OsCERK1 Contributes to Cupric Oxide Nanoparticles Induced Phytotoxicity and Basal Resistance against Blast by Regulating the Anti-Oxidant System in Rice

CuO NPs (cupric oxide nanoparticles) are widely used in various fields due to their high electrical conductivity, electronic correlation effect, and special physical property. Notably, CuO NPs have good application prospects in agricultural production because of its antifungal activity to prevent crop diseases. However, the increasing release of CuO NPs into the environment has resulted in a serious threat to the ecosystem, including plants. Previous studies have reported the toxicity of CuO NPs on rice, but little is known about the underlying molecular mechanisms or specific genes involved in the response to CuO NPs. In this study, we found that the rice well-known receptor Chitin Elicitor Receptor Kinase 1 (OsCERK1), which is essential for basal resistance against pathogens, is involved in CuO NPs stress in rice. Knockout of OsCERK1 gene resulted in enhanced tolerance to CuO NPs stress. Furthermore, it was revealed that OsCERK1 reduces the tolerance to CuO NPs stress by regulating the anti-oxidant system and increasing the accumulation of H2O2 in rice. In addition, CuO NPs treatment significantly enhances the basal resistance against M. oryzae which is mediated by OsCERK1. In conclusion, this study demonstrated a dual role of OsCERK1 in response to CuO NPs stress and M. oryzae infection by modulating ROS accumulation, which expands our understanding about the crosstalk between abiotic and biotic stresses.


Introduction
With the application of nanotechnology in many industries, it increases the potential exposure level of nanoparticles in the environment, and also raises concerns about their accumulation in crops and humans through crop consumption. CuO NPs (cupric oxide nanoparticles) possess high electrical conductivity, electronic correlation effect, and special physical and chemical properties, so they are widely used in gas sensors, catalysts, high temperature conductors, solar energy converters, cosmetics, medicine, and other fields [1]. With the wide use of CuO NPs, the exposure of CuO NPs to the environment and humans has increased rapidly [2]. Studies have shown that CuO NPs were detected in water, soil, and air [3]. After absorption, CuO NPs cause various degrees of toxicity to plants and animals [4][5][6], and even induce liver and intestine cancer in humans [3], which seriously threatens human health.
shown as mean with SD based on at least three repetitions.

Basal Resistance Gene OsCERK1 Is Involved in Response to CuO NPs Stress in Rice
To investigate whether OsCERK1 is involved in the response to CuO NPs stress, the expression level of OsCERK1 in the rice seedlings treated with 50 mg/L CuO NPs was detected. The results showed the expression level of OsCERK1 in the CuO NPs-treated rice seedlings was significantly higher than that in the control ( Figure 1B). It was known that OsCERK1 exerts an important role in mediating chitin-induced ROS burst and MAPK activation [18]. To further investigate whether the activity of OsCERK1 was changed in response to CuO NPs in rice, the MAP kinases activity, and the ROS accumulation in the CuO NPs treated rice seedlings were determined. The results showed that the phosphorylation levels of MAP kinases were higher in the seedlings with CuO NPs treatment than that without CuO NPs ( Figure 1C). Meanwhile, chitin-induced ROS accumulation was increased in the seedlings under CuO NPs stress condition ( Figure 1D). The increased MAP kinases activity and ROS accumulation may be caused by the up-regulated expression of OsCERK1 upon CuO NPs treatment. All these results suggested that the LysM receptor-like kinase OsCERK1 is involved in the response to CuO NPs stress in rice. The MAPKs activities in the rice seedlings treated with CuO NPs were higher than those of the control. The total proteins were extracted from Nipponbare (NIP) seedlings treated with CuO NPs at 3, 7, and 14 days to perform western blot assay with antiphospho-p44/42 anti-body. The relative intensities of the blots were quantified by ImageJ with actin The MAPKs activities in the rice seedlings treated with CuO NPs were higher than those of the control. The total proteins were extracted from Nipponbare (NIP) seedlings treated with CuO NPs at 3, 7, and 14 days to perform western blot assay with anti-phospho-p44/42 anti-body. The relative intensities of the blots were quantified by ImageJ with actin as an internal control. (E) Reactive oxygen species (ROS) generation was effectively induced by chitin in the CuO NPs treated rice seedlings. The bars are shown as means ± SD of three replications. The student's t-test was used to analyze the data and generate p value.

