70, but we did find the E3 ligase OsZFP1. The association of OsDjA6 with OsZFP1 was further confirmed by Co‐IP assay in N. benthamiana (Fig. 5). Recent studies have shown that molecular chaperones can assist with the ubiquitin/proteasome system for protein quality control in eukaryotic cells (Esser et al., 2004; Howarth et al., 2007). We therefore hypothesize that OsDjA6 may bring substrate proteins to OsZFP1 in order to regulate cell homeostasis through chaperone‐assisted proteasomal degradation. Further studies are needed to determine the biochemical association between possible targets of OsDjA6 and OsZFP1. In addition, generation of the rice OsZFP1 mutant will help researchers to determine the function of OsZFP1 in rice immune responses.

Experimental Procedures

Plant materials and growth conditions

Rice seeds were surface sterilized with 75% ethanol for 1 min, transferred to 2% sodium hypochlorite for 0.5 h, washed with distilled water three times and then placed on half‐strength Murashige and Skoog (MS) medium. Plants were transplanted into soil after germination, and maintained in a growth chamber at 27 °C and 80% relative humidity under a photoperiod of 12 h light/12 h dark.

RNA extraction and gene expression analysis

Total RNA was isolated from rice leaves using TRIzol® Reagent (Invitrogen), and treated with RNase‐free DNase I (Qiagen) to remove genomic DNA. RNA concentration and purity were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). One microgram of DNaseI‐treated total RNA was used to synthesize the first‐strand cDNA with a reverse transcriptase (Promega). Target gene‐specific primers were designed according to the Universal Probe Library. qRT‐PCR was performed on a MyiQ™2 two‐colour real‐time PCR detection system (Bio‐Rad) using SYBR Premix Ex Taq (Perfect Real Time; TaKaRa). The expression levels of target genes were normalized to the internal control, i.e. the rice ubiquitin gene OsUBQ. Primers for qRT‐PCR assays are given in Table S1.

Subcellular localization in rice protoplasts

To generate the OsDjA6‐PYBA1155 construct, the full length of the OsDjA6 ORF from TG394 leaf cDNA was amplified using primers OsDjA6‐GFP‐F/R. The fragment was cloned into the PYBA1155 vector after digestion with BglII and HindIII. Rice protoplasts were transfected with 1.0 µg of PYBA1155 or OsDjA6‐PYBA1155 plasmid, respectively, by the PEG method (Zhang et al., 2011). The transfected rice protoplasts were observed with a confocal laser scanning microscope after 24 h (Zeiss LSM Confocal, Oberkochen, Germany).

Protein extraction, immunoblot analysis and immunoprecipitation assays

Plasmids containing HA or cLUC tag constructs were transiently co‐expressed in N. benthamiana using the agroinfiltration method. Protein extraction, immunoblot and immunoprecipitation were performed as described previously (Yoo et al., 2007; Zhang et al., 2011). In brief, total proteins were extracted with 0.1 mL of native protein extraction buffer [50 mm Tris‐HCl, pH 7.5, 0.5 m sucrose, 1 mm MgCl2, 10 mm ethylenediaminetetraacetic acid (EDTA), 5 mm dithiothreitol (DTT), protease inhibitor cocktail] and incubated at 4 °C for 20 min. Samples were centrifuged at 15 000 g at 4 °C for 15 min. Supernatants were incubated with 0.25 mg of pre‐washed Pierce anti‐HA magnetic beads (Thermo Fisher Scientific) at room temperature for 2 h with mixing. Following incubation, beads were washed several times with pre‐cooled TBS‐T buffer (20 mM Tris‐HCl, pH 7.5, 0.1 M NaCl, 0.05% Tween‐20). Finally, 50 mm NaOH was used to elute the HA‐tagged protein. Total proteins (input) or immunoprecipitated proteins were separated using 10% sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a nitrocellulose membrane (Thermo Fisher Scientific). HA and cLUC fusion proteins were detected by western blot with anti‐HA and anti‐cLUC primary antibodies, respectively.

Plasmid construction

The DNA constructs used in this study were generated following standard molecular biology protocols or one‐step cloning (Toyobo). Primers for DNA constructs are listed in Table S1.

Rice blast inoculation and disease resistance evaluations

Isolates C9240 and RO1‐1 of M. oryzae used in this study were cultivated as described previously (Park et al., 2012). For punch inoculation, leaves from 6‐week‐old rice seedlings were punched with a mouse ear, and 10 μL of a spore suspension (5.0 × 105 spores/mL) were added to the wounding site, and then wrapped with transparent tape. Lesions were photographed and measured at 9–12 days after inoculation. Quantification of M. oryzae sporulation on lesions was performed according to a procedure described previously (Ding et al., 2012). The relative fungal biomass was determined using DNA‐based qPCR as reported previously (Akamatsu et al., 2013). In brief, the relative fungal biomass was calculated from the ratio of M. oryzae Pot2 DNA and rice genomic ubiquitin DNA in the inoculated leaves. For spray inoculation, 3‐week‐old seedlings were spray inoculated with a spore suspension (1.0 × 105 spores/mL) in 0.05% Tween‐20, and the infected plants were kept in a high‐humidity chamber; disease phenotypes were evaluated at 6–7 days after inoculation (Liu et al., 2002).

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's website.

Table S1 Primers used in this work.

Click here for additional data file. (17.3KB, docx)

Acknowledgements

This study was supported by the Natural Science Foundation of China (31471737), the China Postdoctoral Science Foundation (2016M590161) and the China Scholarship Council (201503250011).

Contributor Information

Xuli Wang, Email: wangxuli@caas.cn.

Guo‐Liang Wang, Email: wang.620@osu.edu.

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Table S1 Primers used in this work.

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