Niclosamide inhibits leaf blight caused by Xanthomonas oryzae in rice

Rice leaf blight, which is caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), results in huge losses in grain yield. Here, we show that Xoo-induced rice leaf blight is effectively controlled by niclosamide, an oral antihelminthic drug and molluscicide, which also functions as an anti-tumor agent. Niclosamide directly inhibited the growth of the three Xoo strains PXO99, 10208 and K3a. Niclosamide moved long distances from the site of local application to distant rice tissues. Niclosamide also increased the levels of salicylate and induced the expression of defense-related genes such as OsPR1 and OsWRKY45, which suppressed Xoo-induced leaf wilting. Niclosamide had no detrimental effects on vegetative/reproductive growth and yield. These combined results indicate that niclosamide can be used to block bacterial leaf blight in rice with no negative side effects.

effective and economical chemical treatment for rice leaf blight has not been established, although research and development are ongoing.
Enhancing plant genetic resistance is an effective method for controlling bacterial leaf blight disease. A number of studies have identified plant genes that confer resistance against Xanthomonas bacteria. At least 38 bacterial leaf blight resistance gene (R genes), designated in series from Xa1 to Xa38, have been identified 16,17 . Six of these genes have been cloned (Xa1, xa5, xa13, Xa21, Xa3/Xa26, and Xa27), and six additional genes have been physically mapped (Xa2, Xa4, Xa7, Xa30, Xa33, and Xa38) [16][17][18][19][20][21][22] . Two major classes of R genes, receptor kinase (RLK) and nucleotide-binding site leucine rice repeat (NBS)-LRR, are involved in disease resistance in rice. Xa21 is the first R gene of the RLK class to be cloned with a broad spectrum of resistance, and Xa1 is an R gene of the NBS-LRR class 23 , which is the largest R gene class conferring resistance against bacteria, fungi, and viruses 24 . Other R genes, including xa5 and xa13, encode proteins such as a small subunit of the transcription factor IIA (TFIIAγ ) and a plasma membrane protein, respectively 25,26 . These data have been used to develop a breeding program for bacterial leaf blight-resistant rice. The introduction of resistance genes xa5, xa13, and Xa21 into new cultivars conferred a high level of resistance against Xoo 27 , indicating that germplasm screening against bacterial leaf blight and breeding-mediated transfer of resistant genes to target cultivars can generate new rice cultivars with increased resistance to bacterial leaf blight disease. Large-scale experiments, including microarrays, were used to isolate genes related to bacterial leaf blight; a large number of candidate genes was selected, although their functions were not clearly identified 28,29 . A transgenic approach was employed to protect rice from bacterial leaf blight and fungal blast. For example, transgenic rice overexpressing cecropin B (an antibacterial peptide from Bombyx mori) showed increased resistance to bacterial leaf blight 30 . The introduction of multiple bacterial blight resistance genes (Xa4, xa5, xa13, and Xa21) into rice conferred resistance to six Xoo bacteria 31 . Rice transformants overexpressing WRKY30 are resistant to Xoo 32 and the fungal pathogen Magnaporthe oryzae 33 , and those overexpressing WRKY45 displayed markedly enhanced resistance to bacterial leaf blight and fungal blast disease [34][35][36] . These results suggest that transgenic approaches can be effectively utilized to develop disease-resistant rice.
Although several biological, chemical, and genetic control approaches have yielded improvements in rice protection from bacterial leaf blight, there was still a need for an effective approach that would provide large-scale protection. Therefore, we focused our current efforts on screening known chemicals for their effects on rice leaf blight. We are interested in the discovery of a master regulator that effectively functions to protect animals and plants from infectious diseases mediated by bacteria, fungi, and/or parasites. Therefore, we first considered infectious diseases of humans. We selected two drugs that cure infectious diseases in humans, auranofin [3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate] and niclosamide [5-chloro-N-(2-chloro-4nitrophenyl)-2-hydroxybenzamide], for further analysis. However, because auranofin is much more expensive than niclosamide, we focused our initial screen on niclosamide. Niclosamide has been widely used since 1960 to treat gastrointestinal tapeworm infections in both humans and animals. Recent studies show that niclosamide has antiviral activity against the severe acute respiratory syndrome virus 37 , anti-anthrax toxin properties 38 , and anti-neoplastic activity 39 . Niclosamide strongly induces LC3-positive autophagosomes 40 , inhibits the Wnt/ Frizzled pathway 41 , suppresses the autonomous notch-signaling pathway 42 , and inhibits mTOR signaling 43 . Niclosamide uncouples mitochondrial oxidative phosphorylation 44 and thereby slows cell growth. These combined results indicate that niclosamide has various curative effects on humans and animals. We reasoned that niclosamide might affect plant disease responses by modulating disease signaling pathways as a broad spectrum regulator. Therefore, we investigated the effect of niclosamide on bacterial leaf blight in rice.