Culture and CuO NPs Treatment of Rice Seedlings
The wild-type Oryza sativa subsp. Japonica cultivars Nipponbare and TP309, and transgenic rice plants were used as experimental materials. The oscerk1 mutant, which was generated in Nipponbare background using CRISPR/cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) as previously described [28].
Rice seeds were immersed in the 9 cm dishes with water at 37 • C for 1 day in the dark, then germinated on the wet paper for 4-5 days at 25 • C under 16  For CuO NPs treatment, the rice seedlings were cultured in nutrient solutions with 0 mg/L and 50 mg/L CuO NPs, which was known to significantly inhibit the growth of rice [29]. After 14 days, the roots and shoots of rice seedlings were collected to quantitatively determine the plant height, fresh biomass, and indexes of the anti-oxidative system. All of these experiments were performed in triplicate.

Rice Seedling Infection Assay
Rice seedling infection assays were performed with 5 × 10 5 mL −1 conidial suspension as previously described [30]. The M. oryzae strain En2-2, which was isolated from a paddy field in Enshi, Hubei province, China [31], was cultured on OA plates at 28 • C in dark conditions for 7 days to collect conidia [32]. After spray inoculation with conidia, the rice seedlings were cultured under dark condition for 24 h, and then transferred into an incubator with a 12 h light/12 h dark cycle for 6 days at 22 • C. Disease symptoms of infection assays were assessed on 7 days post inoculation and shown by the images. The relative lesion area was calculated by ImageJ. The infection assays were repeated three times with similar results

Quantitative RT-PCR Analysis
Total RNA was extracted from the rice leaves using Trizol (Invitrogen, 15596-026) and reversely transcribed using M-MLV enzyme (Beyotime, D7176L-1) according to the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) was applied to determine relative gene expression levels using Hieff ® qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China). The primers for qRT-PCR analyses were shown in the Supplemental  Table S1. The OsUbiquitin gene (LOC_Os03g13170) was used as an internal control for normalization. The relative transcript abundances were calculated with the 2 −∆∆CT method based on the abundance levels in control samples.

MAPK Assay
The rice seedlings were collected after CuO NPs treatment for 1, 3, 7, and 14 days, then ground in liquid nitrogen and homogenized in an extraction buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 1% NonidetP-40, 1 mM EDTA, 1 mM PMSF, and 1× protease inhibitor cocktail (Roche, Basel, Switzerland). Then, the total proteins were resolved in 10% SDS-polyacrylamide gel. Phosphorylation of MAPK proteins was detected by immunoblotting with anti-phospho-p44/42 MAPK antibody (Cell Signaling Technology, 4370s). The Actin protein was used as a control. Similar results were obtained from two biological replicates.

Measurement of ROS
For measurement of ROS, the upper leaves of the treated rice seedlings were cut to 0.25 cm 2 circles, and then floated in sterilized water overnight at room temperature to recover from wounding stress. The leave circles were placed in a 1.5 mL tube containing 1 µL horseradish peroxidase, 100 µL L-012 (Wako, Tokyo, Japan), and elicitor (8 nM hexa-N566 acetyl-chitohexaose). Double distilled H 2 O served as control. The luminescence was recorded continuously at 10 s intervals for 20 min by a Glomax 20/20 luminometer (Promega, Beijing, China). Three replications were carried out with similar results.

Measurement of Hydrogen Peroxide (H 2 O 2 ) Content
The levels of H 2 O 2 were examined according to the commercial kits (Solarbio, Beijing, China) with the manufacturer's protocol [33]. About 0.1 g of rice seedlings was ground and blended in 500 µL of lysate. Then the homogenate was centrifuged at 4 • C for 10 min at 8000× g. The supernatant was mixed with the same account of H 2 O 2 detection reagent to detect the absorbance at 415 nm. The standard curve was utilized to analyze the content of H 2 O 2 . And H 2 O 2 content was calculated according to the manufacturer's instructions. Three replications were carried out with similar results.

