Osmotic stress activated protein kinase 5 (SAPK5) positively regulates immune response and blast resistance in rice


 Background

The sucrose non-fermenting 1 (SNF1) related protein kinase 2 (SnRK2) family proteins are plant-specific Ser/Thr protein kinases that are mainly involved in response to abiotic stress. However, their regulation roles on immune response and disease resistance in crops are largely unknown.
Results

In this study, we report the characterization of a rice SnRK2 family member, osmotic stress activated protein kinases 5 (SAPK5), on plant immune response. The SAPK5 protein is localized in cytoplasm and possesses kinase activity. The kinase activity of SAPK5 is promoted upon treatment with chitin, a conserved pathogen-associated molecular pattern (PAMP). Knockout of SAPK5 compromises rice immune response and resistance against the fungal pathogen Magnaporthe oryzae.
Conclusions

Our study demonstrates that SAPK5 positively regulates immune response and disease resistance in rice and provides new insights into the regulatory roles of SnRK2 proteins on plant immunity.


Background
The sucrose non-fermenting 1 (SNF1) protein kinase family from plant belongs to a class of Ser/Ter protein kinase which is named as SNF1 in yeast, AMP activated protein kinase (AMPK) in mammals and SNF1-related protein kinase (SnRK) in plants and green algae (Mao et al. 2019). The proteins in this family are evolutionarily conserved and can be divided into three subfamilies, namely SnRK1, SnRK2 and SnRK3, based on sequence similarity and domain structure (Hrabak et al. 2003). Members from plant SnRK1 subfamily are orthologs of the SNF1/AMPK which mainly regulate cellular sugar/energy homeostasis (Martínez-Barajas and Coello 2020). The proteins form a complex by employing SnRK1α as the catalytic subunit, SnRK1β and SnRK1γ as the regulatory subunits to phosphorylate key enzymes of the related metabolic processes (Martínez-Barajas and Coello 2020). Members from plant SnRK3 subfamily are usually able to interact with calcineurin B-like proteins (CBL) thus also termed as CBLinteracting protein kinases (CIPK) (Yu et al. 2014). The CBL-CIPK/SnRK3 signaling pathway is a Ca²dependent pathway that regulates response to both biotic and abiotic stimuli (Yu et al. 2014). Members from plant SnRK2 subfamily usually participate in response to various abiotic stresses. For examples, SnRK2.3 regulates drought response in Arabidopsis; SAPK8 and TaSnRK2.3 regulate freezing, drought and salt tolerance of rice and wheat respectively (Tian et al. 2013;Tan et al. 2018;Zhong et al. 2020).
Members of SnRK2 subfamily can further be classed into three groups, including the group I of which the kinase activity is not activated by abscisic acid (ABA), the group II of which the kinase activity is not activated or activated weakly by ABA, and the group III of which the kinase activity is strongly activated by ABA (Mao et al. 2019). Ten members from SnRK2 subfamily have been identi ed in rice by homology searching the rice genome databases. They are designated as osmotic stress activated protein kinases (SAPKs) based on their characteristics reveled (Kobayashi et al. 2004). These ten kinases, i.e. SAPK1 through SAPK10, can be divided into three subclasses: subclass I contains SAPK4, 5, 6 and 7, subclass II consists of SAPK1, 2 and 3, and subclass III comprises SAPK8, 9 and 10 (Kobayashi et al. 2004;Kulik et al. 2011). All SAPK members are activated by hyperosmotic stress, and three of these proteins, SAPK8, SAPK9 and SAPK10, are activated by ABA (Kobayashi et al. 2004).
Protein kinases catalyze the phosphorylation of certain proteins by adding phosphate group(s) to substrate(s), whereby regulating various biological processes including plant immunity (Park et al. 2012;Meng and Zhang 2013). As a class of Ser/Thr protein kinases, SnRKs have also been predicted to regulate plant immunity. The SnRK1 subfamily members SnRK1α/β/γ have been linked to resistance against diverse pathogens including virus, bacterium and fungus (Hulsmans et al. 2016). For example, the viral pathogenesis protein AL2/L2 interacts and inhibits the phosphorylation of SnRK1α for decreasing immune response, thus causing enhanced susceptibility of plant in Nicotiana benthamiana (Hao et al. 2003).The rice OSK35, as a homology of SnRK1β, is a key regulator involved in resistance against both fungal and bacterial pathogens (Kim et al. 2015). Expression of the phosphorylated OSK35 protein is able to enhance resistance against rice blast and bacterial leaf blight (Kim et al. 2015). Arabidopsis SnRK1 β/γ-subunits interact with AtHSPRO1 and AtHSPRO2 and regulate disease resistance against bacterial pathogens (Gissot et al. 2006;Murray et al. 2007). SnRK3/CIPK subfamily members are also involved in regulation of plant immunity. TaCIPK10 from wheat confers to enhanced strip rust resistance through interacted with and phosphorylated TaNH2, which is homologous to AtNPR3/4 ). Arabidopsis CIPK6 functions as a negative regulator of immunity against the bacterial pathogen (Sardar et al. 2017). In rice, OsCIPK14/15 likely play crucial role in the PAMP induced defense signaling pathway (Kurusu et al. 2010).
In the SnRK2 subfamily, two Arabidopsis SnRK2s, SnRK2.6/OST1 and SnRK2.8, participate in plant immunity (Melotto et al. 2006;Lee et al. 2015). OST1 functions together with signals such as nitric oxide (NO) and salicylic acid (SA), and has a pivotal role in response to biotic stress (Melotto et al. 2006). SnRK2.8 regulates plant immunity through the key immune regulator NPR1. SnRK2.8 can interact with and phosphorylate NPR1 to promote nuclear import of NPR1 from the cytoplasm (Lee et al. 2015). In rice, some of SnRK2s are involved in plant immunity. These SnRK2s include SAPK3, 5, 7 and 9, as their transcription levels could be induced by bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) infection (Xu et al. 2013). SAPK9 is able to form a protein complex with the molecular chaperones, OsSGT1 and OsHsp90, to regulate rice resistance to bacterial blight . SAPK10 is also involved in bacterial pathogen resistance through phosphorylation WRKY72 to releases its suppression on jasmonic acid (JA) biosynthesis (Hou et al. 2019). However, the role of rice SnRK2s members in fungal pathogen resistance remains largely unknown.
In this study, we report the identi cation of SAPK5 and its regulation on immunity in rice. SAPK5 posseses kinase activity and the 123th aspartic acid residue is essential for its kinase activity. Loss function of SAPK5 leads to compromised immune response and decreased resistance against Magnaporthe oryzae (M.oryzae) in rice. Furthermore, the phosphoryaltion of SAPK5 is involved in chitin induced immune response. Our study reveals that the SnRK2 family member SAPK5 positively regulates immunity and blast resistance in rice.