Here, we show that niclosamide blocks rice leaf wilting mediated by Xoo bacteria, both locally and systemically, without negatively affecting plant growth. The results suggest that niclosamide may be used to prevent rice leaf blight caused by bacterial pathogen attack.

Results
Niclosamide inhibits Xoo bacterial growth. To evaluate the functional effect of niclosamide on bacterial blight, we first examined its effect on the growth of Xoo bacteria using three different strains, PXO99, 10208, and K3a, and Xanthomonas axonopodis pv. glycines (Xag). The results showed that growth of the Xoo strains was completely inhibited by 5 μ g/ml niclosamide, whereas Xag growth was inhibited by 15 μ g/ml niclosamide (Fig. 1a). Xag is a pathogen that causes bacterial leaf pustule disease in soybean 45 . We also examined the effect of niclosamide on the growth of the fungal pathogen Magnaporthe oryzae. Niclosamide did exert an inhibitory effect on the growth of 12 M. oryzae strains, although the inhibition was considerably weaker than that against Xoo (Fig. 1b).
We also tested the effects of parthenolide, a sesquiterpene lactone with anti-tumor activity, on the growth of the Xoo strains PXO99 and 10208. Parthenolide failed to inhibit the growth of either Xoo strain at a concentration of 5 μ g/ml, although it slightly inhibited bacterial growth at 50 μ g/ml (Fig. 1c). We also tested the effect of niclosamide on the growth of three E. coli strains, Top10, Rosseta2, and DH10b, which served as Gram-negative control bacteria. At lower concentrations, niclosamide had no effect on the growth of these E. coli strains, although it slightly inhibited growth at 50 μ g/ml (Fig. 1d).
To determine the minimum inhibitory concentration (MIC) of niclosamide on PXO99 and 10208 growth, we tested concentrations ranging from 0-5 μ g/ml. Both Xoo strains could grow in the presence of 4 μ g/ml niclosamide, but not 5 μ g/ml niclosamide ( Fig. 1e; left panel). We further narrowed the MIC of niclosamide from 4-5 μ g/ml. As shown in Fig. 1e, the growth of both PXO99 and 10208 was completely inhibited by 4.2 μ g/ml niclosamide ( Fig. 1e; right panel), indicating that 4.2 μ g/ml niclosamide is the MIC for both Xoo strains.
Rice bacterial blight is blocked by niclosamide. We examined whether niclosamide blocks bacterial blight in rice. For this experiment, we grew the rice cultivar Nipponbare in a growth chamber for 12 weeks (before bolting), and subjected the plants to pathogen and niclosamide treatment. First, to determine the minimum concentration of niclosamide that would block bacterial blight, we examined the niclosamide dosage effect on disease responses to the representative Xoo strain PXO99. We inoculated PXO99 onto Nipponbare using the leaf-clipping method, sprayed the plants with different niclosamide concentrations, and examined the phenotypes of plants treated with pathogen only or pathogen plus niclosamide. Leaf wilting did not develop in plants treated with ≥ 8 μ g/ml niclosamide (Fig. 2a). We also estimated lesion development by measuring lesion length. Lesion development also was significantly inhibited by 8 μ g/ml niclosamide, although a small lesion was still detected at this concentration (Fig. 2b). Lesion development gradually declined with increasing niclosamide concentrations (Fig. 2b). We also examined the levels of bacterial growth, and found that population levels of Xoo bacteria declined with increasing niclosamide concentrations (Fig. 2c).