Measurement of Activities of Anti-Oxidant Enzymes
The anti-oxidant enzyme activities, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), were quantified using the commercial kits (Solarbio, Beijing, China) as previously described [33]. Approximately 0.1 g nitrogen ground sample was added into 1 mL precooled corresponding extraction solution in the kit. And the absorbance of the reaction products was detected. The activities of anti-oxidant enzymes were standardized as following descriptions: one unit of activity of SOD was defined as the amount of enzyme that caused the inhibition of photoreduction of 4-nitroblue tetrazolium chloride by 50%. One unit of POD activity was defined as the amount of enzyme that caused an increase in absorbance at 470 nm of 0.001 per minute. One unit of CAT activity was defined as the amount of enzyme that caused an increase in absorbance at 470 nm of 0.001 per minute. Similar results were obtained from three replications.

Investigation of Physiological Features
The rice seedlings treated with 50 mg/L CuO NPs for 14 days were collected. After thoroughly rinsing with sterilized water, the physiological features of the rice seedlings, including shoot length, root length, and root number were determined. The fresh biomass was measured after drying the surface of rice with filter paper. The data was shown as the mean with standard deviation (SD) based on ten replications. The relative inhibition rate of fresh biomass was calculated based on the formula: inhibition rate (%) = [fresh biomass (control) − fresh biomass (CuO NPs)]/fresh biomass (control) × 100%. Similarly, the relative inhibition rates of shoot length, root length, root number, and the third leaf length were calculated according to the formula.

Data Analysis
Relative expression values were calculated using the 2 −∆∆CT method. The Students' t-test was used for performing all statistical analyses and generating p value. The data was shown as mean with SD based on at least three repetitions.

Basal Resistance Gene OsCERK1 Is Involved in Response to CuO NPs Stress in Rice
To investigate whether OsCERK1 is involved in the response to CuO NPs stress, the expression level of OsCERK1 in the rice seedlings treated with 50 mg/L CuO NPs was detected. The results showed the expression level of OsCERK1 in the CuO NPstreated rice seedlings was significantly higher than that in the control ( Figure 1B). It was known that OsCERK1 exerts an important role in mediating chitin-induced ROS burst and MAPK activation [18]. To further investigate whether the activity of OsCERK1 was changed in response to CuO NPs in rice, the MAP kinases activity, and the ROS accumulation in the CuO NPs treated rice seedlings were determined. The results showed that the phosphorylation levels of MAP kinases were higher in the seedlings with CuO NPs treatment than that without CuO NPs ( Figure 1C). Meanwhile, chitin-induced ROS accumulation was increased in the seedlings under CuO NPs stress condition ( Figure 1D). The increased MAP kinases activity and ROS accumulation may be caused by the upregulated expression of OsCERK1 upon CuO NPs treatment. All these results suggested that the LysM receptor-like kinase OsCERK1 is involved in the response to CuO NPs stress in rice.

OsCERK1 Regulates Phytotoxicity of CuO NPs Stress to Rice
To investigate the biological role of OsCERK1 in the response to CuO NPs stress in rice, we determined the effect of CuO NPs on the oscerk1 mutant, which was created using CRISPR-Cas9 system, and its wild-type [28]. The growth of rice seedlings was significantly inhibited by CuO NPs (Figure 2A). Upon CuO NPs treatment for 14 days, the fresh biomass of the wild-type was significantly reduced by 64%, while that in the oscerk1 mutant only decreased by 43% ( Figure 2B). Moreover, the growth of the third leaf (the white arrow) of the wild-type was inhibited by CuO NPs stress, while the inhibitory phenotype was partially restored by the knockout of OsCERK1 gene (Figure 2A,F). Furthermore, CuO NPs treatment strongly inhibited root growth in rice ( Figure 2D,E). After CuO NPs treatment, the root lengths of the oscerk1 mutants were significantly longer than those of the wild-type ( Figure 2D), and the root numbers of the oscerk1 mutants were more than those of the wildtype ( Figure 2E), suggesting that knockout of OsCERK1 gene resulted in higher tolerance to CuO NPs stress than the wild-type. All these results showed that the knockout of OsCERK1 alleviated the toxic effect of CuO NPs in rice, indicating that OsCERK1 negatively regulates the tolerance of rice to CuO NPs stress.
as an internal control. (E) Reactive oxygen species (ROS) generation was effectively induced by chitin in the CuO NPs treated rice seedlings. The bars are shown as means ± SD of three replications. The student's t-test was used to analyze the data and generate p value.

