Establishment Of Efficient Apple Protoplasts Expression System Based On Pro-BIUTNT Promoter And Its Application In Immune Research

The discovery of elite genes and in-depth analysis of specific signal transduction mechanism of apple are inseparable from the high-efficiency and precise regulated expression technology of target genes based on apple cells.At present,main research means in the field of apple research can be summarized as the stable genetic transformation system based on homologous cells and the heterologous genetic transformation system.Homologous not only has long technical cycle and low efficiency,but also causes great inconvenience in the field of researching modification of some key proteins at a specific time,especially after transcription.As for the heterologous transformation system,although we can refer to the developed research methods of model plant,the actual biological function of the target protein may not be revealed precisely due to the great difference on cell physiological activity and regulatory mechanism in heterologous cells.As a powerful technology,protoplast transient expression has been widely used in model plants including Arabidopsis,but it has not been reported in the field of apple research.In this study,protoplasts were isolated from callus of‘Orin'and‘Zihong'apple,and a high-efficiency transient expression system of apple protoplast was established based on Pro-BIUTNT promoter.Partial molecular mechanism of apple immune resistance was studied.The PTI resistance pathways of apple were preliminarily revealed,including specific interaction between BAK1 and FLS2 induced by flg22 and MKK7-MAPK6-WRKY33 signaling.The ETI resistance pathways of apple were also proved,including the degradation of AXR2 mediated by AvrRpt2 and NAA mediated and AvrRpt2 mediated cleavage of RIN4.The apple specific immune resistance of the autologous cleavage of RIN4 and the feedback degradation of AvrRpt2 mediated by NAA and RIN4 were revealed for the first time.The main results are as follows:1.More than 1×10~6 protoplasts were obtained from one gram fresh callus.The highest yield of protoplasts was harvested from callus cultured for 10 days.The highest yield of protoplasts isolated from‘Orin'and‘Zihong'callus were up to 3.68×10~6 and 4.07×10~6protoplasts from one gram fresh callus,respectively.Due to aging,hard,brittle and dense texture with the dark red color on the surface and fade to yellow inside,we could not obtain any protoplasts isolated from the‘Zihong'callus cultured for 20 or 25 days.2.Although the promoter of cauliflower mosaic virus(CaMV)35S promoter can effectively drive target gene expression in many crops,its transient expression activity in apple protoplasts is weak,which cannot meet the needs of biochemical research related.We confirmed that Pro-BIUTNT,natural promoter of ubiquitin gene UBQ10(ubiquitin 10)of Arabidopsis,has a higher expression activity than CaMV 35S promoter in apple protoplasts.Taking the MdERF98,MdERF1,MdERF2,MdERF3a,MdERF6a,MdWRKY29,MdWRKY33,MdBAK1,MdFLS2,MdEIL2 as the tested genes,the activity of CaMV 35S and Pro-BIUTNT promoter for driving the expression of these tested genes in apple protoplasts were detected respectively.The result showed that strong target protein signal of these tested genes were detected driven by Pro-BIUTNT,while that of CaMV 35S were not detected or were very weak.Taking MdERF1,MdWRKY33 and MdFLS2 as tested genes,the effects of culture time and expression time on the expression level of target genes were also tested.The results showed that the highest expression level of the target protein was obtained in protoplasts isolated from callus cultured for 6 days,and the highest expression level was obtained in protoplasts expressed for 6?8 h.Pro-BIUTNT also promote the high expression of GFP and GFP fusion protein in apple protoplasts and tobacco mesophyll cells,and the fluorescence signal level was significantly stronger than that of CaMV 35S.3.Different in apple protoplasts,CaMV 35S performed higher transient expression activity in Arabidopsis protoplasts,and there was no significant difference between CaMV 35S and Pro-BIUTNT promoter.Moreover,the transient expression activity of the two promoters in Arabidopsis protoplasts was higher than that in Apple protoplasts,which may be related to the difference of interspecific protein expression regulation mechanism determined by genetic background.4.Pro-MdBIUTNT and Pro-BIUTNT-2(1539 bp and 2501 bp,respectively),the promoter of apple orthologous gene of Arabidopsis UBQ10 gene,also performed higher expression activity than CaMV 35S in apple protoplasts.Among them,two segments of Pro-MdBIUTNT with 539 bp and 739 bp(upstream from the ATG translation start site)possessed stronger activity.5.Considering that ribose-1,5-diphosphate carboxylase/oxygenase(Rubisco)is the most abundant protein in plant leaves,we detected the constitutive expression activity and photoinduced expression activity of the promoters Pro-MdRP-1,Pro-MdRP-2(natural promoter of Rubisco,length of 1972 bp and 3050 bp,respectively)and promoters Pro-MdRC-1 and Pro-MdRC-2(natural promoter of Rubisco activase,length of 2025 bp and 2542 bp,respectively).The result showed that the constitutive expression activity of the promoter of Rubisco related genes in apple protoplasts were low,and the expression level was slightly increased after light treatment.6.Partial PTI and ETI resistance mechanisms of apple were further confirmed based on the transient expression of apple protoplast.In terms of PTI resistance,we confirmed the conservation of the specific interaction between At BAK1 and At FLS2 induced by flg22 in apple cells.Based on the result of in vitro kinase assay,we found that At MKK7ac could modify MdWRKY33 and transduce phosphokinase signal mediated by phosphorylating MdMAPK6,and MdMAPK6 could improve the protein stability of MdWRKY33 independent from At MKK7ac.In terms of ETI resistance,NAA treatment or co-expression of AvrRpt2 could significantly reduce the stability of AXR2 protein in‘Orin'apple protoplast,and the stability of AXR2 P87S mutant was significantly improved,indicating that the mechanism was also conserved in apple cells.By the caspase activity of AvrRpt2,the mechanism of AvrRpt2mediated cleavage of RIN4 is still conserved in apple cells.7.In this study,we also found the apple specific immune resistance mechanisms which were different from that of the model plant for the first time.NAA treatment significantly reduced the protein level of AvrRpt2,indicating that there may exist a specific feedback mechanism in apple cells that mediates the degradation of the pathogen effector protein AvrRpt2 to alleviate the sustained damage of the pathogen to the host,which plays an important role in balancing plant growth and immune process.The presence of RIN4 further reduced the level of AvrRpt2.Another specific cleavage of RIN4 independent from AvrRpt2 was also exist in apple cells,indicating that there may be some regulatory pathways for controlling the dose of RIN4 in apple cells to maintain moderate sensitivity to AvrRpt2.In conclusion,a high-efficiency transient expression system of apple protoplasts was established based on Pro-BIUTNT promoter.Partial conserved PTI and ETI plant resistance mechanisms in apple cells were revealed initially.The apple specific immune resistance of the autologous cleavage of RIN4 and the feedback degradation of AvrRpt2 mediated by NAA and RIN4 were also revealed for the first time.The co-existence of these common and specific regulatory mechanisms not only reflect the importance of conservative immune mechanism between specifics,but also embody the continuous evolution of plants for adapting the environment.Our research not only provides a good technical for supporting the research of apple functional genes and specific signaling pathways,but also provides some theoretical and technical reference in understanding the specific mechanism of apple immune resistance.