SAPK5 protein mainly distributes in cytoplasm
It is well-known that the SnRK2 family proteins are involved in abiotic stress response (Kulik et al. 2011;Mao et al. 2019). However, the investigation of their roles in biotic stress is limited (Mao et al. 2019). In rice, the transcription levels of SAPK5, a member of SnRK2 family, can be induced by pathogen infection (Xu et al. 2013), suggesting that SAPK5 might be a regulator involved in rice immunity. However, the molecular characteristics of SAPK5 and its roles in rice immune response and disease resistance are still remain to be elucidated.
To characterize SAPK5, we rstly introduced a plasmid containing a gene expression cassette (35S::GFP-SAPK5) into rice protoplasts by PEG mediated transformation method to determinate the subcellular distribution of SAPK5 protein. As results shown in gure 1, green uorescence signals can be mainly detected in cytoplasm in the cells expressing GFP-SAPK5 protein, whereas they were ubiquitously detected in the cells expressing GFP protein. These results indicate that SAPK5 is a cytoplasmic protein.
The 123 th aspartic acid residue is essential for SAPK5 kinase activity SnRK2 members are suggested to be protein kinases which mainly relies on their phosphorylation (Kulik et al. 2011;Shinozawa et al. 2019). To ensure the protein kinase activity of SAPK5, we rstly expressed and puri ed the GST and GST-SAPK5 proteins from E.coli strain BL21 respectively and carried out an in vitro kinase activity assay. We found that a speci c band was detected by anti phos-tag antibody, suggesting that GST-SAPK5 is a protein kinase possessing the autophosphorylation ability (Fig. 2). Then, we analyzed SAPK5 protein with PROSITE (https://prosite.expasy.org/prosite.html) and identi ed a predicated kinase active site, the 123 th aspartic acid residue (D), located in the predicted kinase domain.
We then arti cially replaced the 123 th D of SAPK5 with leucine acid residue (N) and puri ed the GST-SAPK5 D123N protein. When performed the similar kinase assay on GST-SAPK5 D123N , no bands were detected by anti phos-tag antibody (Fig. 2). These results suggest that SAPK5 is a functional protein kinase. Moreover, the newly identi ed aspartic acid residue in the kinase domain is a novel residue that essential for its kinase activity.

