Negative Regulation Of Epigenetic Factors NbALKBH9 And NbJMJ16 Study On Resistance Of Host Plants To Tomato Spotted Wilt Virus

Tomato spotted wilt virus（TSWV）is a plant segment negative sense stranded RNA virus,which is a typical representative member of Orthotospovirus and belongs to Bunyavirales.It is listed as one of the world’s top ten plant viruses because of its extremely serious harm.N6-methyl adenine（m⁶A）modification is an important RNA modification that exists widely in eukaryotes such as mammals,insects,plants and yeasts.It dynamically and reversively regulates RNA metabolism and processing,growth and development,stress response and directional differentiation of stem cells,etc.ALKBH is a specific demethylase dependent on Fe²⁺andα-ketoglutarate,which can catalyze demethylation of nucleic acid substrates.Although the important effect of m⁶A modification on viral infection has been widely reported in many animal viruses,it has been rarely reported in plant viruses,and the molecular mechanism by which plant m⁶A modification system regulates the m⁶A modification level of viral RNA to affect viral infection remains unclear.In this study,we focused on the presence of m⁶A modification in TSWV genomic RNA to analyze the pathogenic molecular mechanism of TSWV from a new perspective of viral epigenetics.In addition,TSWV nucleocapsid N was used as bait to screen the host factors interacting with TSWV N,providing new clues for understanding the molecular mechanism of host plant-virus interaction.The specific research contents of this paper are as follows:1.The host plant m⁶A demethylase ALKBH9B negatively regulates tomato spot wilt virus infection.In order to investigate whether there is m⁶A modification in TSWV genomic RNA,the genomic RNA of viral particles was extracted from TSWV infected N.benthamiana and analyzed by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.The results showed that there was high abundance of m⁶A modification in TSWV genomic RNA.The ALKBH family genes are important components of the m⁶A demethylase complex.The ALKBH9B gene knockout mutant has been reported in Arabidopsis thaliana to inhibit the accumulation of Alafafa Mosaic Virus（AMV）,and the homologous gene NbALKBH9B of N.benthaliana was cloned by homologous comparison analysis.The effect of gene silencing on virus infection was evaluated by the fluorescence intensity of TSWV minireplicon reporter gene e GFP using TRV mediated gene silencing system.It was found that when the NbALKBH9B gene was silenced,the accumulation of e GFP protein of TSWV miniplicon was increased,and the expression levels of the minireplicon g RNA,ag RNA and m RNA were also significantly increased,suggesting that silencing NbALKBH9B gene could promote the replication and transcription of TSWV minireplicon.CRISPR/Cas9 knockout transgenic plants of NbALKBH9B gene also had a similar effect on TSWV infection.Western blot was used to detect the accumulation level of TSWV N protein in the silenced NbALKBH9B plants.Compared with the silenced GUS control plants,the expression level of N protein in the silenced NbALKBH9B plants was significantly increased.TSWV was further inoculated into atalkbh9b T-DNA mutant.Western blot was used to detect the expression level of N protein in the mutant.Compared with the wild type（Col-0）,the accumulation of N protein in the mutant was significantly increased.These results suggest that,unlike the antiviral effect of m⁶A modification on AMV,m⁶A modification plays a positive role in the regulation of TSWV which is beneficial to virus infection.2.Host plant lysine specific demethylase JMJ16 negatively regulates TSWV infectionNucleocapsid N is one of the main structural proteins of TSWV,and also an important component of the assembly of ribonucleoprotein（RNP）complex,which is involved in the regulation of viral replication and transcription.The yeast two-hybrid system was used to screen the N interacting proteins from the c DNA library of N.benthaliana and construct the N interacting protein network map,which is of great significance for the further analysis of the function of N gene and the study of the mechanism of virus infection.Firstly,a total of8 candidate genes interacting with N were obtained by screening host factors from the c DNA library of N.benthaliana yeast using N as bait protein.The function of lysine specific demethylase JMJ16 was further studied.JMJ16 is a specific H3K4 demethylase protein containing the JMJ-C domain in Arabidopsis thaliana.After TRV-mediated silencing of Nb JMJ16 gene in N.benthaliana,the fluorescence intensity of reporter gene e GFP of TSWV minireplicon system was used to confirm that silencing of Nb JMJ16 could promote the infection of TSWV.Western blot analysis showed that the protein accumulation of reporter gene e GFP in TSWV minireplicon system was significantly increased.At the same time,TSWV was inoculated on silenced Nb JMJ16 plants.Western blot analysis showed that N protein accumulation in system leaves was significantly increased.In conclusion,Nb JMJ16 negatively regulates TSWV infection in N.benthaliana.