OsCERK1 Regulates Phytotoxicity of CuO NPs Stress to Rice
To investigate the biological role of OsCERK1 in the response to CuO NPs stress in rice, we determined the effect of CuO NPs on the oscerk1 mutant, which was created using CRISPR-Cas9 system, and its wild-type [28]. The growth of rice seedlings was significantly inhibited by CuO NPs (Figure 2A). Upon CuO NPs treatment for 14 days, the fresh biomass of the wild-type was significantly reduced by 64%, while that in the oscerk1 mutant only decreased by 43% ( Figure 2B). Moreover, the growth of the third leaf (the white arrow) of the wild-type was inhibited by CuO NPs stress, while the inhibitory phenotype was partially restored by the knockout of OsCERK1 gene (Figures 2A,F). Furthermore, CuO NPs treatment strongly inhibited root growth in rice ( Figures 2D,E). After CuO NPs treatment, the root lengths of the oscerk1 mutants were significantly longer than those of the wild-type ( Figure 2D), and the root numbers of the oscerk1 mutants were more than those of the wild-type ( Figure 2E), suggesting that knockout of OsCERK1 gene resulted in higher tolerance to CuO NPs stress than the wild-type. All these results showed that the knockout of OsCERK1 alleviated the toxic effect of CuO NPs in rice, indicating that Os-CERK1 negatively regulates the tolerance of rice to CuO NPs stress.  The growth of the oscerk1 mutant and its wild-type Nipponbare (NIP) treated with CuO NPs for 14 days. The relative inhibition rates of fresh biomass (B), shoot length (C), root length (D), root numbers (E), and the third leaves' length (F) of the oscerk1 mutants and its wild-type treated with CuO NPs for 14 days. The relative inhibition rate of fresh biomass was calculated based on the formula: inhibition rate (%) = [fresh biomass (control) − fresh biomass (CuO NPs)] / fresh biomass (control) × 100%. Similarly, the relative inhibition rates of shoot length, root length, root number, and the third leaf length were calculated according to the formula. The bars are shown as means ± SD of ten replications. The student's t-test was used to analyze the data and generate a p value.

OsCERK1 Regulates Anti-Oxidative System in Response to CuO NPs Stress
In plants, the anti-oxidative system plays important roles during abiotic stress [34]. Moreover, CuO NPs treatment leads to oxidative damage in rice seedlings [35]. To further determine the functions of OsCERK1 in response to CuO NPs stress, the H 2 O 2 levels in the oscerk1 mutant and the wild-type seedlings treated with CuO NPs for 14 days were determined. The result showed that CuO NPs treatment highly induced the accumulation of H 2 O 2 in the rice seedlings ( Figure 3A). Compared with the wild-type, the accumulation of H 2 O 2 in the oscerk1 mutant was significantly reduced. The relative increasing rate of H 2 O 2 content in the wild-type seedlings was higher than that in the oscerk1 mutant ( Figure 3A), suggesting that knockout of OsCERK1 reduced the accumulation of H 2 O 2 triggered by CuO NPs. oscerk1 mutant was lower than that in the wild-type, the activities of anti-oxidant enzymes in the CuO NPs treated rice seedlings were determined. The results showed that CuO NPs treatment significantly down-regulated the activities of the peroxidase (POD) and catalase (CAT), which are H2O2-scavenging enzymes, in all rice seedlings (Figures 3B,D). In addition, the rate of relative inhibition rates of POD and CAT activities in the wild-type seedlings were significantly higher than those in the oscerk1 mutant ( Figures 3B,D). Furthermore, the activity of superoxide dismutase (SOD), which catalyzes the dismutation of superoxide anion (O 2− ) into oxygen and hydrogen peroxide, was significantly up-regulated by the CuO NPs treatment in the wild-type ( Figure 3C). In contrast, there was no significant change in the SOD activity in the oscerk1 mutant under CuO NPs stress condition ( Figure 3C). These results indicated that knockout of OsCERK1 relieved the negative effects of CuO NPs on the antioxidant system of rice. In conclusion, OsCERK1 negatively modulates the tolerance of rice to CuO NPs stress by down-regulating the activities of CAT and POD, and up-regulating the activity of SOD, leading to the accumulation of H2O2.  In the same manner, the relative increasing rate of SOD activity was calculated with the formula. The relative inhibition rate of POD activity was calculated as follows: relative inhibition rate (%) = [POD activity (control) − POD activity (CuO NPs)]/POD activity (control) × 100%. Similarly, the relative inhibition rate of CAT activity was calculated with the formula. The bars are shown as means ± SD of three replications. Data was analyzed with student's t-test to obtain p value.
To explore why the content of H 2 O 2 accumulation induced by CuO NPs stress in the oscerk1 mutant was lower than that in the wild-type, the activities of anti-oxidant enzymes in the CuO NPs treated rice seedlings were determined. The results showed that CuO NPs treatment significantly down-regulated the activities of the peroxidase (POD) and catalase (CAT), which are H 2 O 2 -scavenging enzymes, in all rice seedlings ( Figure 3B,D). In addition, the rate of relative inhibition rates of POD and CAT activities in the wild-type seedlings were significantly higher than those in the oscerk1 mutant ( Figure 3B,D). Furthermore, the activity of superoxide dismutase (SOD), which catalyzes the dismutation of superoxide anion (O 2− ) into oxygen and hydrogen peroxide, was significantly up-regulated by the CuO NPs treatment in the wild-type ( Figure 3C). In contrast, there was no significant change in the SOD activity in the oscerk1 mutant under CuO NPs stress condition ( Figure 3C).
These results indicated that knockout of OsCERK1 relieved the negative effects of CuO NPs on the antioxidant system of rice. In conclusion, OsCERK1 negatively modulates the tolerance of rice to CuO NPs stress by down-regulating the activities of CAT and POD, and up-regulating the activity of SOD, leading to the accumulation of H 2 O 2 .