We next examined the effect of niclosamide on rice disease responses to Xoo bacteria after treatment with PXO99. First, Nipponbare rice was inoculated with PXO99 by the leaf-clipping method. After incubation for four days until leaf blight lesions with a length of 2 cm developed, the plants were sprayed with 8 μ g/ml niclosamide (Fig. 3). Leaf blight was completely blocked in niclosamide-treated leaves (Fig. 3a). Lesion development also was completely blocked in niclosamide-treated leaves (Fig. 3b), and PXO99 growth was inhibited (Fig. 3c).
Niclosamide has a systemic effect on rice disease response. We next investigated whether niclosamide could move from the site of local application to distant tissues and subsequently inhibit PXO99-mediated leaf wilting. We inoculated half of the leaves of Nipponbare plants with PXO99 and covered them with polythene bags. Then, we sprayed the non-inoculated leaves with niclosamide and examined leaf blight in both local and systemic leaves. Leaf wilting and lesion development were completely inhibited in PXO99-inoculated leaves that had not been treated with niclosamide ( Fig. 4a,b). PXO99 growth also was significantly inhibited in the inoculated leaves (Fig. 4c).
Next, we examined the long-distance movement of niclosamide by extracting niclosamide from niclosamide-treated leaves and untreated systemic leaves. Niclosamide levels gradually increased in systemic leaves ( Fig. 5a,b), indicating that niclosamide can systemically move from niclosamide-treated local leaves to untreated distal leaves.
Niclosamide induces the expression of defense-related genes. We next examined whether niclosamide protects PXO99-infected plants through the induction of pathogen-related gene expression. We treated the rice cultivar Nipponbare with different niclosamide concentrations for 24 h, and then extracted total RNA from niclosamide-treated and untreated control plants. We performed qRT-PCR analysis to examine the transcript levels of seven defense-related genes, including OsPR1, OsPR10, OsWRKY45, OsWRKY62, OsWRKY71, OsHI-LOX, and OsACS2. The results showed that OsPR1, OsPR10, OsWRKY45, OsWRKY62, OsWRKY71, and OsHI-LOX were induced by niclosamide treatment, and that transcript expression levels increased with increasing niclosamide levels (Fig. 6). However, OsACS2 expression was only slightly increased 30 min after treatment with 40 μ g/ml niclosamide, which suggests that its expression was not affected by niclosamide (Fig. 6).
The pathogenesis-related genes OsPR1, OsPR10, OsWRKY45, OsWRKY62, and OsWRKY71 are induced by high salicylate (SA) levels [46][47][48][49] ; therefore, we measured free SA and glucosyl-SA levels in the leaves of niclosamide-treated rice plants. SA levels increased in response to niclosamide treatment (Fig. 7a,b), indicating that niclosamide induces the expression of pathogenesis-related genes by increasing the levels of SA and SA-conjugates. Niclosamide has no effect on rice growth and development. We examined the effect of niclosamide on rice growth and development from the vegetative stage to seed maturation. Three-week-old rice plants were treated with 8 μ g/ml niclosamide at 4-day intervals and the phenotypic characteristics of niclosamide-treated and -untreated plants were examined at 80 days after planting. Plant height and leaf characteristics were not altered by niclosamide treatment, although the contents of chlorophyll and other compounds (SPAD values) were slightly reduced in niclosamide-treated plants ( Fig. 8a-g). Seed characteristics such as color, number, and weight were not affected by niclosamide treatment (Fig. 8h-l), indicating that niclosamide does not have any detrimental effects on rice growth, development, and grain yield.

Discussion
Xoo-mediated leaf blight is one of the most devastating rice diseases worldwide. For the past two decades, substantial efforts have been made to identify and isolate bacterial blight-resistance genes. In this study, we used an alternative chemical approach to protect rice from Xoo infection.
More than 30 drugs, including antibiotics, have been utilized to protect crops from pathogen attack. Some of these have broad-spectrum bactericidal and fungicidal activity, whereas others specifically target bacteria or fungi. Oxytetracycline and streptomycin are commonly used antibiotics in humans and plants 15 , suggesting that some types of human drugs can positively control diseases in plants. Therefore, we screened human drugs for their ability to prevent rice leaf blight disease. Our long-term goal is to identify a master regulator that inhibits pathogenic disease in both plants and humans.
