The Functional Analysis Of CaSYT5 In Pepper’s Response To Ralstonia Solanacearum Or High-temperature-high-Humidity Challenge

Synaptotagmins is a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons and endocrine tissues.Thought to be exclusive to animals,synaptotagmins have recently been characterized in Arabidopsis thaliana,in which comprised five subfamilies.At present,most researches of SYT protein family are focusing on SYTA.SYTA’s function is mainly to repair the damaged cell membrane and regulate the endocytosis and exocytosis of the vesicle cell and the transportation between cells for viral movement protein.However,the other functions of SYT in plantare rarely reported.In this study,we found that CaSYT5,one of the SYT protein family members in pepper,play important regulation roles in the pepper plants to response to high temperature（40℃）-high humidity（90%）or Ralstonia soanacearum infection.The main results are as follows:（1）A full-length cDNA containing the C2 functional domain was screened from the pepper cDNA library.It’s 2198bp in length,contains a 1698 bp open reading frame（ORF）.It shared 93%,94%,93%,82%,79%,78%,77%,77%,76%,73%and 68%deduced amino acid sequence identities with SlSYT5（Solanum lycopersicum,XP₀04242744.1）,StSYT5（Solanum tuberosum,XP₀06359490.1）,SiSYT5（Sesamum indicum,XP₀11078421.1）,InSYT5（Ipomoea nil,XP₀19183803.1）,RcSYT5（Ricinus communis,XP₀15581849.1）,CmSYT5（Cucumis melo,XP₀08457176.1）,CsSYT5（Cucumis sativus,XP₀04139289.1）,JrSYT5（Juglans regia,XP₀18811223.1）,AtSYT5（Arabidopsis thaliana,NP₁72041.2）,OsSYT5（Oryza sativa Japonica Group,XP₀15650839.1）,respectively.Sience the highest squence identity with Arabidopsis thaliana SYT5 is 73%,so it was named CaSYT5.（2）CaSYT5 was found to be distributed on the nucleus and cell membrane by subcellular localization.The gene promoted the necrosis of HR（hypersensitive response）cell caused by Agrobacterium tumefaciens infection,and induced the up-regulation of the marker gene related to the physiological defense response in pepper plants.（3）We found that heat shock response element（HSE）,element responsive to ethylene（ERE）,Auxin response element（AuxRR）,light response element（G-box）were harbored in the promoter of CaSYT5 by the analysis of CaSYT5 promoter.The results of fluorescent quantitative analysis showed that the transcript level of CaSYT5 was induced by the treatment of Ralstonia solanacearum inoculation or high temperature-high humidity.In addition,the transcript level of CaSYT5 were induced by exogenous plant hormone eabscisic acid（ABA）,ethephon（ETH）and Methyl jasmonate（MeJA）.However,the transcript level of CaSYT5had no significant effect by the exogenous plant hormone salicylic acid（SA）.This indicates that CaSYT5 may have function in response to Ralstonia solanacearum effection or high temperature-high humidity and JA,ET and ABA-induced signaling pathway.（4）The result of virus-induced gene silencing（VIGS）experiment showed that the transcript level of CaSYT5 in the pepper leaves of CaSYT5 silencing was significantly reduced by the treatment of Ralstonia solanacearum inoculation or high temperature-high humidity compared with that in control plants,accompanied by down-regulation of immunity-or thermo tolerance-associated CaABR1,CaPR1,CaACO and CaHSP24.（5）The result showed that transient overexpression 35S:CaSYT5-GPF in pepper leaves induced significant Hypersensitivere sponse-mediated cell death and H2O2 accumulation which were assessed by staining with trypan blue and DAB,respectively.The higher ion leakage,furthermore,was detected to measure the severity of cell death compared with the control.The qPCR result showed that the transcript level of marker gene CaABR1,CaPR1,CaACO and CaHSP24 were increased continuously during transient overexpression of CaSYT5.In conclusion,the results of this experiment indicate that CaSYT5 has regulation effect in the response to Ralstonia solanacearum inoculation and high-temperature-high-humidity stress,and may play an important role in the mechanism of resistance mediated by ABA,ET and JA signaling pathway in pepper plants.