Disruption of SAPK5 compromise rice basal immune response
To further investigate the genetic function of SAPK5 in rice immune response, we generated SAPK5 loss of function mutants, named as SAPK5KO, using CRISPR/CAS9 technology (Fig.3). The SAPK5KO plants exhibited signi cantly reduced tiller number, while other agronomic traits, including plant height, spikelet number per panicle, thousand seed weight, primary branch number per panicle and secondary branch number per panicle, were not obviously affected (Table 1).
Two genes, the OsKS4 and OsNAC4, are used as markers to evaluate the downstream immune response (Zhang et al. 2015). We thus examined the transcriptional expression levels of these two genes in SAPK5KO plants, in which SAPK5 is knocked out through CRISPR-based editing, post inoculation with the PAMP chitin. In wild type plants, the transcriptional levels of OsKS4 and OsNAC4 were induced about 100and 200-folds after chitin treatment, in comparison with the control (Fig. 4). However, the increased folds of these two genes in SAPK5KO plants were almost less than half of those in wild type plants (Fig. 4), indicating that immune response was inhibited in the plants with SAPK5 knocked out. These results suggest that SAPK5 is required for full immunity of rice plants and loss function of SAPK5 compromises rice basal immune response against.

SAPK5 positively regulates blast resistance in rice
Since our results showed that knock out of SAPK5 compromises chitin induced immune response in rice plants, we predicted that SAPK5 may regulate fungal pathogen resistance of rice. To verify our hypotheses, we inoculated the SAPK5KO transgenic and wild type plants with M. oryzae. Four-week-old SAPK5KO plants and the wild type were challenged with blast isolate Zhong10-8-14 by punch-inoculation method. The lesion length and fungi biomass for each sample were measured and compared 7 days post inoculation (dpi). In accordance with the dampened immune response, SAPK5KO plants presented longer lesions and accumulated more fungi DNA than the control (Fig. 5), indicating that loss function of SAPK5 largely reduced rice resistance to the blast fungi. Our results clearly showed that SAPK5 is indispensable for normal immune response and fungal pathogen resistance for rice.

