Validamycin A enhances the interaction between neutral trehalase and 14‑3‑3 protein Bmh1 in Fusarium graminearum

Trehalase is considered the main target of the biological fungicide validamycin A, and toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization and molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects when compared to wild-type PH-1. When exposed to validamycin A, high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol (DON) production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, Co-immunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1-100μg/mL. Besides, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol (HOG) pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmhs are involved in growth, responces to external stresses and virulence, and validamycin A enhanced the interaction between FgNth and FgBmh1in F. graminearum.

Characterization and Functional Analysis of trehalase Related to Chitin Metabolism in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Glyphodes pyloalis Walker (G. pyloalis) is a serious pest on mulberry. Due to the increasing pesticide resistance, the development of new and effective environmental methods to control G. pyloalis is needed. Trehalase is an essential enzyme in trehalose hydrolysis and energy supply, and it has been considered a promising target for insect pest control. However, the specific function of trehalase in G. pyloalis has not been reported. In this study, two trehalase genes (GpTre1 and GpTre2) were identified from our previous transcriptome database. The functions of the trehalase in chitin metabolism were studied by injecting larvae with dsRNAs and trehalase inhibitor, Validamycin A. The open reading frames (ORFs) of GpTre1 and GpTre2 were 1,704 bp and 1,869 bp, which encoded 567 and 622 amino acid residues, respectively. Both of GpTre1 and GpTre2 were mainly expressed in the head and midgut. The highest expression levels of them were in 5th instar during different development stages. Moreover, knockdown both of GpTre1 and GpTre2 by the dsRNAs led to significantly decreased expression of chitin metabolism pathway-related genes, including GpCHSA, GpCDA1, GpCDA2, GpCHT3a, GpCHT7, GpCHSB, GpCHT-h, GpCHT3b, GpPAGM, and GpUAP, and abnormal phenotypes. Furthermore, the trehalase inhibitor, Validamycin A, treatment increased the expressions of GpTre1 and GpTre2, increased content of trehalose, and decreased the levels of glycogen and glucose. Additionally, the inhibitor caused a significantly increased cumulative mortality of G. pyloalis larvae on the 2nd (16%) to 6th (41.3%) day, and decreased the rate of cumulative pupation (72.3%) compared with the control group (95.6%). After the activities of trehalase were suppressed, the expressions of 6 integument chitin metabolism-related genes decreased significantly at 24 h and increased at 48 h. The expressions of GpCHSB and GpCHT-h, involved in chitin metabolism pathway of peritrophic membrane in the midgut, increased at 24 h and 48 h, and there were no changes to GpCHT3b and GpPAGM. These results reveal that GpTre1 and GpTre2 play an essential role in the growth of G. pyloalis by affecting chitin metabolism, and this provides useful information for insect pest control in the future.

Search for streptomycetes with antagonistic activity to the aggressive Fusarium sp. strain AC

The antagonistic activity of 126 Streptomyces strains against the fungus Fusarium sp. AC was studied. The greatest extent of mycelium growth was inhibited by S. geldanamicininus 3K9, which produces validamycin A.

Rapid Contamination Detection in Validamycin A Production by HS-SPME/GC-MS

Background: Validamycin A (Val-A) is one of the most widely used agricultural antibiotics in East Asia especially for controlling rice sheath blight disease. Fermentation contamination of the industrial Val-A producing strain is a common occurrence. Methods: Fermentation culture of S. hygroscopicus 5008 has a special smell that could be distinguished from other tainted samples. The change of the volatiles in untainted and tainted samples was characterized using headspace solid phase microextraction (HS-SPME) combined with gas chromatography- mass spectrometry (GC-MS). Results: Seventy-one volatile compounds (including alkanes, amines, alcohols, esters, aldehydes and others) were identified and there were significant differences in the composition of volatiles among different samples. Principal component analysis (PCA) based on the GC-MS data was used to identify the important volatile compounds that contributed to the differentiation of the fermentation samples under different fermentation stages, as well as among different pollution species and fermentation media. Contamination could be discovered in time irrespective of the stage of fermentation and the contaminating bacteria in broth. Conclusion: It is the first report to detect contamination by volatile compounds in the antibiotic fermentation and it was proved that HS-SPME/GC-MS is an effective contamination detection method in Val-A production.

Validamycin A Induces Broad-Spectrum Resistance Involving Salicylic Acid and Jasmonic Acid/Ethylene Signaling Pathways

Validamycin A (VMA) is an aminoglycoside antibiotic used to control rice sheath blight. Although it has been reported that VMA can induce the plant defense responses, the mechanism remains poorly understood. Here, we found that reactive oxygen species (ROS) bursts and callose deposition in Arabidopsis thaliana, rice (Oryza sativa L.), and wheat (Triticum aestivum L.) were induced by VMA and were most intense with 10 μg of VMA per milliliter at 24 h. Moreover, we showed that VMA induced resistance against Pseudomonas syringae, Botrytis cinerea, and Fusarium graminearum in Arabidopsis leaves, indicating that VMA induces broad-spectrum disease resistance in both dicots and monocots. In addition, VMA-mediated resistance against P. syringae was not induced in NahG transgenic plants, was partially decreased in npr1 mutants, and VMA-mediated resistance to B. cinerea was not induced in npr1, jar1, and ein2 mutants. These results strongly indicated that VMA triggers plant defense responses to both biotrophic and necrotrophic pathogens involved in salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) signaling pathways and is dependent on NPR1. In addition, transcriptome analysis further revealed that VMA regulated the expression of genes involved in SA, JA/ET, abscisic acid (ABA), and auxin signal pathways. Taken together, VMA induces systemic resistance involving in SA and JA/ET signaling pathways and also exerts a positive influence on ABA and auxin signaling pathways. Our study highlights the creative application of VMA in triggering plant defense responses against plant pathogens, providing a valuable insight into applying VMA to enhance plant resistance and reduce the use of chemical pesticides. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The Inhibitory Effect of Validamycin A on Aspergillus flavus

Aspergillus flavus is one of the most common isolates from patients with fungal infections. Aspergillus infection is usually treated with antifungal agents, but side effects of these agents are common. Trehalase is an essential enzyme involved in fungal metabolism, and the trehalase inhibitor, validamycin A, has been used to prevent fungal infections in agricultural products. In this study, we observed that validamycin A significantly increased trehalose levels in A. flavus conidia and delayed germination, including decreased fungal adherence. In addition, validamycin A and amphotericin B showed a combinatorial effect on A. flavus ATCC204304 and clinical isolates with high minimum inhibitory concentrations (MICs) of amphotericin B using checkerboard assays. We observed that validamycin A and amphotericin B had a synergistic effect on A. flavus strains resistant to amphotericin B. The MICs in the combination of validamycin A and amphotericin B were at 0.125 μg/mL and 2 μg/mL, respectively. The FICI of validamycin A and amphotericin B of these clinical isolates was about 0.25–0.28 with synergistic effects. No drug cytotoxicity was observed in human bronchial epithelial cells treated with validamycin A using LDH-cytotoxicity assays. In conclusion, this study demonstrated that validamycin A inhibited the growth of A. flavus and delayed conidial germination. Furthermore, the combined effect of validamycin A with amphotericin B increased A. flavus killing, without significant cytotoxicity to human bronchial epithelial cells. We propose that validamycin A could potentially be used in vivo as an alternative treatment for A. flavus infections.