Research On The Syntaxin Protein MdSYP121 And Ethylene Negatively Regulate Apple Resistance To Botryosphaeria Dothidea

Apple ring rot is caused by the pathogenic fungus Botryosphaeria dothidea.It could suppress the growth of plant,cause apple fruit rot and reduce the yield and quality of apple.Apple ring rot severely restricts the sustainable development of the apple industry.At present,the pathogenic mechanism of B.dothidea is still unclear,which prevents the research on the mechanism of apple disease resistance.Therefore,research on the pathogenic mechanism of B.dothidea has great significance and practical application value on mining apple disease resistance genes,revealing the mechanism of disease resistance and breeding new disease-resistant varieties.In the process of pathogen infection,plant is usually accompanied by the transport of resistant substances and the changes of hormone levels.Membrane transport systems and ethylene signal transduction pathway play important roles in plant immune processes.Membrane transport primarily acts on the exchange of matter and information between plants and the environment.When stimulated by the environment,plants can response to various stresses through transporting resistive substances and replenishing cell wall components.The specific regulation mechanism of membrane transport system in resistance to B.dothidea is still unknown.Ethylene is usually used as a virulence factor of pathogens in biological stress and a signal molecule of defense response to immune response.Meanwhile,a large amount of ethylene was produced when apple infected by B.dothidea and caused apple ring rot.Therefore,the role of the ethylene signal transduction pathway in resistance to B.dothidea is also urgently exploited.In this study,apple was used as test materials.Firstly,it was identified that the apple syntaxin protein MdSYP121 played an important role in regulating the defense to B.dothidea by affecting the SA signaling transduction pathway and oxidation-reduction reactions.Secondly,the results showed that the interaction between apple and pathogen produced a large amount of ethylene and caused the decay of fruit.Overexpressing the ethylene receptors increased the sensitivity to ethylene in plants and reduced the resistance to B.dothidea.The main results for this research are as follows:?1?MdSYP121 played a negatively role in regulating apple resistance to B.dothidea.In this study,the RNA sequencing?RNA-Seq?analysis of apple after inoculation with B.dothidea showed that Md SYP121 was located in the center of the protein interaction network.It was associated with a variety of disease resistance mechanisms and may play an important role in resistance to B.dothidea.After inoculation with B.dothidea,the lesions of MdSYP121silenced calli were smaller than in control,while the lesions of MdSYP121 overexpressing transgenic calli and Arabidopsis were larger.These results indicated that MdSYP121negatively regulated the resistance to B.dothidea in apple calli.The qRT-PCR results showed that when inoculated with B.dothidea,the expression levels of SA related genes EDS1,PAL,PAD4,PR1,PR5,and NPR1 were higher in Md SYP121 silenced calli than in control.The expression of these genes in MdSYP121 overexpressing lines was lower.MdSYP121 might affect the resistance to B.dothidea through influencing the SA mediated resistance signaling pathway.?2?RNA-Seq analysis of cotrol and MdSYP121 silenced calli lines with or without B.dothidea infection revealed that MdSYP121 played the main role in regulating oxidation-reduction process?GO:0055114?and oxidoreductase activity?GO:0016491?for resistance to B.dothidea.APX,CAT,POD and GST genes were closely related to redox reaction.The qRT-PCR results showed that,after inoculation with B.dothidea,the expression of these genes was strongly induced and significantly higher than the control.The activity of the four oxidation-reduction enzymes was also increased.It was suggested that MdSYP121may regulate the resistance of plants to B.dothidea together with a set of reactive oxygen biotic stress genes.?3?MdSYP121 normally formed SNARE complex and played important roles in plants.In this study,subcellular localization analysis revealed that MdSYP121 and MdSNAP33 were located on the plasma membrane.Through bimolecular fluorescence complementation?BiFC?and pull down tests,MdSYP121 can interact with MdSNAP33 and form a binary target membrane?t?-SNARE complex on the plasma membrane.In addition,MdSYP121 formed a SNARE complex together with MdSNAP33 in apple by using the SDS-resistance test.?4?A large amount of ethylene was produced when apple infected by B.dothidea and resulted an increase morbidity degree of apple ring rot.Along with the days increasing,the release of ethylene gradually increased,and the lesion area gradually expanded after inoculated with B.dothidea.Ethylene release and lesion area were difference in different development stages.The amount of ethylene released and the area of lesions at 60 days after bloom flowering were lower,as fruit development they were higher between 100 to 160 days after bloom flowering,and relatively small at 180 days after bloom flowering.qRT-PCR results showed that the B.dothidea induced the expression of ethylene synthesis-related genes.MdACS5A and MdACS5B genes were usually induced by stress.The expression levels of MdACS5A and MdACS5B genes induced by B.dothidea significantly increased the biosynthesis of ethylene at 140 and 160 days after bloom flowering.Further studies showed that compared with untreated fruits,ethylene release was smaller and the expression levels of MdPR2,MdPR4,MdPR5,MdPR8 genes were higher in apple fruits at 60 days after bloom flowering after innoculated with B.dothidea;ethylene release was increased and the expression of PR genes was lower in apple fruits at 140 and 160 days after bloom flowering after innoculated with B.dothidea.According to the results,the synthesis of ethylene was increased in apples with the infection of B.dothidea.The ethylene could prevent the expression of PR genes and reduce the apple disease resistance to B.dothidea.?5?Overexpressing apple ethylene receptor genes enhanced plant sensitivity to ethylene.The apple ethylene receptor genes?MdEIN4,MdETR1,MdETR2 and MdERS1?were transformed into Arabidopsis thaliana,respectively.A.thaliana‘Triple response'analysis showed that the elongation of hypocotyls was inhibited when ACC was present.The hypocotyls length of MdEIN4 overexpressing transgenic seedlings was 0.67-fold compared with wild type,and the length of hypocotyls in other overexpressing transgenic lines of MdETR1,MdETR2,MdERS1 were similar to wild type,indicating that MdEIN4 is more sensitive to ethylene in apple.?6?Apple ethylene receptors played negative roles in regulating apple resistance to B.dothidea.In the present study,Md EIN4,MdETR1,MdETR2 and MdERS1 overexpressing A.thaliana transgenic plants were inoculated with B.dothidea.It was found that lesion area and mycelial growth in A.thaliana transgenic lines were higher than in wild type and ein2mutants,and they were greatest in MdEIN4 overexpressing transgenic lines.qRT-PCR results showed that the expression of PR genes in MdEIN4 overexpressing transgenic plants was the lowest with or without B.dothidea inoculation.When treated with CoCl₂,the lesion area was reduced,and the expression of the PR genes was increased in MdEIN4 overexpressing transgenic lines.We speculated that the sensitivity of MdEIN4 Arabidopsis transgenic plant to ethylene was enhanced,and may reduce the resistance to B.dothidea by affecting the expression level of PR genes.