BTB-BACK Domain E3 Ligase MdPOB1 Suppresses Plant Pathogen Defense Against Botryosphaeria Dothidea By Ubiquitinating And Degrading MdPUB29 Protein In Apple

Apple ring rot is one of the most destructive apple diseases worldwide,and it is primarily caused by Botryosphaeria dothidea(B.dothidea).Apple is the main commercial crop in China.In the process of apple production,apple ring rot can cause a drop in fruit yield and quality,leading losses in economic.Therefore,it is of great significance to study the resistance mechanism towards ring rot in apple fruits to improve the yield and quality.Ubiquitination,has been shown in previous studies,plays pivotal roles in plant stress responses,and it is mediated by a three-step enzymatic cascade,which consists ubiquitin activating enzymes E1,ubiquitin conjugating enzymes E2,and ubiquitin ligases E3.Among them,E3 determines the specific identification of target proteins,thus promoting the ubiquitination of downstream target proteins.In this study,we reported that apple POZ/BTB CONTAINING-PROTEIN 1(MdPOB1),a BTB-BACK domain E3 ligase protein,functioned as a suppresser in apple pathogen defense against B.dothidea.Both in vivo and in vitro assays were conducted to explore the resistance mechanism to apple ring rot of MdPOB1 protein.The main works and results are as follows:1.MdPOB1 inhibits plant pathogen defense against B.dothidea in apple.Bioinformatics analysis showed that MdPOB1(MD07G1143200)contained a conserved BTB-BACK domain and was highly homologous with AtPOB1.MdPOB1 overexpressed and expression-inhibited apple calli were obtained using Agrobacterium-mediated transformation.And MdPOB1 transiently transformed apple fruits were obtained by fruit injections.After infection with B.dothidea,it was found that both overexpressed apple calli and apple fruits decreased the defense against B.dothidea,while expression-inhibited apple calli and apple fruits increased the defense against B.dothidea.The results indicated that MdPOB1 could inhibit plant pathogen defense against B.dothidea in apple.2.MdPOB1 protein interacts with and degrades MdPUB29 protein by ubiquitination.To explore the resistance mechanism to B.dothidea of MdPOB1 protein,an apple plant U-box E3 ligase MdPUB29,which was interacted with MdPOB1 protein,was screened by yeast two-hybrid assay.The interaction between MdPOB1 and MdPUB29 was further verified by pull-down and bimolecular fluorescence complementation experiments.In vivo ubiquitination assays showed that MdPOB1 could promote the degradation of MdPUB29 through ubiquitin/26 S proteasome pathway.3.MdPUB29 enhances plant pathogen defense against B.dothidea in apple.Bioinformatics analysis showed that MdPUB29(MD01G1010900)contained a conserved U-box domain and was highly homologous to AtPUB29.Subcellular localization showed that MdPUB29 was located in the nucleus.MdPUB29 overexpressed and expression-inhibited apple calli and ectopic expressed Arabidopsis were obtained by Agrobacterium-mediated transformation.After infection with B.dothidea,it was found that both overexpressed apple calli and transgenic Arabidopsis enhanced the defense against B.dothidea,while expressioninhibited apple calli decreased the defense against B.dothidea.These results indicated that MdPUB29 could enhance plant pathogen defense against B.dothidea in apple.4.MdPOB1 decreases plant pathogen defense against B.dothidea in a MdPUB29-dependent manner in apple fruits.To further study the resistance mechanism to B.dothidea of MdPOB1 protein,transiently transformed apple fruits were obtained by fruit injections.After infection with B.dothidea,it was found that apple fruits that inhibited the expression of MdPOB1 alone increased the defense against B.dothidea,while apple fruits that inhibited the expression of MdPUB29 alone and inhibited both expression of MdPOB1 and MdPUB29 decreased the defense gainst B.dothidea.The results indicated that MdPOB1 decreases plant pathogen defense against B.dothidea in a MdPUB29-dependent manner in apple fruits.