CuO NPs Enhance the Basal Resistance against M. oryzae in Rice
Due to the role of CuO NPs in regulating the accumulation of ROS in rice, we postulated that CuO NPs stress may affect basal resistance against M. oryzae in rice. To confirm this hypothesis, the wild-type Oryza sativa subsp. Japonica cultivars Nipponbare and TP309 were inoculated with the M. oryzae wild-type strain En2-2 after treated with CuO NPs for 14 days. The seedling infection assay showed that compared with the control, the blast lesions formed on the rice seedlings with CuO NPs treatment were smaller ( Figure 4A,D). Consistent with the observation of disease symptoms, the relative lesion areas calculated by ImageJ under CuO NPs were smaller than those in the control ( Figure 4B,E). In addition, the relative expression levels of the PR genes OsPAL1 and OsNAC4 were up-regulated more than four folds in the Nipponbare seedlings in the response to M. oryzae inoculation after treated with CuO NPs for 14 days ( Figure 4C). Similarly, in the TP309 seedlings, the relative expression levels of the OsPAL1 and OsNAC4 were also up-regulated more than three folds ( Figure 4F). In conclusion, all these results demonstrated that CuO NPs treatment significantly enhances the basal resistance against M. oryzae in rice. CAT activities (D) in the oscerk1 mutant treated with CuO NPs for 14 days. The relative increasing rate of H2O2 content was calculated based on the formula: relative increasing rate (%) = [H2O2 content (CuO NPs) − H2O2 content (control)] / H2O2 content (control) × 100%. In the same manner, the relative increasing rate of SOD activity was calculated with the formula. The relative inhibition rate of POD activity was calculated as follows: relative inhibition rate (%) = [POD activity (control) − POD activity (CuO NPs)] / POD activity (control) × 100%. Similarly, the relative inhibition rate of CAT activity was calculated with the formula. The bars are shown as means ± SD of three replications. Data was analyzed with student's t-test to obtain p value.

CuO NPs Enhance the Basal Resistance against M. oryzae in Rice
Due to the role of CuO NPs in regulating the accumulation of ROS in rice, we postulated that CuO NPs stress may affect basal resistance against M. oryzae in rice. To confirm this hypothesis, the wild-type Oryza sativa subsp. Japonica cultivars Nipponbare and TP309 were inoculated with the M. oryzae wild-type strain En2-2 after treated with CuO NPs for 14 days. The seedling infection assay showed that compared with the control, the blast lesions formed on the rice seedlings with CuO NPs treatment were smaller (Figures 4A,  D). Consistent with the observation of disease symptoms, the relative lesion areas calculated by ImageJ under CuO NPs were smaller than those in the control (Figures 4B,E). In addition, the relative expression levels of the PR genes OsPAL1 and OsNAC4 were upregulated more than four folds in the Nipponbare seedlings in the response to M. oryzae inoculation after treated with CuO NPs for 14 days ( Figure 4C). Similarly, in the TP309 seedlings, the relative expression levels of the OsPAL1 and OsNAC4 were also up-regulated more than three folds ( Figure 4F). In conclusion, all these results demonstrated that CuO NPs treatment significantly enhances the basal resistance against M. oryzae in rice.  Disease symptoms (D), the relative lesion area (E), and relative expression levels of PR genes (F) of Oryza sativa subsp. Japonica cultivars TP309 rice seedlings inoculated with M. oryzae after treated with CuO NPs for 14 days. The relative lesion area was calculated by ImageJ. The relative transcript abundances were calculated using the 2 −∆∆CT method based on the abundance levels in the control samples. The Ubiquitin gene (LOC_Os03g13170) was used as the internal control for normalization. The bars are shown as means ± SD of three replications. The student's t-test was used to analyze the data and generate p value.

