| Rice false smut(RFS)is a significant disease which is caused by the pathogenic fungus Villosiclava virens(anamorph:Ustilaginoidea virens)in China.The pathogen infects rice to form Rice false smut balls(RFSBs)and accumulates variety of my cotoxins.Ustiloxin is one main kind of metabolite in RFSBs.Among them,ustiloxin A is accounted for about 80%of the total content of known ustiloxins in RFSBs.The toxins can not only inhibit the growth of rice,but also poisoned people and animals.For evaluating the effects and risks of ustiloxin on the growth and development of plants and animals,the formation time of all known ustiloxins and the dynamic distribution of ustiloxin A in the course of infection were measured firstly.Then,the effects of ustiloxin A on the gene expression in the development of rice spikelets were studied,as well as the effects on the growth and development of Arabidopsis root,central cell and endosperm including cytoskeleton in them.In addition,the toxicity of ustiloxin A on animal growth were analyzed by using human tumor cells and zebrafish as research models.The main results were as follows:(1)Monomers of ustiloxin A,B and G were obtained from RFSBs via ultrasonic extraction,resin enrichment,reversed phase silica gel,Sephadex gel column chromatography and semi preparation high performance liquid chromatography.(2)The technique of artificial inoculation coupled with UPLC-ESI-HRMS was introduced to detect the accumulation dynamics of ustiloxins.The accumulation time of six known ustiloxins were initiated 5-9 days post inoculation(dpi),before the formation of RFSBs.The content of ustiloxin A in each spikelet increased from 6.0 ng/spikelet at 5 dpi when hypha of U.virens grew on the surface of the filament to 14157.1 ng/spikelet at 25 dpi when chlamydospores matured.Two accumulation peaks of ustiloxin A per dry weight(DW)were 1164.3 μg/g and 1321.2 μg/g at 15 dpi and 25 dpi,respectively.A RNA-seq result showed that 146 genes were differentially expressed in rice spikelets treated with 50 μg/ml of ustiloxin A for 3 hours.Among differential expressed genes(DEGs),OsHSP70 genes,which is related to microtubule dynamic,in Protein processing in endoplasmic reticulum pathway were upregulated,And resistance related genes,including OsTGA2 gene in salicylic acid pathway and OsLOX2 gene in Linoleic acid metabolism pathway,were downregulated.It indicate that ustiloxin A may affect the microtubule dynamic and resistance in rice to help the pathogenicity of U.virens.(3)F-actin labeled Arabidopsis line was generated by Agrobacterium tumefaciens mediated transformation(ATMT)in col-0 background.The effects of ustiloxin A on the growth and development of seedlings and the cytoskeleton in root epidermal cells were studied by using stereoscope,fluorescence microscope and laser confocal microscope.The results showed that ustiloxin A could not only disassemble microtubule,but also inhibit elongation of root and hypocotyl,morphogenesis of root epidermal cell,and affect growth of root hair in Arabidopsis.In addition,live-cell imaging was used to study the effects of ustiloxin A on the cytoskeleton structure and dynamics of central cell and free-nuclear endosperm,as well as the effects on the development of free-nuclear endosperm.The results were as follows:the microtubule in the central cells was disassembled by ustiloxin A,but the F-actin in central cells wasn’t disassembled;the number of F-actin in central cells of treated group increased compared with that of the control;the inward movement of F-actin stopped in central cell of treated group;after treatment with ustiloxin A,the fluorescence signal of tubulin in the free-nuclear endosperm was transferred from the original radial microtubule system(RMS)to polygon,circularity or spot depending on time and dose;the mitosis in central cell was also inhibited at the same time.(4)The antiproliferative activities of ustiloxin A and B were evaluated in human tumor cell lines MDA-MB-231,MGC803,A549 and HepG2.Both ustiloxin A and B showed antiproliferative activity in the concentration range from 1.5625 μmol/L to 25μmol/L.Among the four cell lines,human breast cancer cell line MB-MDA-231 is most sensitive to the antiproliferative activity of them.The antiproliferation EC50 of ustiloxin A to 4 cell lines was lower than that of ustiloxin B,indicating ustiloxin A has higher antiproliferation activity.In the test dose range,only 25 μmol/L ustiloxin A showed cytotoxicity to human lung cancer cell A549.The results above showed that ustiloxin A and B were not toxic to human tumor cells,but could significantly inhibit their proliferation in a certain dose range.(5)The effects of ustiloxin A on embryo development,motility and gene expression were studied and evaluated by using zebrafish as a model.The results showed that the effects of ustiloxin A on the hatching rate,mortality,body length and heart rate of zebrafish embryo/larva were positively correlated with the dose.In 25 μ m ustiloxin A treated group,the hatching rate of zebrafish embryos decreased by 11.41%,mortality increased by 14.27%,body length shortened by 0.12cm,heart rate of larvae increased from 79.16±2.90/30s of the control group to 88.47±1.25/30s,and swimming speed of zebrafish larvae at light period decreased by about 60%under the light dark alternate stimulation.The expression of 339 genes in zebrafish larvae treated with 25 μM ustiloxin A were significantly changed.Among them,jdp2,which is related to cardiac homeostasis,was significantly downregulated.DNA photolyase genes Cry5 and cry-dash were significant upregulated.Eight human diseases-related pathways such as Legionellosis pathway were significantly alternated,and Nine metabolism related pathways such as drug metabolism cytochrome P450 pathway were significantly down regulated.The results showed that ustiloxin A had significant toxicities on the development and growth of zebrafish embryos and larvae.In this study,the accumulation of ustiloxin A in the pathogenesisic process of U.virens were measured,what enriched the understanding of the role of ustiloxins play in the pathogenic processesis.Here,it was also clarified that ustiloxin can inhibit the growth and development of endosperm and root by affecting the structure and dynamics of cytoskeleton and nuclear division.This study also confirmed that acute exposure to ustiloxin A could affect the growth and development of early life zebrafish,Danio rerio.The results of this study provides data support for the comprehensive and scientific evaluation of the impacts of ustiloxin on food safety and animal health. |
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