Niclosamide was initially characterized as an oral antihelminthic drug and molluscicide 36 . Several recent studies report that niclosamide is active against cancer cells 39,50,51 , which indicates that niclosamide has broad-spectrum disease control activity. Therefore, we examined whether niclosamide had inhibitory activity against rice leaf blight, and found that the leaf blight symptoms were suppressed by niclosamide through inhibition of Xoo growth and lesion development (Figs 2 and 3). Niclosamide moved systemically from the local application site to Xoo-inoculated distal leaves, and inhibited lesion development and leaf wilting by inhibiting Xoo growth in distal leaves (Fig. 4). We examined the effect of niclosamide on the growth of three Xoo strains, and found that it inhibited their growth (Fig. 1a,e). Next, we evaluated whether niclosamide induced the expression of the defense-related genes OsPR1, OsPR10, OsWRKY45, OsWRKY62, OsWRKY71, and OsHI-LOX, and detected higher transcript levels for these genes (Fig. 6). These results clearly indicate that niclosamide blocks the development of rice leaf blight by directly inhibiting Xoo bacterial growth (Figs 2c and 3c) and/or by inducing defense-related gene expression (Fig. 6). Niclosamide also inhibited Xag growth, which causes bacterial leaf pustule disease in soybean 45 , although its inhibitory effect against Xag was weaker than that against Xoo strains (Fig. 1a). This suggests that niclosamide may inhibit the growth of select bacterial pathogens of other crops and thereby protect them from disease. Jasmonate (JA) and SA function in plant defense pathways by inducing the expression of numerous genes 52 . Treatment of rice leaves with benzothiadiazole or probenazole induced SA accumulation and increased resistance to bacterial blight and fungal blast caused by Xoo and M. grisea, respectively 13,14,34 . Our qRT-PCR analysis showed that niclosamide induced the expression of SA-dependent genes in rice leaves (Fig. 6). We investigated whether niclosamide induced defense mechanisms directly by functioning like a phytohormone, or indirectly via SA-mediated signaling pathways. We measured the levels of free SA and its conjugate glucosyl-SA, and found that their levels were higher in niclosamide-treated leaves than in control leaves (Fig. 7). This indicates that increase in SA level can contribute to the blockage of Xoo lesion development by niclosamide.
WRKY proteins are involved in JA response pathways in rice. OsWRKY30 triggers the expression of JA-responsive genes and JA accumulation, and provides rice with resistance to fungal pathogens Rhizoctonia solani and M. grisea 33 . OsWRKY45 expression regulates JA accumulation and resistance to the rice blast fungus M. grisea 53 . Our results showed that the expression of OsHI-LOX, a JA-responsive gene, was induced in niclosamide-treated leaves (Fig. 6). This indicates that JA-dependent defense signaling pathways can also contribute to the blockage of Xoo lesion development by niclosamide. Pathogens are sensitive to the activities of certain classes of drugs. The antibiotic streptomycin, which is commonly used in animals and humans, protects crops from some bacterial and fungal pathogens 15 . Niclosamide serves as an oral antihelminthic drug, molluscicide, and anti-tumor agent in human disease; therefore, we reasoned that niclosamide might have beneficial effects on bacterial and fungal diseases of rice. Niclosamide inhibited growth of the fungal pathogen M. oryzae, although this effect was much weaker than that against pathogenic Xoo bacteria (Fig. 1b). This suggests that niclosamide may inhibit the growth of other fungal pathogens and thereby protect plants from fungal diseases. Benzothiadiazole and probenazole induce SA pathway-mediated defense responses in plants by acting as chemical inducers 13,14,34,54-57 , leading to strong resistance against bacterial and fungal pathogens. Niclosamide induces the expression of both SA-and JA-responsive genes in rice (Fig. 6). This indicates that niclosamide may also be effective against fungi due to JA. Further study of the effects of niclosamide against other fungal pathogens will indicate whether the compound can protect plants from diseases caused by fungal pathogens.
