Abstract
Pathogen attack and abiotic stress affect grain yields in rice fields worldwide. Rice leaf blast is caused by the fungal pathogen Magnaporthe oryzae, whereas rice leaf blight is caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Rice leaf blast and blight, the two most destructive diseases in rice, result in huge yield losses. We therefore tried to identify a chemical that could be utilized as an inhibitor of rice leaf blast and blight. Here, we show that both bacterial-induced rice blight and fungal-induced rice blast can be controlled by auranofin, an anti-rheumatoid arthritis and anti-cancer drug used in humans. Auranofin directly inhibited the growth of four Xoo strains, including PXO99, KACC10208, 1209, and 1308. In addition, auranofin effectively controlled the growth of 10 M. oryzae strains, including KACC46531, 46532, 46534, 46535, 46536, 46538, 46540, 46541, 46542, and 46544, although its effect on M. oryzae was weaker than that on Xoo. These results suggest that auranofin can be applied to rice to block both rice leaf blight and blast caused by Xoo and M. oryzae, respectively.
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Key words: Auranofin, Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae, Rice leaf blight, Rice leaf blast
INTRODUCTION
Rice blast, which is caused by the fungus
Magnaporthe oryzae, is one of the most destructive diseases of cultivated rice (
Ford et al. 1994;
Talbot and Foster 2001).
M. oryzae uses a hemibiotrophic infection strategy. After infecting a rice plant, a germ tube from
M. oryzae differentiates into a specialized infectious structure known as the appressorium and generates enormous turgor pressure inside the melanized appressorium (
Howard et al. 1991;
De Jong et al. 1997). After penetration, the thin peg differentiates into bulbous and lobed infectious hyphae, resulting in the formation of blast lesions (
Heath et al. 1990,
1992;
Tucker and Talbot 2001). Rice blight, which is caused by
Xanthomonas oryzae pv.
oryzae (
Xoo) (
Ishiyama 1992), a phytopathogenic Gram-negative bacterium, results in grain yield losses.
Xoo enters the plant through wounds or hydathodes, multiplies in the epitheme, moves to the xylem vessels, and undergoes active multiplication, resulting in blight disease symptoms in leaves.
Since rice leaf blast and blight are two of the most destructive rice diseases worldwide, leading to severe yield losses (
Hu et al. 2008;
Liu et al. 2014), many approaches have been taken to protect rice from blast and blight. Five different strategies, including chemical control, biological control, improved cultivation techniques, introducing genetic resistance, and disease forecasting, are widely used to control these diseases. However, although several approaches have yielded improvements in the protection of rice plants facing leaf blight and blast, there is still a need for an effective approach to controlling these diseases.
Numerous chemicals have been tested for their effects on controlling bacterial leaf blight and fungal leaf blast. For example, chlorine bleaching powder, broad-spectrum antibiotics including benzylpenicillin, ampicillin, kanamycin, streptomycin, chloramphenicol, and Sino Bionic, and the plant activator benzothiadiazole have been shown to block bacterial leaf blight (
Chand et al. 1979;
Karthikeyan and Gnanamanickam 2011;
Khan et al. 2012). Probenazole protects rice from blast caused by
M. grisea (
Iwai et al. 2007). Streptomycin, oxytetracycline, gentamycin, and oxolinic acid have also been used to control several bacterial and fungal diseases (
McManus et al. 2002). However, an effective and economical chemical treatment for rice leaf blight and blast has not been established, although research and development are ongoing.
We are interested in discovering a master regulator that could successfully be used to protect animals and plants from infectious diseases mediated by bacteria, fungi, and/or parasites. We first considered infectious diseases in humans, because rice leaf blight and blast are also infectious diseases. We selected two drugs that cure infectious diseases in humans: niclosamide [5-chloro-
N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide] and auranofin [3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate]. We initially focused on niclosamide because auranofin is much more expensive than niclosamide. We demonstrated that niclosamide blocks
Xoo-mediated leaf wilting in rice without negatively affecting plant growth (
Kim et al. 2016). Niclosamide has been widely used since 1960 to treat gastrointestinal tapeworm infections in both humans and animals as an oral antihelminthic drug and molluscicide. Niclosamide also has various curative effects, including antiviral activity, anti-anthrax toxin properties, and anti-tumor activity, in humans and animals (
Wu et al. 2004;
Balgi et al. 2009;
Chen et al. 2009;
Zhu et al. 2009;
Osada et al. 2011). Auranofin is used to treat rheumatoid arthritis. Auranofin also has potential therapeutic value for treating various diseases, including cancer, HIV/AIDS, and parasitic and bacterial infections (
Tejman-Yarden et al. 2013), as well as neurodegenerative disorders including Parkinson’s disease and Alzheimer’s (
Roder and Thomson 2015). Auranofin functions through inhibiting the activity of reduction/oxidation enzymes such as thioredoxin reductase, which are essential for maintaining proper intracellular levels of reactive oxygen species in pathogens, thereby inducing cellular oxidative stress and intrinsic apoptosis in the pathogens to combat diseases (
Marzano et al. 2007;
Pessetto et al. 2013;
Fan et al. 2014;
Fiskus et al. 2014). These findings strongly suggest that auranofin might also have bactericidal and fungicidal activity against the pathogens that cause rice leaf blast and blight diseases, although this drug is more expensive than niclosamide. Therefore, we recently investigated the activity of auranofin against
