Functional Identification Of An Agrobacterium Tumefaciens O-demethylase And Its Effect On Pathogenicity

Agrobacterium tumefaciens is a plant pathogen that can cause crown gall tumors in a variety of plants.Some plant-derived signaling molecules,such as phenolic compounds,can directly affect Agrobacterium tumefaciens interactions with the host.On the one hand,phenolic compounds affect the growth of bacteria;on the other hand,they act as Agrobacterium tumefaciens signals to the host,which can activate the expression of vir gene and affect the pathogenicity of Agrobacterium tumefaciens.In this study,atu1420,a gene involved in phenolic metabolism,was found in Agrobacterium tumefaciens.Atu1420 is predicted to be O-demethylase,which is highly homologous to the structure of LigM in Sphingobium sp.SYK-6.This study mainly reveals the function of O-demethylase Atu1420 from the following aspects.First,O-demethylase was heterologous expressed and purified,and the biochemical activity of Atul420 was identified.It was predicted by the molecular dock model that Atu1420 could generate binding and binding active amino acid sites with acetosyringone(AS),which were similar to the binding sites of LigM and vanillic acid(VA).The purified Atu1420 protein was incubated with AS in vitro.HPLC-UV analysis showed that Atu1420 had the biochemical activity to degrade AS.Subsequently,Δatu1420 was obtained by unmarked counterselectable homologous recombination,and the Hbatu1420 strain was obtained by the replacement plasmid.By measuring the growth curves of Agrobacterium tumefaciens C58,Δatu1420 and Hbatu1420,it was found that atu1420 gene deletion did not affect the normal growth of Agrobacterium tumefaciens,and atu1420 alleviated the inhibition of VA on the growth of Agrobacterium tumefaciens.After adding AS to Agrobacterium tumefaciens culture medium for a period time,HPLC analysis showed that atu1420 deletion significantly reduced the activity of Agrobacterium tumefaciens to degrade AS.qPCR results revealed that VA and IAA(Indole-3-acetic acid)can positively regulate the expression of atu1420 gene.In addition,through the construction of a molecular docking model and differential scanning fluorescence assay(DSF),it was identified that VA might bind to transcription suppressor Atul419 to regulate the expression of atu1420 gene,thus affecting the degradation activity of atu1420 on AS.The expression of atu1420 gene is regulated by VA and IAA,and its catalytic degradation ability of AS will be affected.and the expression of AS-induced vir gene will also be affected.The residual amount of AS in C58 and Δatz1-420 cultures treated with VA or IAA and AS was determined by HPLC.It was found that after addition VA or IAA,the residual amount of AS in C58 culture media decreased,and VA or IAA improved the degradation ability of atu1420 to AS.The content of AS in the culture medium Δatu1420 changed slightly,and the residual amount was still relatively high,vir gene expression was then detected by qPCR and 3galactosidase activity assay in C58,Δatu1420 and Hbatu1420 carriers of promotervirB::lacZ gene fusion plasmid.It was found that atu1420 deletion significantly enhanced the expression of AS-induced vir gene.VA inhibited the expression of AS-induced vir gene in Agrobacterium tumefaciens C58 and Δatu1420,especially in Δatu1420 strain,while atu1420 can alleviate VA inhibition of AS-induced vir gene expression.Furthermore,IAA also inhibited the expression of AS-induced vir gene in C58,possibly because IAA enhanced the transcription of atu1420 gene and thus enhanced the degradation of atu1420 to vir gene inducer AS.Deletion of atu1420 could partially alleviate the inhibition of IAA on vir gene expression but does not remove the inhibition,suggesting that improving the activity of atu1420 on AS degradation is one of the ways that IAA can inhibit the expression of vir gene.Agrobacterium tumefaciens,as a tool bacterium for plant transgenic,is still very inefficient in its mediated genetic transformation.Therefore,a binary vector system was used to detect the transient transformation of tobacco mediated by Agrobacterium tumefaciensΔatu1420.and atu1420 deletion was found to enhance the transient transformation effect of Agrobacterium tumefaciens.To investigate the effect of atu1420 deletion on host pathogenicity,the Agrobacterium tumefaciens C58 and Δatu1420 were inoculated on the surface of different plant tissues and compared their tumorigenesis.The study found that the tumors formed on the surface of different plant tissues infected by Agrobacterium tumefaciens varied greatly,and the atu1420 deletion produced more tumors on carrot root discs and kalanchoe leaves than the wild-type strain,this suggests that atu1420 may affect the tumorigenesis of Agrobacterium tumefaciens in a complex way.Based on the above studies,the role of Atu1420 in the interaction between Agrobacterium tumefaciens and the host is flexible and complex.By studying the function of Atu1420,which can degrade AS,this study provides a theoretical basis for further exploring its degradation mechanism and cloning related degradation genes,and it broadens our understanding of the regulatory network of Agrobacterium tumefaciens and host interaction.In addition,this study confirmed the application potential of Atu1420 in the improvement of transgenic engineered bacteria,which provided a new protein target for further improving the efficiency of gene transfer and transforming more efficient transgenic engineered bacteria.