Chitin treatment elevates the phosphorylation level of SAPK5
Since SAPK5 can be phosphorylated in vitro (Fig. 2), we speculated that phosphorylation of SAPK5 might also be associated with its mediated immune response and fungal pathogen resistance. Thus, we intend to test the basal immune response of SAPK5 at post-translational level. The Myc-SAPK5 transgenic rice plants were used and the phosphorylation level of SAPK5 upon chitin treatment was detected at 0, 5, 15, 30, 60 and 120 min respectively by western blot using anti phos-tag antibody. The result indicated that SAPK5 was quickly phosphorylated within 5 min post chitin treatment and the phosphorylation status continued at the remaining time points (Fig. 6). This result suggest that, in addition to transcription level, SAPK5 is also very likely regulate rice immunity through a post-translational mechanism. Discussion SAPK5 is an active protein kinase and the 123th aspartic acid residue is essential for its kinase activity Plant SnRK family consists of three subfamilies, SnRK1, 2 and 3 (Kulik et al. 2011). All the members of this family are protein kinases that can phosphorylate their substrates to regulate various biological processes (Kulik et al. 2011;Yu et al. 2014;Martínez-Barajas and Coello 2020). For example, plant protein kinase SnRK1 phosphorylates AL2 proteins from three subgroups of virus, resulting in a delay in viral DNA accumulation and symptom appearance (Shen et al. 2014). Arabidopsis SnRK2 subfamily members SnRK2.5, 2.6 and 2.10 interact with and phosphorylate the mRNA decapping complex member VARICOSE, controlling root development and response to salt (Kawa et al. 2020). The SnRK3 member CIPK23 interacts with and phosphorylates the voltage-gated inward K + channel (AKT1) required for K + acquisition in Arabidopsis to regulate low K + response (Li et al. 2006). In rice, in-gel kinase assay with MBP as substrate have revealed that all SnRK2 members, SAPK1 through 10, possess kinase activity (Kobayashi et al. 2004). However, their conserved kinase activity site(s) have not been identi ed. Interestingly, the 123th aspartic acid residue is responsible for the kinase activity of SAPK5 (Fig. 2), not only demonstrating that SAPK5 is an active protein kinase but also suggesting that in other SnRK2 members, the conserved residues corresponding to the 123th aspartic acid residue of SAPK5 are essential for their kinase activities.
SAPK5 is essential for rice to ght against M. oryzae Emerging researches have revealed that some of SnRK2 proteins function in plant immunity regulation. In Arabidopsis, SnRK2.6/OST1 and SnRK2.8 confer to bacterial pathogen resistance (Melotto et al. 2006;Lee et al. 2015). Studies also have suggested that the rice SnRKs, including SAPK3, 5, 7, 9 and 10, are likely involved in resistance regulation against bacterial pathogen (Xu et al. 2013;Hou et al. 2019;Zhang et al. 2019). However, it is unknown whether SnRKs regulate resistance against fungal pathogens. Our study reveals that rice plant in knocking out of SAPK5 showed compromised immune response compared with the wild type plants (Fig. 4). Moreover, when challenged with the fungal pathogen M. oryzae, the rice plants with SAPK5 knocked out are more susceptible than the wild type (Fig. 5). Thus, SAPK5 is a novel immunity regulator which is essential for rice to ght against fungal pathogen M. oryzae.

Phosphorylation cascades mediated by SAPK5 is likely required for plant immune response
Protein phosphorylation is an important event as a post-transcriptional modi cation way that involved in plant immunity (Park et al. 2012). Since phosphorylation of a certain substrate is usually executed by protein kinases, thus the proper activation of protein kinases is pivotal for plant immunity. In plant, the mitogen-activated protein kinase (MAPK) cascades pathway is well know as it's a critical signaling module for immunity (Meng and Zhang 2013). For example, OsMAPKKKε/OsMAPKKK18 are phosphorylated and activated by OsRLCK185 upon chitin perception in rice Yamada et al. 2017). The activated OsMAPKKKε/OsMAPKKK18 then phosphorylate OsMKK4/5, and OsMAPK3/6 are subsequently phosphorylated by OsMKK4/5 thus to burst immune response Yamada et al. 2017). Previous studies also found that the SnRK2 family protein SnRK2.8 modulate plant immunity through phosphorylation of NPR1 to facilitate its nuclear importation from cytoplasm (Lee et al. 2015). Our study reveals that SAPK5 is able to be phosphorylated upon chitin treatment within 5 min (Fig. 6). The phosphorylation status of SAPK5 sustained at least 120 min (Fig. 6) or likely even more longer thus to guarantee the su cient immune response. Although there is no evidence to indicate that NPR1 or other PR proteins are the directly targets of SAPK5 at this time, our present results also suggest that the phosphorylation signaling mediated by SAPK5 may play important roles in regulation of rice immunity and the phosphorylation of SAPK5 is likely a key switcher for immunity regulation.