OsCERK1 Contributes to the CuO NPs-Modulated Basal Resistance against M. oryzae in Rice
OsCERK1 is essential for fungal chitin-driven immune responses and contributes to basal resistance against M. oryzae in rice [22]. To determine whether CuO NPs-modulated basal resistance is dependent on OsCERK1 in rice, the oscerk1 mutant and its wild-type Nipponbare were inoculated with the M. oryzae wild-type strain En2-2 after treated with CuO NPs for 14 days. The rice seedling infection assay showed that the blast lesions formed on the seedlings with CuO NPs treatment were significantly smaller than that without CuO NPs ( Figure 5A). In contrast, compared with the wild-type, the inhibition rate of the relative lesion area caused by CuO NPs in the oscerk1 mutant was significantly lower ( Figure 5B). The rice seedling infection assay results revealed that the susceptibility of the oscerk1 mutant to M. oryzae was comparable with or without CuO NPs treatment. In addition, the relative expression levels of the PR genes OsPAL1 and OsNAC4 were significantly up-regulated in all rice seedlings in response to M. oryzae inoculation after treated with CuO NPs for 14 days (Figure 5C,D). Compared with the wild-type, CuO NPs treatment had less effect on relative expression levels of OsPAL1 and OsNAC4 in the oscerk1 mutant ( Figure 5C,D). All these results suggested that OsCERK1 is required for the CuO NPs-modulated basal resistance against M. oryzae in rice.

OsCERK1 Contributes to the CuO NPs-Modulated Basal Resistance against M. oryzae in Rice
OsCERK1 is essential for fungal chitin-driven immune responses and contributes to basal resistance against M. oryzae in rice [22]. To determine whether CuO NPs-modulated basal resistance is dependent on OsCERK1 in rice, the oscerk1 mutant and its wild-type Nipponbare were inoculated with the M. oryzae wild-type strain En2-2 after treated with CuO NPs for 14 days. The rice seedling infection assay showed that the blast lesions formed on the seedlings with CuO NPs treatment were significantly smaller than that without CuO NPs ( Figure 5A). In contrast, compared with the wild-type, the inhibition rate of the relative lesion area caused by CuO NPs in the oscerk1 mutant was significantly lower ( Figure 5B). The rice seedling infection assay results revealed that the susceptibility of the oscerk1 mutant to M. oryzae was comparable with or without CuO NPs treatment. In addition, the relative expression levels of the PR genes OsPAL1 and OsNAC4 were significantly up-regulated in all rice seedlings in response to M. oryzae inoculation after treated with CuO NPs for 14 days (Figures 5C,D). Compared with the wild-type, CuO NPs treatment had less effect on relative expression levels of OsPAL1 and OsNAC4 in the oscerk1 mutant ( Figures 5C,D). All these results suggested that OsCERK1 is required for the CuO NPs-modulated basal resistance against M. oryzae in rice. The relative lesion area was calculated by ImageJ. The relative inhibition rate of the relative lesion area was calculated based on the formula: inhibition rate (%) = [the relative lesion area (control) − the relative lesion area (CuO NPs)] / the relative lesion area (control) × 100%. The relative transcript abundances were calculated using the 2 −ΔΔCT method with Ubiquitin gene as the internal control. The bars are shown as means ± SD of three replications. The student's t-test was used to analyze the data and generate p value. Disease symptoms (A), the relative lesion areas (B), relative expression levels of PR genes OsPAL1 (C) and OsNAC4 (D) of the oscerk1 mutant and its wild-type Nipponbare (NIP) inoculated with M. oryzae after treated with CuO NPs for 14 days. The relative lesion area was calculated by ImageJ. The relative inhibition rate of the relative lesion area was calculated based on the formula: inhibition rate (%) = [the relative lesion area (control) − the relative lesion area (CuO NPs)]/the relative lesion area (control) × 100%. The relative transcript abundances were calculated using the 2 −∆∆CT method with Ubiquitin gene as the internal control. The bars are shown as means ± SD of three replications. The student's t-test was used to analyze the data and generate p value.

