Transcriptomics And Proteomics Analysises Of Jujube's Responses To Infection By And Recovery From Candidatus Phytoplasma Ziziphi

Ziziphus jujuba Mill.belongs to the Family of Rhamnaceae,which is an important fruit tree that originated in China,where it has been cultivated for more than 4,000 years and over 700 cultivars exist.Jujube witches' broom(JWB),caused by a phytoplasma,devastates jujube tree growth and production in Asia.Although host responses to phytoplasmas are studied at the phenotypic,physiological,and biochemical levels,it remains unclear how a host plant responds at the molecular level during the early stages of infection.To understand the response of the jujube tree to JWB infection,leaves were sampled at different times during the phytoplasma infection.We analyzed changes in the transcriptome,proteome and phytohormone levels in response to grafting a phytoplasma-infected scion onto a susceptible cultivar of jujube.The large-scale,multi-omics data set allowed identification of jujube genes and proteins that respond early to phytoplasma infection.Further,we explored the correlation between the phytoplasma-responsive transcriptome and proteome.Jujube shoots(Ziziphus jujuba Mill.‘Huizao')infected by phytoplasma were excised and grown in vitro before treatment with tetracycline.RNA-Seq and i TRAQ analyses of samples treated for different lengths of time were performed during recovery from jujube witches' broom(JWB)symptoms and diminishing presence of phytoplasma.Phytoplasma was not detected by PCR in the shoots after 6 and 9 months of tetracycline treatment(MTT).Leaves and shoots infected by JWB recovered to normal morphology after 9 MTT.A total of 25067 unigenes were mapped to jujube genome reference sequences.In the first infection stage(0-2WAG),a total of 582 jujube genes were differentially regulated but no visible symptoms appeared.Quite a few DEGs related to abscisic acid(ABA)and cytokinin(CTK)were down-regulated,while some related to jasmonic acid(JA)and salicylic acid(SA)were up-regulated,DEGs related to plant-pathogen interaction were also up-or down-regulated.In the second infection stage(37-39WAG),witches' broom symptoms were visible,and a total of 4373 DEGs were identified.Genes involved in biosynthesis and signal transduction of ABA,brassinosteroid(BR),CTK,ethylene(ET),and auxin(IAA),GA,JA and SA,plant-pathogen interaction,flavonoid biosynthesis genes were significantly regulated,suggesting that jujube trees activated defense factors related to SA,JA,ABA and secondary metabolites to defend against phytoplasma infection.By the third infection stage(48-52WAG),serious symptoms occurred and 3386 DEGs were identified.Most DEGs involved in biosynthesis and signal transduction of JA,SA and GA were up-regulated,while those relating to ABA were down-regulated.Genes involved in plant-pathogen interaction were up-or down-regulated,while phenylpropanoid and flavonoid biosynthesis genes were significantly up-regulated.Meanwhile,DEGs involved in photosynthesis,chlorophyll and peroxisome biosynthesis,and carbohydrate metabolism were down-regulated.These results suggested that phytoplasma infection had completely destroyed jujube trees' defense system and had disturbed chlorophyll synthesis and photosynthetic activity in the infected leaves at the late stage,resulting in yellow leaves and other JWB symptoms.At 37 WAG,289 DEPs were observed.Among the upregulated DEPs at 37 WAG were proteins related to JA-induced protein,flavonoid biosynthesis and phenylalanine metabolism.Fourteen DEGs/DEPs were shown to share similar expression trends by correlation analyses,and most of these were involved in flavonoid biosynthesis,phenylalanine metabolism and phenylpropanoid biosynthesis.Moreover,phytoplasma infection resulted in reduced auxin content and increased JA content during the early stage of phytoplasma infection.At 48 WAG,753 DEPs were detected.Of the 395 downregulated proteins,LOX2 were decreased by sixfold,and two JA-induced proteins were greatly reduced.Moreover,DEPs involved in carbon metabolism,biosynthesis of amino acids and carbon fixation in photosynthetic organisms were also down-regulated.At 48 WAG,most of 98 DEGs/DEPs of similar expression trends in the correlation analyses were involved in carbon metabolism,carbon fixation in photosynthetic organisms and photosynthesis.RNA-Seq and i TRAQ analyses identified 26,790 genes and 6184 proteins from jujube shoot samples.There were 3345,444 and 1389 differentially expressed genes(DEGs),and 69,20,and 16 differentially expressed proteins(DEPs)between the 0 and 3 MTT,the 3 and 6 MTT,and the 6 and 9 MTT samples,respectively.The largest number and the greatest changes of DEGs and DEPs were for those related to alpha-linolenic acid metabolism,jasmonate biosynthesis,and jasmonate induced protein-like(JIPs).JA content increased by the third month,slightly decreased at the sixth month,and finally increased again at the ninth month.The research avenues explored here showed that genes in the JA biosynthesis pathway,proteins that respond to JA(JIPs),and JA content itself were concurrently regulated during JWB recovery.Alpha-linolenic acid metabolism pathway had higher Rich Factor in comparisons between the 3 and 6 MTT and the 6 and 9 MTT samples during JWB recovery,which suggesting JA played vital roles during JWB recovery.The results in this study will help us to understand the roles JA plays in host-phytoplasma interactions during recovery and infection of JWB,which can serve as the foundation for further phytoplasma pathogenesis and response mechanism studies...