The biochemical function and physiological action of niclosamide in human health and disease have been elucidated. Niclosamide inhibits glucose uptake, mitochondrial oxidative phosphorylation, and anaerobic metabolism in parasitic helminths 44 . It also inhibits transcription and DNA binding of the NFκ Β pathway, and increases ROS levels to induce apoptosis in acute myelogenous leukemia cells 50 . Recent work reports that niclosamide inhibits Pseudomonas aeruginosa quorum sensing 58 . These results suggest that niclosamide controls signaling networks of prokaryotes and eukaryotes by modulating metabolic and signaling pathways. Currently, we do not know the mechanism underlying niclosamide-mediated inhibition of Xoo growth and induction of SA-and JA-dependent plant defense responses. Future work will examine the effects of niclosamide on glucose metabolism, the transcriptome and proteome, and quorum sensing to identify the mechanism by which niclosamide inhibits bacterial leaf blight in rice. Further investigations of the effects of niclosamide on mitochondrial oxidative phosphorylation, changes in transcriptome and ROS levels, and changes in hormone signals (including SA and JA) also are required to fully characterize its functions as a broad-spectrum signaling regulator in plants.
The use of antibiotics and chemicals in plant agriculture is a subject of some concern. The use of antibiotics and chemicals in open fields over large expanses of land may increase the frequency of antibiotic resistance in gene pools, or possible chemical accumulation in plant tissues and the food chain. Niclosamide is a teniacide, which is effective against cestodes such as tapeworms that infect humans and many other animals (although it is not effective against as pinworms and roundworms). Niclosamide also is used as a piscicide. Recent work reported that it appeared to be safe and well tolerated in humans, even when used in mass treatment campaigns in several countries, although it has known adverse effects including nausea, retching, abdominal pain, light headedness, pruritus, vomiting, and dizziness 59 . There are no previous reports of niclosamide effects on plant growth, other organisms, or the food chain. We cannot state that niclosamide treatment of agricultural crops is safe for other organisms or for the food chain because research in this area is still lacking. Therefore, further studies on the function and stability of niclosamide under natural environmental conditions and when applied to crop plants are required to determine whether niclosamide has adverse effects on other organisms and human health when applied to plants.
In conclusion, the results presented herein indicate that niclosamide protects rice plants from bacterial leaf blight by inhibiting Xoo growth, inducing SA accumulation, and/or by inducing the expression of defense-related gene pathways. Further functional studies on niclosamide-mediated growth inhibition of bacterial and fungal pathogens, and elucidation of its role in plant signaling pathways, will provide information about the mechanism underlying niclosamide activity in plants. Field tests evaluating the effects of niclosamide application on rice plants will provide information on whether it has adverse effects on other organisms or the food chain. Examining the effect of niclosamide on Xoo bacterial growth. The effect of niclosamide on the growth of Xoo strains PXO99, 10208, and K3a, and a Xag strain, and the MIC of niclosamide against the PXO99 and 10208 strains were investigated. Xoo and Xag strains were obtained from the Rural Development Administration, Korea. Bacterial strains were cultivated in peptone-sucrose broth containing 15 μ g/ml of the antibiotic cephalexin (Sigma-Aldrich), and grown to an optical density of 1.0 at 600 nm. Then, 2 μ l of each culture was grown on agar medium containing different concentrations of niclosamide (0− 50 μ g/ml) to examine the effects of niclosamide on bacterial growth. Inoculated plates were incubated at 28 °C for 48 h and then photographed. The same method was used to determine whether parthenolide had bactericidal activity against the PXO99 and 10208 strains. The bactericidal activity of niclosamide on three different E. coli strains, Top10, Rosseta2, and DH10b, also was examined. The E. coli strains were cultivated in LB medium to an optical density of 1.0 at 600 nm. Then, 2 μ l of each culture was grown on agar medium containing different concentrations of niclosamide. Inoculated plates were incubated at 37 °C for 24 h and photographed.
To determine the MIC, 2 μ l of each culture were inoculated onto peptone-sucrose agar (PSA) medium containing 0− 5.0 μ g/ml niclosamide. Bacterial growth was not detected at a concentration of 5.0 μ g/ml niclosamide. Thus, to determine a more specific MIC value, the MIC of niclosamide against both Xoo strains was narrowed down from 4.0 to 4.8 μ g/ml. The lowest concentration of niclosamide (4.2 μ g/ml) with no visible growth was taken as the MIC.

Effect of niclosamide dosage on rice disease responses.