Discussion
With the wide application, CuO NPs pose a potential threat to cause serious toxicity to crops. In rice, CuO NPs could decrease the photosynthetic performances, increase the generation of ROS, and inhibit the growth of seedlings [36]. However, the molecular mechanisms underlying the adverse effects of CuO NPs have not yet been revealed in detail.
In this study, we found that a LysM receptor-like kinase OsCERK1 negatively regulates the tolerance to CuO NPs stress. Further analyses revealed that OsCERK1 regulates the antioxidant system and increases the accumulation of H 2 O 2 , thus reducing the tolerance of rice to CuO NPs stress. In addition, we found that CuO NPs treatment significantly enhances the OsCERK1-mediated basal resistance against M. oryzae in rice. Taken together, this study demonstrated that OsCERK1 is involved in the phytotoxicity of CuO NPs and CuO NPs-modulated basal resistance against blast by regulating the activities of anti-oxidant enzymes and inducing the accumulation of ROS ( Figure 6).

Discussion
With the wide application, CuO NPs pose a potential threat to cause serious toxicity to crops. In rice, CuO NPs could decrease the photosynthetic performances, increase the generation of ROS, and inhibit the growth of seedlings [36]. However, the molecular mechanisms underlying the adverse effects of CuO NPs have not yet been revealed in detail. In this study, we found that a LysM receptor-like kinase OsCERK1 negatively regulates the tolerance to CuO NPs stress. Further analyses revealed that OsCERK1 regulates the anti-oxidant system and increases the accumulation of H2O2, thus reducing the tolerance of rice to CuO NPs stress. In addition, we found that CuO NPs treatment significantly enhances the OsCERK1-mediated basal resistance against M. oryzae in rice. Taken together, this study demonstrated that OsCERK1 is involved in the phytotoxicity of CuO NPs and CuO NPs-modulated basal resistance against blast by regulating the activities of anti-oxidant enzymes and inducing the accumulation of ROS ( Figure 6). OsCERK1 is a critical immune receptor of rice, which is triggered by chitin and responds to fungal pathogens, such as the causal agent of rice blast disease M. oryzae [22]. Chitin induces oligomerization of receptors OsCEBiP and OsCERK1 to promote the phosphorylation of OsCERK1, which in turn phosphorylates and activates downstream components, thus inducing MAPK activation and ROS burst [21]. This study demonstrated that OsCERK1 is not only required for the resistance to pathogens, but also is critical for the tolerance to abiotic CuO NPs stress in rice. Similarly, AtCERK1 positively regulates disease resistance and abiotic salinity stress tolerance in Arabidopsis [25], which suggested that CERK1 plays an important role in the crosstalk between biotic and abiotic stress. In this study, we found that the expression of OsCERK1 is up-regulated by CuO NPs stress, leading to increase the activities of MAPK and ROS accumulation ( Figure 1). Furthermore, knockout of OsCERK1 relieved the negative impact of CuO NPs on rice growth. All these results suggested that OsCERK1 negatively regulates the tolerance to CuO NPs in rice, which is a new function of OsCERK1. OsCERK1 is a critical immune receptor of rice, which is triggered by chitin and responds to fungal pathogens, such as the causal agent of rice blast disease M. oryzae [22]. Chitin induces oligomerization of receptors OsCEBiP and OsCERK1 to promote the phosphorylation of OsCERK1, which in turn phosphorylates and activates downstream components, thus inducing MAPK activation and ROS burst [21]. This study demonstrated that OsCERK1 is not only required for the resistance to pathogens, but also is critical for the tolerance to abiotic CuO NPs stress in rice. Similarly, AtCERK1 positively regulates disease resistance and abiotic salinity stress tolerance in Arabidopsis [25], which suggested that CERK1 plays an important role in the crosstalk between biotic and abiotic stress. In this study, we found that the expression of OsCERK1 is up-regulated by CuO NPs stress, leading to increase the activities of MAPK and ROS accumulation ( Figure 1). Furthermore, knockout of OsCERK1 relieved the negative impact of CuO NPs on rice growth. All these results suggested that OsCERK1 negatively regulates the tolerance to CuO NPs in rice, which is a new function of OsCERK1.
