Roels Of N-and C-terminal Of Arabidopsis P4-ATPases ALAL And ALA7 In Mycotoxin Detoxification

The secondary metabolite deoxynivalenol(DON),produced by Fusarium graminearum,acts as a virulence factor that is essential for the infection.Fusarium head blight(FHB)and maize ear rot are important diseases,caused by F.graminearum,leading to great economic losses.With high thermostability,DON is probably the best known and most common contaminant of grains and processed foods.Consuming by humans and animals,DON can provokes acute and chronic disease symptoms,including vomiting,anorexia,indigestion and decreased immune function.In addition,DON displays carcinogenicity and teratogenicity,producing a potential risk to the health of humans and animals.Cinnamyl Acetate(CIA),a low molecular weight substances produced by Verticillium dahliae,which are toxic to plants.V.dahliae is the primary causal agent of Verticillium wilt that causes tremendous losses worldwide.The pathogen has a wide host range in plants and is difficult to be controlled.Toxins secreted by V.dahliae are considered to be a key chemical factor that causes Verticillium wilt.On the other hand,plants have evolved numerous defense mechanisms to resist the attack of a wide variety of toxins,including mycotoxin detoxification and toxin compartment.However,the mechanisms of plant tolerance to DON and CIA are still unclear.Previous research of our team found that ALA1 contributes to both vacuolar transport and tolerance of DON in Arabidopsis;ALA7 plays an important role in transporting CIA to vacuole,increasing Arabidopsis' tolerance to CIA and resistance to V.dahliae.ALA1 and ALA7 belong to P4-ATPases subfamily,which is associated with lipid asymmetry,vesicle formation and protein transport.In order to study the roles of ALA1 and ALA7 in the detoxification of these mycotoxins,the N-and C-terminus of ALA1 and ALA7 were analyzed in this paper.The main results are as follows:1.Deletion of C-terminus of ALA1 reduces tolerance of Arabidopsis to DONTo clarify the role of N-and C-terminus of ALA1 in tolerance to DON,the N-or C-terminus of ALA1 were deleted,respectively.DON can inhibit the germination of Arabidopsis seeds.There was no significant difference in the root length between wild type Arabidopsis and Arabidopsis thaliana with N-terminus deleted.However,the deletion of C-terminus resulted in a shorter root length like that of the loss-of-ALA1-function mutant(ala1-12),indicating that the C-terminus of ALA1 played an important role in the tolerance of Arabidopsis to DON.2.Deletion of N-and C-terminus of ALA1 decreases the transport of Don to the vacuoleThe observation of laser confocal microscopy showed that the deletion of N-and C-terminus of ALA1 decreased the accumulation of DON in the vacuole,which is consistent with the decreased distribution of DON in ala1-12,indicating that N-terminus and C-terminus of ALA1 play an important role in the transport of DON to the vacuole.3.Deletion of N-and C-terminus of ALA7 reduces the tolerance of Arabidopsis to CIACIA had little effect on the germination of Arabidopsis seeds,but it could cause leaf yellow.The yellowing level of leaves of transgenic Arabidopsis expressing N-or C-terminus defected ALA7,similar to loss-of-ALA7-function mutant(ala7-24),is significantly worse than that of wild type,indicating that the deletion of N-and C-terminus of ALA7 could decrease the tolerance of Arabidopsis to CIA.4.Deletion of N-terminus of ALA7 affect the transport of CIA to the vacuoleConfocal microscopy observation showed that like mutant ala7-24,the deletion of N-terminus of ALA7 decreased the accumulation of CIA in vacuoles,while the deletion of C-terminus did not affect the accumulation of CIA in vacuoles.It is suggested that N-terminus of ALA7 plays an important role in the transport of CIA to vacuole.5.Screening of proteins potentially interacted with ALA1 or ALA7Yeast library screening and bimolecular fluorescence complementation experiments showed that proteins potentially interacted with ALA1,include CARK1,WNK10,CDC48,GPK1,NRAMP4,ABCB27,APG8 H,CCP2,Rab A2 D,SEH1H,GLTP,GRF2 and Rab B1 C.It is speculated that the transport of DON mediated by ALA1 may be involved in protein phosphorylation,ubiquitination and vesicle transport.As to ALA7,its potential interaction protein is VAMP724,a SNARE,suggesting that the transport of CIA by ALA 7 is related to SNARE mediated vesicle transport.