Xoo and
M. oryzae.
Here, we show that auranofin inhibits the growth of Xoo and M. oryzae, which cause rice leaf blight and blast, respectively, suggesting that auranofin can be applied to rice to prevent rice leaf blast and blight caused by bacterial and fungal pathogen attack.
MATERIALS AND METHODS
Chemicals
Stock solutions of niclosamide (Sigma-Aldrich) and auranofin (Sigma-Aldrich) were made by dissolving the chemicals in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/mL. The stock solutions were diluted in distilled water before use.
Examining the effects of auranofin on Xoo bacterial growth
The effects of auranofin on the growth of Xoo strains PXO99, KACC10208, 10209, and 10308 were investigated. The Xoo strains were obtained from the Rural Development Administration, Korea. The bacterial strains were cultivated in peptone-sucrose broth containing 15 μg/mL of the antibiotic cephalexin (Sigma-Aldrich) and grown to an optical density of 1.0 at 600 nm. A 2 μL aliquot of each culture was grown on agar medium containing different concentrations of auranofin to examine its effects on bacterial growth. Inoculated plates were incubated at 28°C for 2 days and photographed.
Examining the effects of auranofin on M. oryzae growth
M. oryzae strains were also obtained from the Rural Development Administration, Korea. To examine the effects of auranofin on fungal growth, 10 M. oryzae strains (KACC46531, 46532, 46534, 46535, 46536, 46538, 46540, 46541, 46542, and 46544) were inoculated onto potato dextrose agar (Difco) medium containing different concentrations of auranofin (0, 5, 15, and 50 μg/mL). The plates were incubated at 28°C for 3 days and photographed.
Examining the effects of auranofin on Gram-negative bacterial growth
The bactericidal activity of auranofin on three different E. coli strains, Top10, Rosseta2, and DH10b, was also examined. The E. coli cells were cultivated in LB medium to an optical density of 1.0 at 600 nm. A 2 μL aliquot of each culture was grown on agar medium containing different concentrations of auranofin. The inoculated plates were incubated at 37°C for 24 hours and photographed.
RESULTS
Auranofin inhibits Xoo bacterial growth
We previously reported that niclosamide completely inhibits the growth of
Xoo strains at a concentration of 5 μg/mL (
Kim et al. 2016). Therefore, we were interested in identifying additional human drugs that could be utilized to prevent rice diseases. Among the drugs used to treat infectious diseases in humans, we chose auranofin for further study because it is widely used to treat various human diseases including parasitic and bacterial infections, AIDS, cancer, and neurodegenerative disorders (
Tejman-Yarden et al. 2013).
To evaluate the functional effect of auranofin on rice leaf blight, we examined its effect on the growth of
Xoo bacteria using four different strains: PXO99, KACC10208, 10209, and 10308. Specifically, we tested its effect on bacterial growth in PSA medium containing different concentrations of auranofin (0, 5, 15, and 50 μg/mL). The growth of all four
Xoo bacterial strains was completely inhibited in the presence of 5 μg/mL auranofin (
Fig. 1A), which is similar to the effect of niclosamide (
Fig. 1B), indicating that auranofin successfully blocks the growth of
Xoo bacteria.
Next, to evaluate the functional effect of auranofin on leaf blast, we examined the effect of auranofin on the growth of the fungal pathogen
M. oryzae. We used 10
M. oryzae strains, including KACC46531, 46532, 46534, 46535, 46536, 46538, 46540, 46541, 46542, and 46544. This experiment was also carried out using PSA medium containing different concentrations of auranofin (0, 5, 15, and 50 μg/mL). We found that auranofin exerted an inhibitory effect on the growth of all 10
M. oryzae strains (
Fig. 2), although the inhibitory effect of this drug was considerably weaker than that against
Xoo (
Fig. 1A).
However, the inhibitory effect of auranofin on the growth of
M. oryzae (
Fig. 2) was much stronger than that of niclosamide (
Kim et al. 2016). Indeed, the inhibitory effect of auranofin on the growth of
M. oryzae at 5 μg/mL was similar to that of 50 μg/mL niclosamide, indicating that its effect is approximately 10-fold stronger than that of niclosamide. In fact, among the 10
M. oryzae strains examined, the growth of three strains, KACC46534, 46535, and 46544, was completely inhibited by 50 μg/mL auranofin (
Fig. 2), whereas all of the strains still grew in the presence of 50 μg/mL niclosamide (
Kim et al. 2016), indicating that auranofin is more effective against leaf blast-causing fungal pathogens than niclosamide.