The antifungal activity of CuO NPs have been revealed [14][15][16]. For instance, CuO NPs trigger resistance in tobacco against the soil-borne fungal pathogen Phytophthora nicotianae [37]. However, little is known about the effect of CuO NPs on rice resistance. In this study, we found that CuO NPs significantly enhance resistance against M. oryzae but inhibit growth in rice. Several studies have highlighted that oxidative damage, such as lipid peroxidation, may be the main cause of CuO NPs toxicity to plants [6]. In duckweed, CuO NPs treatment increases H 2 O 2 by 56% and •OH by 57% [17]. In Arabidopsis, CuO NPs (2 to 100 mg/L) induces the generation of singlet oxygen ( 1 O2) and hydrogen peroxide (H 2 O 2 ) in the roots and leaves [38]. In addition, the activities of CAT and SOD of the rice leaves treated with 250 mg/L CuO NPs were decreased, while the activity of the SOD in the rice roots exposed to 125 mg/L CuO NPs significantly increased [18]. Similarly, this study found that upon CuO NPs treatment, the activities of CAT and POD were significantly decreased, and the SOD activity was increased, resulting in H 2 O 2 accumulation in rice. The induced ROS accumulation might be one of the important reasons why CuO NPs treatment significantly enhances the basal resistance against M. oryzae in rice. In contrast, the activities of these antioxidant enzymes and the accumulation of H 2 O 2 in the oscerk1 mutant with or without CuO NPs treatment were comparable. Moreover, the inhibited growth of rice seedlings caused by CuO NPs has been partially restored by the knockout of OsCERK1. These results indicated that OsCERK1 participates in the response to CuO NPs stress by regulating the accumulation of H 2 O 2 . It was well known that both the invasion of pathogens and CuO NPs stress could induce a burst of ROS in rice [39,40]. The reduced ROS accumulation might be one of the important reasons why the oscerk1 mutant shows compromised resistance to M. oryzae and less inhibition of growth in response to CuO NPs stress. Based on these results, we inferred that in response to CuO NPs stress signal, OsCERK1 regulates the antioxidant system in rice, resulting in an increase in ROS accumulation, inhibition of growth, and enhanced resistance against M. oryzae in rice. In addition, these results suggested that in order to safely and properly use CuO NPs as a fungicide to prevent rice blast in the future, the phytotoxicity of CuO to rice has to be considered.
Plants face a variety of stresses in the environment. Naturally, stress signaling in plant cells is a sophisticated network. However, most studies focused on the plant response to abiotic stresses and pathogens separately in the past few centuries [41][42][43]. With the changes in climate, increasing environmental pollution, and the constant pressures of diseases and pests, the interactions between the plant response to abiotic stresses and pathogens have been attracting extensive attention worldwide. Recent studies revealed that SiO 2 NPs can induce systemic acquired resistance in Arabidopsis [44], indicating that there is a crosstalk between the response to nanomaterials and disease resistance in plants. In this study, we revealed that there is a crosstalk among the signal transduction pathways of blast resistance and stress response induced by CuO NPs, with the pattern recognition receptors (PRRs) OsCERK1 as the core module. OsCERK1, which is located on the surface of rice cells, may percept not only the signals from pathogenic fungi, but also the abiotic stress signals from the environment. The results of this study revealed the molecular mechanism of OsCERK1 regulating ROS accumulation to inhibit growth and enhance blast resistance in response to CuO NPs stress in rice, as well as expanded our understanding of the crosstalk between different environmental signals.

Conclusions
This study aimed to explore the molecular mechanism of rice response to CuO NPs. The results revealed that CuO NPs treatment up-regulates the expression level of OsCERK1. Moreover, OsCERK1 modulates tolerance to CuO NPs in addition to basal resistance in rice. Further analyses revealed that OsCERK1 regulates the activities of anti-oxidant enzymes to induce the accumulation of ROS in the response to CuO NPs stress and contributes to CuO NPs-modulated resistance against M. oryzae in rice. Our results expand our understanding of the crosstalk between different environmental signals and provide important theoretical bases for the sustainable application of CuO NPs in plants.