Functional Analysis Of A Fusaric Acid Resistant Gene In Arabidopsis Thaliana

Fusarium wilt is a worldwide soil-borne disease caused by Fusarium oxysporum.At present,there are various economic crops reported to be seriously harmed by Fusarium wilt.As a typical soil-borne pathogen,Fusarium oxysporum secretes cell wall degrading enzymes when infecting the root of the host plant.At the same time,it produces penetrating hyphae,extending to the vascular system of host.While the hyphae absorbs water and nutrients of the plant,it also discharges various phenolic metabolites produced by its own metabolism into the parasitic environment.Current research indicates that fusaric acid(FA)is the main pathogenic factor in the phenolic acid metabolite of Fusarium oxysporum.It alters the permeability of cell membranes,causing normal metabolic disorders,decreased cell vitality and loss of defense function.On the one hand,these toxicities inhibit the growth of other microorganisms in the soil environment,which makes Fusarium oxysporum superior in competition with other microorganisms.On the other hand,it destroys the plant defense response,causing foliar chlorosis,premature defoliation of lower leaves,extensive necrosis in vascular tissue,wilting,and,ultimately,plant death.Therefore,the research on the control of Fusarium wilt is mainly to study how plants withstand high concentration of FA.The researchers found a class of proteins:FUSC(Fusaric acid resistance protein),which allows bacteria withstand high concentrations of FA in the Gram-negative bacteria.But how FUSC helps cells resist FA remains unanswered.So,is there a protein similar to FUSC in plants to make them resistant to fusaric acid?We compared the predicted functional domain of FUSC in the NCBI database and found that there was a protein,AtPHA1,which belongs to P-HYDROXYBENZOIC ACID EFFLUX PUMP SUBUNIT-RELATED superfamily and has the high sequence identity with functionally characterized FUSC protein AaeB of K.oxytoca.Then,we selected AtPHA1 and AtPHA2 as research objects and screened single-gene T-DNA insertion mutants and double-gene mutants of phal and pha2.By using tissue staining,fluorescent protein localization and molecular biology,we obtained the following findings:1.Both PHA1 and PHA2 are proteins localized by the plasma membrane;We used the method of transient transformation of Arabidopsis suspension cells to study the sublocation of PHA1 and PHA2 and found that the fusion proteins PHA1-GFP and PHA2-GFP colocalized with the plasma membrane localization protein SCAMP-RFP,so we speculate that PHA1 and PHA2 are also proteins localized by the plasma membrane.2.FA inhibits the expression of PHA1 and PHA2,and this inhibition is time-and concentration-dependent;We used qRT-PCR to study the expression levels of PHA1 and PHA2 at different concentrations of FA and different times of FA treatment.The results showed that the expression levels of PHA1 and PHA2 decreased with the prolongation of the time of FA treatment and decreased with the increase of FA concentration.3.FA inhibits the growth of Arabidopsis,and induces the increased production of ROS,but neither PHA1 nor PHA2 could resist these effects of FA;FA induces a large amount of intracellular ROS production,and the accumulation of ROS destroys the normal metabolism of cells.We studied changes in root growth and ROS accumulation in different mutants and WT(wild-type)under FA treatment.The results showed that under FA treatment,the growth of root was inhibited and DAB staining was deepened,but there was no significant difference in these phenotypes between mutants and WT.4.PHA1 expresses mainly in guard cells;PHA2 widely expresses in the root,leaf,flower and pod;We found that there was no difference in the phenotype of root growth inhibition and ROS production under FA treatment between mutants and WT,we speculated the reason maybe that PHA1 and PHA2 did not function in the roots.Therefore,we studied the organization of PHA1 and PHA2.In this study,we used both histochemical staining and qRT-PCR.The results showed that PHA1 was mainly expressed in leaves except for weak expression in hypocotyls.Amplifying the leaves after GUS staining,we found that PHA1 was only expressed in guard cells.PHA2 is widely expressed in tissues such as roots,leaves,flowers and pods.In flowers,PHA2 is mainly expressed at the top of bracts,filaments and stigma;in pods,PHA2 is mainly expressed at the base and at the top.5.FA could not induce stomatal opening or closing,but it can inhibit stomatal movement;Since PHA1 is mainly expressed in the guard cells,we studied the stomatal movement under FA treatment.The results showed that the FA treatment alone did not change the stomatal aperture;the stomal closure process under FA and ABA co-treatment was slower than the process under ABA treatment alone;the opening process slowed down under FA treatment.6.The mutant of PHA1 impairs FA's ability to block the closure of stomatal,or FA's ability to block the closure of stomatal may depend on PHA1.Comparing the stomatal closure process of phal and WT,the stomatal closure process of pha2 and dm,we found that after the mutation of PHA1,the stomatal closure process slowed down under ABA treatment,and the inhibition rate of FA on the stomatal closure process also decreased.Therefore,the process by which FA hinders the closing of the pores may be achieved by PHA1.Taken together,we hypothesize that PHA1 may play a role in maintaining the efflux of ions,the function of PHA1 allows cells to resist the toxicity of FA to inhibit ion flux.Related research on the function of PHA1 can provide a theoretical basis for the study of plant resistance to Fusarium wilt.