The Molecular Mechanism Of HbWRKY40 In Regulation Of The Resistance Of Hevea Brasiliensis To Colletotrichum Gloeosporioides

It has been reported that WRKY transcription factor is involved in many life activities,such as plant growth and stress responses.It has been proved that WRKY transcription factor is a positive or a negative regulator related to many defense responses by knock-out,silencing or overexpression assays of WRKY transcription factor,but the mechanism of WRKY family in regulation of Hevea brasiliensis defense response is unclear.In the rubber tree genome,a total of 111 genes encode HbWRKY transcription factors.By analyzing the transcriptome data of rubber tree leaves which inoculated with Colletotrichum gloeosporioides and q RT-PCR verification,we found that the expression levels of the four genes LOC110657646,LOC110673911,LOC110640762,and LOC110652339 have up-regulated obviously.Overexpression of these four genes in tobacco caused the accumulation of reactive oxygen species(ROS)in tobacco leaves.Particularly,LOC110640762 cause the accumulation of ROS in tobacco leaves to be about six times compare with the control.Therefore,LOC110640762 was selected for further reaearch.1.The length of LOC110640762 c DNA is 975 bp,and encodes a protein of approximately 35.8 k Da.It contains a WRKY DNA binding domain.Analysis of its amino acid sequence and homology revealed that LOC110640762 had the highest homology with At WRKY40 in Arabidopsis thaliana and Nt WRKY40 in Nicotiana tabacum L.,so it was then named HbWRKY40.2.The subcellular localization of HbWRKY40 in rubber tree protoplasts is in the nucleus.3.In the leaves of rubber trees,the plant hormones induce the expression of HbWRKY40.The expression of HbWRKY40 was highly induced by SA,and its expression level is about 15 times compared with the control.4.Overexpression of HbWRKY40 in Arabidopsis caused enhanced resistance to gray mold.5.HbWRKY40 was overexpressed in rubber tree protoplasts for RNA-Seq.Results showed that after overexpression of HbWRKY40,the abundance of 145 genes was up-regulated and 6 genes were down-regulated,12 of which contained HbWRKY40 binding site like W-box.Expression levels of those 12 genes have significantly changed after SA treatment of leaves.These 12 genes are identified as potential genes that HbWRKY40 directly regulates in the process of responding to biological stress.6.In order to study whether HbWRKY40 directly binds to the promoter region of these 12 potential genes to regulate their expression,the DNA-protein complex was pulled down with Anti-GFP magnetic beads by chromatin immunoprecipitation method,and analyzed by CHIP-q PCR after elution.It was shown that HbWRKY40 directly bound to the promoter region of 10 genes of the 12 potential genes,indicating that these 10 genes are downstream target genes of HbWRKY40.In summary,HbWRKY40 is a positive regulator of rubber tree in regulation of resistance to Colletotrichum gloeosporioides.