Transcriptomic Analysis Of BTH-induced Wound Healing Of Potato Tuber And The Screening And Validation Of Interaction Proteins With StCDPK10

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester(BTH)is the structural and functional analogue of salicylic acid,which could induce disease resistance of various postharvest fruits and vegetables.Our previous studies found that the mechanism of BTH promoting the wound healing of potato tubers was related with phenylpropanoid metabolism and reactive oxygen species(ROS)metabolism,however,the detailed action model was unclear.In this study,the microtuber of "Longshu No.3" was used as materials and treated with BTH.The main metabolic pathways involving in the wound healing were analyzed by transcriptome and the effect of BTH treatment on ROS generation mediated by CDPK was clarified.The StCDPK10 was used as the bait protein to screen the c DNA library.Meanwhile,the interaction relationship between StCDPK10 and its target protein St Rbohs were verified.The results show that:1.Transcriptomic analysis shows that 1957 differentially expressed genes(DEGs)were enriched in BTH-induced wound healing tissues of potato tuber,which mainly involved in the various metabolic pathways including plant-pathogen interaction,plant hormone signaling and phenylpropane biosynthesis.The DEGs number in the plant-pathogen interaction pathway was the greatest.Rboh-dependent ROS production related with Ca2+-mediated signal play an important role in this pathway,and the transcripts level of StCDPK10,St Rboh B and St Rboh C in this pathway were also induced the most by BTH treatment.2.BTH treatment promoted the intracellular Ca2+ concentration in healing tissues,while the total Ca2+ content was not affected.Bioinformatics analysis shows that StCDPKs were distributed unevenly on 12 chromosomes,and different CDPK kinases have relatively conserved structures,and these genes are expressed in different parts of tuber,such as tuber cortex and pith.After BTH treatment,the transcription level of StCDPKs in healing tissues were differently upregulated,among which StCDPK10 was the most significant upregulation.BTH treatment also increased the activity of CDPK kinase and H2O2 content in healing tissues.Over-expression of StCDPK10 caused a marked increase in H2O2 content and suberin deposition.3.StCDPK10 was used as bait protein to screen the(split-ubiquitin)yeast two-hybrid c DNA library.The 15 interacting proteins were obtained from yeast two-hybrid c DNA library including dirigent protein,caeyl-3-O-methyltransferase,cytochrome P450 98A2,stearoyl-acyl carrier protein desaturase,mitochondrial uncoupling protein,NADH dehydrogenase,NAD-dependent malate dehydrogenase and 6-phosphofructose kinase.The 15 interacting proteins obtained from split-ubiquitin yeast two-hybrid c DNA library contained peroxiredoxin 2B,peroxidase 12,glutathione s-transferase,calcium-linked proteins,caffeyl-O-methyltransferase,NADH dehydrogenase,ATP synthase,MAPK kinase and ABC transporter.4.Interaction results showed that StCDPK10 interact with St Rboh B and St Rboh C,and the interaction between StCDPK10 and St Rboh B is located in the plasma membrane,while the interaction with St Rboh C is located in the nucleus.St Rbohs protein has a relatively conserved domain,namely the EF-hand domain at N-terminal region and the binding sites of FAD,NADPH-ribose and NADPH-adenine at C-terminal region.The interaction between EF-hand domains of St Rboh B and St Rboh C and StCDPK10 showed that StCDPK10 interacted with St Rboh B and St Rboh C at N-terminal EF-hand domains.In conclusion,the wound healing of potato tubers induced by BTH involves in plant-pathogen interaction,plant hormone signaling and phenylpropane biosynthesis.The transcription of StCDPK10,St Rboh B and St Rboh C in plant-pathogen interaction pathway showed the highest level.After treating potato tubers with BTH,the cellular Ca2+ concentration,CDPK activity and H2O2 content of healing tissues were significantly increased.The result of screening c DNA with StCDPK10 showed that StCDPK10 can interact with multiple substrates.In addition,StCDPK10 also interacts with St Rboh B at membrane and St Rboh C at nucleus.