Function Analysis Of Glycosyltransferase Gene UGT73C4 Involved In Disease Resistance In Arabidopsis Thaliana

Pathogenic bacteria are ubiquitous in nature.Pathogenic bacteria enter plants cells through multiple pathways and can lead to plant diseases.Glycosyltransferases are a very important class of enzymes which can catalyze the glycosylation of plant molecules through transfering the sugar moiety to the acceptor molecules,changing their biological activities and water-solubility for acceptors.Therefore,glycosyltransferases play significant roles in plant growth and environmental adaptation.According to the open-access chip database and published article,the Arabidopsis glycosyltransferase gene UGT73C4 is possibly up-regulated by pathogens and SA.So,this gene may be involved in biotic stress responses.In order to clarify the function of glycosyltransferase UGT73C4,this research was carried out.The wild type Arabidopsis thaliana was treated with the pathogen Pst DC3000 and subjected to the analysis on the expression level of UGT73C4 by qPCR.The results showed that UGT73C4 was up-regulated by pathogens and mostly up-regulated at 24h of treatment.At the same time,the transgenic lines were obtained in which UGT73C4 promoter can drive the GUS gene expression.It was found that the GUS activity was enhanced with the time increase of the pathogenic bacteria treatment on GUS transgenic plants.In addition,UGT73C4 was also induced by salicylic acid(SA).These results indicated that the glycosyltransferase gene UGT73C4 is really induced by the pathogenic bacteria PstDC3000 and SA,and this gene may be involved in plant immune responses.To analyzing the gene function,UGT73C4 knockout plants and overexpression plants were prepared and their leaves were treated with Pst DC3000.We found that the number of propagated pathogens in UGT73C4 overexpressing leaves was much lower than that in wild type and mutant lines.Tobacco transient transformation experiments further demonstrated that UGT73C4 can enhance plant disease resistance.We further explored the mechanism of UGT73C4 in plant immunity.Through DAB and NBT staining experiments,it was found that UGT73C4 overexpressing lines had obviously increased ROS accumulation than wild type plants.SA-associated PR1 and PR2 genes were clearly up-regulation in overexpressed lines.Further,we examined the related genes in the SA pathway and found that the expression levels of EDS1,PAD4,and ICS1 in overexpressing lines were much higher than those in mutant lines and wild-type Arabidopsis after pathogenic bacteria treatment.In order to know whether a change occurred in SA level,we determined the SA accumulation after pathogenic bacteria treatment.We found that both the free SA and the total SA contents were higher in the overexpressing plants than in the wild type and mutant plants.These results suggested that glycosyltransferase UGT73C4 contributes to plant disease resistance likely through the SA pathway.In this study,we also found that the glycosyltransferase gene UGT73C4 was up-regulated by induction of soil-borne fungus Foc699.Thus,we treated the UGT73C4 knockout lines,overexpressing lines and wild Arabidopsis with the GFP-tagged fungus Foc699.The fluorescence of GFP in the roots of overexpressing plants was obviously less than wild type.However,the fluorescence of GFP was clearly stronger in mutants than in wild type.After the plant roots were infected with the soil-borne fungus Foc699 and the plants were then grown in soil,the overexpressing lines were found to be grown to a better state than the mutant and wild-type lines,which proved that the glycosyltransferase UGT73C4 may be also involved in the antifungal process in the underground part of the plants.Jasmonic acid(JA)-associated and anti-fungal-related maker genes were up-regulated to a much higher level in overexpressing plants than in wild type plants and mutant plants.These results supposed that JA pathway is likely involved in the disease resistance in the underground part of the plants mediated by the glycosyltransferase UGT73C4.In addition,the prokaryotic expression of UGT73C4 was explored and the enzyme was purified.UGT73C4 enzymatic activity was analysed towards more than 20 natural compounds including hormones,flavonoids,monolignols,coumarins,Hydroxycinnamic acids.However,no substrate was found to be glycosylated by UGT73C4 at present.In conclusion,through a series of studies on UGT73C4 mutant and overexpression plants,it was confirmed that the glycosyltransferase gene UGT73C4 plays an important role in enhancing the plant resistance to biotic stress,likely through SA and JA related pathways.The detailed molecular mechanism needs further study.