Assay Of Fungal ABCC-type Transporters On Enhancing Tolerance Of Plant To Several Mycotoxins

Aflatoxin B1(AFB1),deoxynivalenol(DON)and cinnamyl acetate(CIA)are toxic metabolites produced by phytopathogenes,Aspergillus flavus and parasiticus,Fusarium graminearum and culmorum,and Verticillium dahlia,respectively.It has been known that CIA is severely detrimental to the growth of plants and thus regarded as a virulence factor of the disease.AFB1 and DON contaminate many agricultural products,such as corn and peanut,and thus cause tremendous health problem in both developing and developed countries.Compartmental detoxification in planta is an important way to remedy this problem.ATP-binding cassette(ABC)transporters play an important role in the detoxification of xenobiotics in plant.They transport glutathione conjugates,formed by covalent linkage to the endogenous glutathione,from the cytosol to the vacuole.However,the relationship between ABC transporters and detoxification of AFB1,DON and CIA is largely unknown.To this end,here,the sensitivity to AFB1 of fifteen Arabidopsis abcc mutants was investigated.In addition,ABC transporters derived from Aspergillus flavus and Magnaporthe oryzae(AfABC and MoMRP)were ectopically expressed in Arabidopsis to assess their potential in mycotoxin detoxification in planta.The main results are as follows: 1.The deletion of ABCCs does not impact the sensitivity of Arabidopsis to aflatoxin B1Treated with AFB1,both Arabidopsis ABCC gene-knockout lines and wild type control exhibited similar sensitivity to the toxin.No significant alteration in root elongations,cotyledon albino and true-leaf chlorosis was found between the mutants and wildtype,suggesting that the deletion of single ABCC gene in Arabidopsis had no significant effect on the tolerance of Arabidopsis to AFB1.2.Expression of AfABC and MoMRP in Arabidopsis enhances the tolerance to aflatoxinsAfABC or MoMRP was constitutively expressed in Arabidopsis thaliana,and transgenic plants were treated with AFB1 or crude aflatoxin.It was found that the true leaves of the transgenic lines were larger than those of the wild type and the roots of the transgenic lines were considerably longer than those of the wild type,demonstrating that the tolerance of transgenic Arabidopsis to aflatoxins was enhanced.Furthermore,AFB1 concentration in AfABC and MoMRP-expressing lines was less than that in the wild type.Accordingly,it was speculated that the enhanced tolerance to AFB1 in transgenic plants was resulted from the increased degradation of the toxin.3.Inhibition of the synthesis of glutathioine(GSH)reduces resistance of AfABC-expressing plant to AFB1To investigate the role of GSH in the resistance of AfABC-enhanced Arabidopsis thaliana to AFB1,glutathione synthesis inhibitor(betionine sulfoximine)was added to the culture medium.Results showed that the resistance to AFB1 conferred by the ectopic expression of AfABC was decreased,indicating that AfABC-enhanced resistance to AFB1 required GSH.4.AfABC and MoMRP does not increase the tolerance of Arabidopsis to CIA and DONNo noticeable increase in resistance to CIA or DON was observed in transgenic AfABC and MoMRP lines compared to the wild type.Moreover,the distribution and accumulation of fluorescence-labeled DON and CIA in the root cells of transgenic plants were similar with that of the wild type control.Based on these results,it is speculated that AfABC and MoMRP may not participate in the transport of DON and CIA into the vacuole.In addition,the distribution of CIA in most abcc mutant cells was not significantly changed,suggesting that ABCC-type transporters of Arabidopsis thaliana may be not involved in the transport of CIA into vacuoles.In conclusion,ectopic expression of AfABC or MoMRP can increase plant resistance to aflatoxin;however,these two genes have little contribution to the tolerance to CIA or DON,suggesting that the transport mechanism of CIA and DON is different from that of AFB1.