To determine the minimum amount of niclosamide needed to inhibit the development of leaf blight in rice, the rice cultivar Nipponbare and Xoo strain PXO99 were used. PXO99 cells were prepared as follows. A single PXO99 colony was suspended in peptone-sucrose broth and plated onto fresh PSA medium. After 2 days of culture at 28 °C, the cells were collected and centrifuged to remove exopolysaccharides. The cells were then resuspended in double-distilled water, producing more than 10 9 cell forming units (cfu) per ml.
The dosage effect of niclosamide on PXO99 lesion development was evaluated by measuring the lesion length (cm) and bacterial growth in the leaves of niclosamide-treated rice. To treat rice with PXO99, plants were grown for 80 days in a greenhouse at 31 °C in the light and 25 °C in the dark under a 16 h photoperiod until the booting stage. The greenhouse humidity was maintained over 90%. The fully expanded uppermost leaves were inoculated with PXO99 by the leaf-clipping method, followed by treatment with various doses (0, 4, 8, 20, and 40 μ g/ml) of niclosamide applied by foliar spraying every 4 days. As a control, mock inoculation was conducted with distilled water. After 16 days, the leaves were photographed and lesion length was measured using ImageJ Software. All data are expressed as the mean values from 15 leaves per treatment. The experiment was repeated five times under the same conditions. To estimate bacterial growth, leaves were cut off the plants and ground with a mortar and pestle. After grinding, each sample was suspended in sterile water and plated onto PSA medium containing 15 μ g/ ml cephalexin. The bacterial population was scored by counting the number of colonies every 4 days after inoculation. The lesion lengths and bacterial populations were expressed as the mean value plus/minus the standard deviation. Niclosamide effects on disease responses in rice. Rice plants were inoculated with Xoo and sprayed with niclosamide. PXO99 cells and plants were prepared as described above. To treat rice with PXO99, the fully expanded uppermost leaves of 80-day-old rice plants were inoculated with PXO99 by the leaf-clipping method. The samples were incubated for 4 days until leaf blight lesions with a length of 2 cm developed, after which the leaves were treated with 8 μ g/ml niclosamide by foliar spraying every 4 days. As a control, mock inoculation was conducted with distilled water. Leaves were photographed after 16 days, and lesion length and bacterial growth estimated as described above. All data are expressed as the mean values of 15 leaves per treatment. The experiment was repeated five times under the same conditions. The lesion length and bacterial population were expressed as the mean value plus/minus the standard deviation. Systemic effect of niclosamide on rice disease responses. To examine the systemic effect of niclosamide on Xoo-mediated leaf blight development, the fully expanded uppermost leaves of 80-day-old rice plants were inoculated with PXO99 by the leaf-clipping as described above. Half of the leaves were completely covered with polythene bags and half were sprayed with 8 μ g/ml niclosamide. The local leaves were then sprayed with 8 μ g/ml niclosamide every 4 days. Both local and systemic leaves were photographed after 16 days. Lesion length and bacterial growth were estimated as described above. The experiment was repeated five times under the same conditions. The lesion length and bacterial population were expressed as the mean value plus/minus the standard deviation.
Quantification of systemically translocated niclosamide. Half of the leaves of 80-day-old rice plants were covered by polythene bags and the remaining systemic leaves were sprayed with 8 μ g/ml niclosamide. After treatment, the bag-covered leaves were harvested after incubation for the indicated time periods. Niclosamide was extracted from 0.5 g of each sample using absolute MeOH, and the niclosamide concentration was determined by HPLC separation and fluorescence detection. The relative niclosamide concentration in each sample was compared with the 100 ppb niclosamide standard. Niclosamide levels were expressed as the mean value plus/ minus the standard deviation.
Determination of salicylic acid (SA) and its conjugate. SA and the SA conjugate glucosyl-SA were extracted from 0.5 g of rice leaves that were treated with 8 μ g/ml niclosamide for 4 h. Samples were collected at five time points (0, 0.5, 1, 2, and 4 h). The concentrations of SA and glucosyl-SA were measured by HPLC as described previously 60 .
Niclosamide effect on rice growth and development. Three-week-old Nipponbare leaves were sprayed with 8 μ g/ml niclosamide at 4-day intervals until seed maturation. The phenotypic characteristics of untreated and niclosamide-treated plants were measured at 70 days after planting. Grain phenotypes were examined after seed maturation.