We also tested the effect of auranofin on the growth of three
E. coli strains, Top10, Rosseta2, and DH10b, which served as Gram-negative control bacteria. At lower concentrations (5 μg/mL), auranofin slightly inhibited bacterial growth (
Fig. 3A). However, 50 μg/mL auranofin greatly inhibited bacterial growth (
Fig. 3A), whereas 50 μg/mL niclosamide only slightly inhibited bacterial growth (
Fig. 3B). These results indicate that auranofin has broad bactericidal activity against Gram-negative bacteria.
DISCUSSION
Various chemicals have been used to protect crops from pathogen attack (
Chand et al. 1979;
McManus et al. 2002;
Iwai et al. 2007;
Karthikeyan and Gnanamanickam 2011;
Khan et al. 2012). Some types of human drugs have positive effects on controlling diseases in plants (
McManus et al. 2002). Therefore, we screened human drugs for their ability to prevent rice leaf blight and blast diseases. We previously demonstrated that niclosamide suppresses
Xoo growth and lesion development in rice (
Kim et al. 2016). Niclosamide also inhibits the growth of
M. oryzae, although this effect is much weaker than that against
Xoo bacteria (
Kim et al. 2016), suggesting that niclosamide can also inhibit the growth of various fungal pathogens, thereby protecting plants from fungal diseases.
Based on this finding, in the current study, we investigated the effect of auranofin, another human drug used to control various diseases including infectious diseases and cancer (
Marzano et al. 2007;
Pessetto et al. 2013;
Fan et al. 2014;
Fiskus et al. 2014;
Roder and Thomson 2015), on
Xoo and
M. oryzae growth. We found that auranofin inhibited the growth of both pathogens (
Figs. 1A and
2). However, its effect on
M. oryzae was much weaker than that on
Xoo. In addition, we found that the effect of auranofin on
M. oryzae was much stronger than that of niclosamide (
Fig. 2,
Kim et al. 2016). These results strongly suggest that auranofin blocks rice leaf blast more efficiently than niclosamide. Moreover, auranofin inhibited the growth of several Gram-negative
E. coli strains (
Fig. 3A), whereas niclosamide had no bactericidal activity on these strains (
Fig. 3B,
Kim et al. 2016). These results indicate that auranofin has broad-spectrum bactericidal activity against Gram-negative bacteria.
We previously found that niclosamide moves systemically from the local application site to distal leaves in plants inoculated with
Xoo and that it inhibits lesion development and leaf wilting by inhibiting
Xoo growth in distal leaves (
Kim et al. 2016). In addition, the blockage of
Xoo-induced lesion development by niclosamide appears to be mediated by salicylate- and jasmonate-dependent defense signaling pathways (
Kim et al. 2016). These findings suggest that auranofin might use a similar mechanism to exert its function in plants. Therefore, similar approaches can be applied to elucidate the mechanism underlying auranofin activity in plants.
In conclusion, the current results suggest that auranofin could be used to protect rice plants from rice leaf blight by inhibiting Xoo growth and from rice leaf blast by inhibiting M. oryzae growth. Auranofin inhibited the growth of M. oryzae more efficiently than niclosamide, suggesting that auranofin is a more promising chemical for use as a protectant against rice leaf blight and blast. Further functional studies of the effects of auranofin against these pathogens will help confirm whether this drug could be used to protect rice plants from diseases caused by bacterial and fungal pathogens. Studies are also needed to determine whether auranofin has adverse effects on other organisms and human health when applied to plants.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center no. PJ01330802), Rural Development Administration, Republic of Korea.
Fig. 1Effects of auranofin on the growth of bacterial pathogens. Xoo strains PXO99, KACC10208, 10209, and 10308 were grown on PSA medium containing different concentrations of auranofin (0–50 μg/mL) (A) or niclosamide (0–50 μg/mL) (B). After incubation at 28°C for 2 days, the plates were photographed.
Fig. 2Effects of auranofin on the growth of fungal pathogens. Ten M. oryzae strains, including KACC46531, 46532, 46534, 46535, 46536, 46538, 46540, 46541, 46542, and 46544, were inoculated onto potato dextrose agar medium containing different concentrations of auranofin (0–50 μg/mL). After incubation at 28°C for 3 days, the plates were photographed.
Fig. 3Effects of auranofin on the growth of E. coli. Gram-negative E. coli strains Top10, Rosseta2, and DH10b were grown on agar medium containing different concentrations of auranofin (0–50 μg/mL) (A) or niclosamide (0–50 μg/mL) (B). Inoculated plates were incubated at 37°C for 1 day and photographed.
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