Trehalose-6-phosphate phosphatase inhibitor: N-(phenylthio) phthalimide, which can inhibit the DON biosynthesis of Fusarium graminearum

https://doi.org/10.1016/j.pestbp.2021.104917Get rights and content

Highlights

  • N-(phenylthio) phthalimide reduces DON pollution.

  • N-(phenylthio) phthalimide as a trehalose-6-phosphate phosphatase inhibitor.

  • Trehalose-6-phosphate phosphatase as a new pesticide target.

Abstract

Fusarium head blight(FHB)caused by Fusarium graminearum species complex (FGSC) is one of the most important diseases around the world. Deoxynivalenol (DON) is a type of mycotoxin produced by FGSC when infecting cereal crops. It is a serious threat to the health of both humans and livestock. Trehalose-6-phosphate phosphatase (TPP), a conserved metabolic enzyme found in many plants and pathogens, catalyzes the formation of trehalose. N-(phenylthio) phthalimide (NPP) has been reported to inhibit the normal growth of nematodes by inhibiting the activity of TPP, but this inhibitor of nematodes has not previously been tested against F. graminearum. In this study, we found that TPP in F. graminearum (FgTPP) had similar secondary structures and conserved cysteine (Cys356) to nematodes by means of bioinformatics. At the same time, the sensitivity of F. graminearum strains to NPP was determined. NPP exhibited a better inhibitory effect on conidia germination than mycelial growth. In addition, the effects of NPP on DON biosynthesis and trehalose biosynthesis pathway in PH-1 were also determined. We found that NPP decreased DON production, trehalose content, glucose content and TPP enzyme activity but increased trehalose-6-phosphate content and trehalose-6-phosphate synthase (TPS) enzyme activity. Moreover, the expression of TRI1, TRI4, TRI5, TRI6, and TPP genes were downregulated, on the contrary, the TPS gene was upregulated. Finally, in order to further determine the control ability of NPP on DON production in the field, we conducted a series of field experiments, and found that NPP could effectively reduce the DON content in wheat grain and had a general control effect on FHB. In conclusion, the research in this study will provide important theoretical basis for controlling FHB caused by F. graminearum and reducing DON production in the field.

Introduction

Fusarium head blight (FHB) is a devastating disease in wheat and other small grain cereals caused by Fusarium graminearum species complex (FGSC). FGSC includes at least sixteen species, and some species have specific geographical distributions (O'Donnell et al., 2008; Yli-Mattila et al., 2009). FGSC causing FHB is mainly composed of F. asiaticum and F. graminearum in China (Zhang et al., 2012; Zhang et al., 2013). FHB not only causes serious loss of yield, but also damages the health of humans and livestock from mycotoxins produced in cereal crops infected with FGSC. At present, type-B trichothecene (TCTC-B) and zearalenone (ZEN) are common mycotoxins produced by FGSC. TCTC-B mainly includes deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15- acetyl-deoxynivalenol (15-ADON) and nivalenol (NIV). Among them, DON contamination is the most serious. DON is an inhibitor of protein synthesis, which has cytotoxic, immunotoxic, carcinogenic, teratogenic and mutagenic effects. In addition, DON has also been reported as an important pathogenic factor of FGSC (Goswami and Kistler, 2005; Li et al., 2019). Therefore, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WTO) have designated it as “the most dangerous naturally occurring food contaminant”. Wheat is the second largest food crop in the world, so it is very important to control mycotoxin contamination with the increasing attention given to food safety.

Trehalose is a non-reducing disaccharide linked by 1α - 1α and a common carbon storage source, which is widely found in bacteria, fungi, plants and invertebrates (Elbein et al., 2003; Avonce et al., 2006). It has a variety of functions: as an osmotic pressure protection agent; as a storage carbon source; as a pressure protection agent to resist cold, hot, drought, oxidation and other stresses; to improve the stability of cell membrane and protein; and to increase the survival rate of dehydration and rehydration of organisms (Singer and Lindquist, 1998a, Singer and Lindquist, 1998b; Kandror et al., 2002; Gancedo and Flores, 2004; Crowe, 2007). Therefore, the trehalose synthesis pathway is particularly important for the normal growth of organisms. Previous studies have shown that there are at least five trehalose biosynthesis pathways in different organisms, and the most widely distributed pathway is catalyzed by trehalose 6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) (Avonce et al., 2006; Tournu et al., 2013). This pathway exists in organisms such as Escherichia coli and Saccharomyces cerevisiae. In S. cerevisiae, trehalose-6-phosphate synthetase (TPS) catalyzes the UDP glucose to produce trehalose-6-phosphate (T-6-P), then T-6-P is catalyzed to trehalose in the presence of TPP. In addition, the trehalose biosynthesis pathway plays an important role in the pathogenicity of fungi. It has been reported that the deletion of the trehalose synthesis gene in four human pathogens and two plant pathogens can cause the decrease of pathogenicity (Dijck et al., 2002; Petzold et al., 2006; Wilson et al., 2007; Lowe et al., 2009; Ngamskulrungroj et al., 2009; Puttikamonkul et al., 2010).