Conclusions
We characterized a rice SnRK2 family member, SAPK5, and elucidated its molecular function in immune response and disease resistance. The SAPK5 protein is an active cytoplasmic kinase and the 123th aspartic acid residue is essential for its kinase activity. Loss function of SAPK5 attenuates chitin induced immune response and presents increased susceptibility to the fungal pathogen M. oryzae. We also found that the phosphorylation of the kinase SAPK5 is promoted upon chitin perception quickly, suggesting that phosphorylation play an important switch role for SAPK5 to function in plant immunity. Our study not only proves that SAPK5 is a novel key factor regulating plant immune response and disease resistance, but also provides new insights on understanding the machinery of SnRK2 family members on plant immunity.

Plant materials and growth conditions
For major agronomic traits analysis, the wild type Teipei309 (TP309) and the SAPK5KO plants were cultivated with three rows (ten plants per row) in the transgenic eld at Sichuan Agricultural University in Wenjiang with three repeats. At maturity stage, four plants in the middle row were selected for agronomic traits analysis. Student's t test was used to compare the differences between WT and SAPK5KO plants and the data were analyzed by Microsoft Excel 2019.
For evaluation of immune response and disease resistance, rice plants were grown in transgenic eld for four weeks and then the leaves were harvested and used.
For protoplasts preparation, rice seedlings were grown in ½ Murashige and Skoog (MS) medium in SoLo cup and incubated in growth chamber for about ten days before being used.

Vectors construction and rice genetic transformation
For construction of plasmids used in the study, the in vitro recombination method was employed by using the ClonExpress MultiS One Step Cloning Kit (P505-d1, Vazyme Biotech, China). The vectors and primers used were listed in Table S1.

Subcellular localization
For subcellular localization analysis, a plasmid expression GFP-SAPK5 fusion protein was transformed into protoplasts prepared from Nipponbare seedlings following the method described previously (Bart et al. 2006). The protoplasts expression GFP protein alone were used as control. Fluorescence was examined under a laser confocal microscopy (NiKon A1 i90, LSCM, Japan) 18 h after transformation.

RNA isolation and RT-qPCR/qPCR
Total RNA was extracted using TRIzol agent (15596026, Invitrogen, USA) following the procedures described by the manufacturer. The RNA was subject to reverse transcription to synthesize rst-strand cDNA by the PrimeScript™ RT reagent Kit with gDNA Eraser (TRR047B, Takara, China) for removal of genomic DNA.
The reverse transcription-quantitative PCR (RT-qPCR) was conducted on a Bio-Rad CFX96 Real-Time System coupled to a C1000 Thermal Cycler (Bio-Rad, Hercules, USA). The reference gene UBIQUITIN 5 (UBQ5) (Zhu et al. 2016) was used as a reference. For DNA based qPCR, the primers speci cally targeting genomic DNA were designed for M. oryzae MoPOT2 and rice UBIQUITIN 5 (OsUBQ5) genes (Li et al. 2017). Primer sequences are listed in Table S1.

Protein expression, puri cation and in vitro phosphorylation assay
The plasmids expression GST, GST-SAPK5 and GST-SAPK5 D123N were seperately transformed into a E.coli strain BL21 for protein puri cation. Bacteria were grown in Luria-Bertani (LB) medium containing 100 μg/ml Ampicillin at 37 °C to OD 600 =0.6. After then, the bacterial cultures were supplemented with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a nal concentration of 1 mM and incubated at 28°C for 8 hours for induction of heterogeneous protein expression. Protein puri cation, immunoblot analysis and in vitro autophosphorylation of the recombinant proteins were performed as described previously . For detection of GST and the fusion proteins, anti-GST antibody was used (ab9106, Abcam, China) and phosphorylation status was detected by western blot with Phos-tag antibody (BTL-104, FUJIFILM, Japan).

