Functional Analysis Of M~6A Demethylases Watermelon ClALKBH2B And ClALKBH4B In Response To CGMMV Infection

Watermelon(Citrullus lanatus,2n=2×=22)is an annual trailing plant in the family Cucurbitaceae.China is the leading country of annual planting area and production of watermelon.However,watermelon has been severely suffered from Cucumber green mottle mosaic virus(CGMMV).N6-Methyladenosine(m~6A)is one of the commonest and the most abundant modification and post-transcriptional regulation in eukaryotic mRNA.Recently,m~6A modification has been discovered to play an vital role in the responses of plants to viruses.m~6A modification is a dynamic and reversible process,including methylation(writers),demethylases(erasers),and recognition(readers).We have detected two watermelon CGMMV-responsive m~6A demethylases genes ClALKBH2B and ClALKBH4B via m~6A-seq and RNA-seq in previous study,and their upstream regulatory mechanisms and roles in CGMMV resistance were further studied.Results as follows:(1)A total of 12 ClALKB proteins were identified from watermelon genome by bioinformatics analysis.These protein length are from 99～697 aa,the molecular weight are from 11.390～75.389 kDa,and the isoelectric point are from 4.70～9.39.Subcellular localization prediction showed that most of watermelon ClALKB proteins are located in nucleus.Transient expression of tobacco indicated that ClALKBH2B is located in the cytoplasm,while ClALKBH4B is located in the nucleus.All of the ClALKB proteins were clustered into 2 subfamilies and have 1-4 conserved structural motifs except ClALKBH8B which has no conserved motif.All of the ClALKB genes contain exons and introns.Some hormone-and stress-responsive cis-acting elements in the promoters of ClALKB genes were identified.The spatio-temporal distribution analysis indicated ClALKB genes are expressed ubiquitously in watermelon.The response patterns of ClALKB genes were varied in CGMMV-resistant and tolerant watermelon in response to CGMMV infection.And the expression of ClALKBH4B in resistant watermelon were induced by CGMMV infection,which suggested ClALKBH4B was probably positive regulation of disease resistance.(2)The accumulation of CGMMV movement protein(MP)and coat protein(CP)coding genes under CGMMV infection were detected when transient overexpression of ClALKBH2B and ClALKBH4B genes in watermelon by qRT-PCR.The results showed the expression of MP and CP genes were significantly decreased on 3 and 5days past infection.It was preliminarily proved that watermelon ClALKBH2B and ClALKBH4B are CGMMV-resistain.The overexpression vectors of ClALKBH2B and ClALKBH4B were constructed and transformed them into watermelon by Agroinfiltraction.At present,some overexpressed transgenic lineshave been obtained,which laid a foundation for the further study of ClALKBH2B and ClALKBH4B in response to CGMMV infection.(3)The promoters of ClALKBH2B and ClALKBH4B were cloned and their activities were detected byβ-glucuronidase(GUS)activity staining.Moreover,yeast one-hybrid screen was performed in further and a total of five candidate upstream transcription factors interacted with ClALKBH2B were obtained,including MYB transcription factor,BTB/POZ and TAZ domain containing protein 2,CBF1interacting corepressor,Telomere-binding family protein,and Transcription factor.A total of 23 candidate upstream transcription factors interacting with ClALKBH4B promoter,including Zinc finger CCCH domain protein,Heat stress transcription factor c-1b,NAC-domain protein,MYB family transcription factor family protein,BZIP-transcription factor,etc.This study will provide further research basis for clarifying the biological functions and transcriptional regulation mechanism of ClALKBH2B and ClALKBH4B in CGMMV response and provide a foundation for further study on the regulatory mechanism of m~6A modification in virus infection in watermelon.