The control of FHB always depends on chemical fungicides. With the increasing resistance of FHB to conventional fungicides in the field, it is of great importance to discover and develop novel fungicides that exhibit inhibitory effects on the fungal growth and DON biosynthesis of FHB pathogens (Chen et al., 2007; Chen et al., 2011; Duan et al., 2014). N-(phenylthio) phthalimide (NPP, C14H9NO2S) (Fig. 1) was reported to be an inhibitor of nematode TPP. This compound conjugated a strictly conserved cysteine residue in the active site of nematode TPP and therefore acted as a suicide inhibitor (Cross et al., 2019). The trehalose biosynthesis pathway has an important role in the normal growth of fungi. Therefore, we investigated whether NPP could also affect the trehalose biosynthesis pathway in F. graminearum. In this study, the effects of NPP in inhibiting mycelial growth, conidia germination, and DON biosynthesis of F. graminearum were determined. We also evaluated the expression of TRI genes (TRI5: trichodiene synthase; TRI6: Zn2His2 transciotion factor; TRI1: C7, 8 oxygenase; TRI4: trichodiene oxygenase) after TPP treatment, which are the key genes associated with DON biosynthesis (McCormick et al., 2006; Nasmith et al., 2011; Subramaniam et al., 2015; Zhang et al., 2015; Tang et al., 2018). In addition, the impacts of NPP on trehalose biosynthesis pathway were evaluated in vitro, including the enzyme activities of TPP and TPS, the content of trehalose, glucose and trehalose-6-phosphate (T-6-P), and the relative expression of TPP and TPS genes. Finally, the effects of NPP on FHB control and DON production were determined in the field.

Section snippets

Chemical compound, fungal strains and culture conditions

Technical-grade NPP (98%) was kindly provided by Aladdin (Shanghai, China). This compound was dissolved in acetone at 10000 μg/mL and stored at 4 °C prior to future use. 43% Tebuconazole aqueous suspension concentrate (SC) was kindly provided by Bayer (Beijing, China). 250 g/L Azoxystrobin SC was kindly provided by Syngenta (Beijing, China).

Ten FGSC strains (G1, G2, G3, G4, G5, G6, G7, G8, G9 and G10) were isolated from the infected wheat ears in the field and stored in the Central Laboratory,

Sensitivity of F. graminearum to NPP

In this study, the sensitivity of the F. graminearum strains to NPP was tested by mycelial growth and conidia germination inhibition methods. For mycelial growth, the EC50 value of F. graminearum strains to NPP were more than 100 μg/mL (Table 2). This suggested that NPP had no good inhibitory effect on mycelial growth of F. graminearum. For conidia germination, the EC50 value of F. graminearum strains to NPP ranged from 34.0715 to 66.7059 μg/mL (Table 3). The results suggested that the

Discussion

FHB caused by FGSC is one of the most serious diseases in wheat production. It not only causes yield reduction, but also contains a variety of Fusarium toxins in the infected grains of wheat, such as the vomitoxin (DON) of trichothecenes, which can cause poisoning of human and livestock and major diseases, and pose a serious threat to food safety. In addition, DON has also been reported as an important virulence factor of FGSC (Goswami and Kistler, 2005; Li et al., 2019). Due to the lack of

Conclusion

In summary, NPP could effectively inhibit the trehalose biosynthesis pathway in PH-1 and reduce the activity of TPP, thus reducing the biosynthesis of trehalose and the raw materials for DON biosynthesis. In addition, NPP could increase the content of T-6-P in PH-1 to affect other regulation pathways. At the same time, it could down-regulate the expression level of TRI genes, thereby inhibit the biosynthesis of DON at the transcriptional level. Field experiments also showed that NPP could

Funding

This work was supported by Zhenjiang Science & Technology Program (Grant No. SH2020008); and Youth Fund Project of Zhenjiang Academy of Agricultural Sciences (Grant No. QNJJ2020002).

Acknowledgements

I appreciate Professor Yiqing Zhuang for his guidance in the whole process of this experiment, Dan Yang of JAAS central laboratorys for assistance with the experiments, as well as every co-author for their joint efforts.

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