Determination of rice immune response induced by chitin
Leaf strips of 4-week-old SAPK5KO plants were treated with chitin (20 μg/mL) for 2 h as described before (Zhou et al. 2016). The expression levels of two PR genes, OsKS4 and OsNAC4, were determined by RT-qPCR and normalized to UBQ5 expression level. Water treatment was used as a control.

Rice blast resistance assay
Rice blast fungal punch inoculation assay was performed as described previously (Li et al. 2017). The lesion length was measured 7 dpi and the relative fungal biomass was measured by genomic DNA based qPCR, primers used were listed in Table S1.

Availability of Data and Materials
All data supporting the conclusions of this article are provided within the article (and its additional les).

Ethics Approval and Consent to Participate
Not applicable.

Consent for Publication
Not applicable.

Competing Interests
The authors declare that they have no competing interests. Zhu X, Yin J, Liang S, Liang R, Zhou X, Chen Z, Zhao W, Wang J, Li W, He M, Yuan C, Miyamoto K, Ma B, Wang J, Qin P, Chen W, Wang Y, Wang W, Wu X, Yamane H, Zhu L, Li S, Chen X (2016) The multivesicular bodies (MVBs)-localized AAA ATPase LRD6-6 inhibits immunity and cell death likely through regulating MVBs-mediated vesicular tra cking in rice. PLOS Genetics 12: e1006311 Figure 1 Subcellular localization of SAPK5 protein. The GFP-SAPK5 fusion protein was expressed in protoplasts prepared from Nipponbare. GFP protein alone was included as a control. Fluorescence was determined 18 h under laser confocal microscope post transformation. The green uorescence represents GFP, Scale bars = 10 μm.

Figure 2
Determination of the kinase activity of SAPK5 protein in vitro. The recombinant proteins GST-SAPK5D123N, GST-SAPK5 and GST alone, were respectively expressed and puri ed from E. coli. The proteins were then incubated in kinase activity assay buffer. After incubation, proteins were subjected to western blot analysis with antibodies indicated. GST-SAPK5D123N is a putative kinase-dead mutant. The phos-tag antibody was used to detect phosphorylation of protein and the GST antibody was used to indicate the amount of each protein loaded. Asterisk indicates the nonspeci c bands.

Figure 3
Generation of SAPK5 knockout rice plants by CRISPR/CAS9. (A) Photograph of two independent SAPK5 knockout (SAPK5KO) lines in rice TP309 (WT). (B) Sequence alignment between the WT and two SAPK5 knockout lines. A twenty nucleotides SAPK5 speci c segment (indicated by wathet) was employed as sgRNA to achieve CRISPR/CAS9-mediated gene knockout. The PAM sequence was shown in blue and the mutations in SAPK5KO plants were presented in red.

Figure 4
Evaluation of chitin-triggered immune response in SAPK5KO rice plants. Leaf strips of 4-week-old plants were treated with 20 μg/mL chitin for 2 h and total RNA for each sample were extracted respectively. The expression levels of two PR genes, OsKS4 and OsNAC4, were determined by RT-qPCR and normalized to UBQ5 level. Water treatment was used as a control. This experiment was biological repeated three times with similar results. Data were obtained from three technical repeats (means ± SD). Asterisks denote signi cant difference as determined by Student's t-test (**, P < 0.01).

Figure 5
Determination on blast resistance of SAPK5KO rice plants. Photographic phenotype (A), lesion length (B) and relative fungal biomass (C) of the two SAPK5KO rice plants inoculated with blast fungal isolate Zhong10-8-14 after 7 days. The relative fungi biomass was measured by genomic DNA based qPCR (MoPOT2/OsUBQ5). This experiment was biologically repeated for three times with similar results. Data were collected from three technical repeats (means ± SD). Asterisks denote a signi cant difference as determined by Student's t-test (**, P < 0.01).