Analysis Of Cherry Stiff Fruit Caused By Cherry Phyllody Phytoplasma Based On Transcriptome And Metabolome

Phytoplasma is a class of prokaryotic parasitic organisms that are cell wall-less and harshly cultured,mainly in the sieve tube cells of plant phloem,as well as in the gut and saliva of prickly insects,and can cause Rice Yellow Dwarf,jujube witches’ broom,Cherry X-Disease,Peanut Witches’ Broom,Sesame Phyllody,Paulownia Witches’ Broom,Sugar Cane White Leaf and many other diseases.The cherry(Prunus pseudocerasus L.G.Don)is one of the four major cherry cultivars and its fruits have a special flavour and are widely grown in the southern mountainous regions of China and are of great economic value.Large-scale occurrences of suspected phytoplasma disease were observed in several cherry growing areas in the Qijiang district of Chongqing,mainly showing the typical symptoms of phyllody,virescence and stiff fruit,accompanied by leaflet clusters,plant decline and death,with disease coverage of up to40%.There are few reports on the molecular mechanisms of phytoplasma diseases in cherries,and there is a gap in research on the mechanisms of phytoplasma diseases causing stiff fruit.In this study,cherry plants affected by phytoplasma were selected as test material and healthy plants were used as controls.The identification and genomic analysis of the pathogenic phytoplsma were carried out using morphological,cytological and molecular biology methods,as well as transcriptomic and metabolomic studies,with the aim of elucidating the molecular mechanism of stiff fruit caused by phytoplasma infestation,and to further study the germplasm of phytoplasma resistance in cherries and the molecular mechanism of stiff fruit.The study aims to elucidate the molecular mechanisms of cherry phytoplasma resistance germplasm and cherry phytoplasma control programmes,and even other phytoplasma diseases,and is of great scientific importance for the further development of the cherry industry in China.The main results of the study are as follows:1.Transcriptome analysis of the molecular mechanisms of stiff fruit formationTranscriptome analysis showed a high proportion of differentially expressed genes in both pathogen resistance and signalling or regulatory functions.A large number of genes within the phytohormone signalling pathway were significantly up-regulated in stiff fruit,with the auxin signalling pathway containing up-regulated expression genes(IAA4,9,14,31,SAUR20,32,71,72,GH3.1,3.17,ARF5,9)and down-regulated expression genes(AUX22,IAA11,12,16,26,26,27,SAUR24,40,50,GH3.9,3.10,ARF3);the salicylate signaling pathway included TGA10,PRB1 and PR1 A,which were significantly up-regulated,and the abscisic acid signaling pathway included PYL4,PYL8,PYL2,PP2 CA and Sn RK2.3,which were up-regulated;the rest of the phytohormone signaling pathways only had individual The rest of the phytohormone signaling pathways were only differentially expressed in individual genes.2.Wide-targeted metabolomic analyses of the molecular mechanisms of fruit formationA high proportion of differentially accumulated metabolites were found in both pathogen resistance and signalling or regulatory functions.The results of metabolomic analysis showed that phenylalanine metabolism and flavonoid synthesis were significantly up-regulated in stiff fruits,with up-regulated metabolites including rhamnoside(Biorobin),5-Aminovaleric acid,ornithine(L-ornithine),astragalin,(+)-Abscisic acid,rutin,etc.;cis-Zeatin,Diethanolamine,Mitraphylline,Myrcene,Indole-3-acetic acid,Xaverta glycosides,etc.were down-regulated.A large number of differential cumulative metabolites have been directly linked to plant resistance,including Schaftoside.Relative quantification of phytohormones showed that stiff fruit had lower auxin content than healthy fruit,while had higher salicylic acid and abscisic acid contents.3.PCR validation and combined histological analysisForty-four differentially expressed genes closely related to phytohormone signalling and plant immune mechanisms,involving salicylic acid signalling pathway,growth hormone signalling pathway,abscisic acid signalling pathway and cytokinin signalling pathway,were selected to validate the reliability of the RNA-seq data.Except for the genes SAUR71 and AREB3,the q RT-PCR data showed the same trend of gene expression as the transcriptome sequencing analysis results(42/44,95.45%),indicating a good reliability of the transcriptome sequencing data and differential expression analysis analysis results of those selected genes.The molecular mechanism of stiff fruit formation is mainly induced by plant biotic stress responses through phytohormone signalling,particularly the salicylic acid,growth hormone and abscisic acid signalling pathways.Notably,there is a significant overlap between differentially expressed genes in response to phytoplasma stress and in response to drought stress.In addition,the levels of phytohormones showed significant differences,with abscisic and salicylic acid levels higher in cherry fruit infected with Phytoplasma than in healthy fruit,while the opposite results were found for zeatin,jasmonic acid and growth hormone.The results were validated by real-time fluorescence quantitative polynucleotide chain reaction(quantitative Real-Time PCR)and included AHK2,IAA4,IAA9,IAA14,IAA31,ARF5,ARF9,GH3.1,GH3.17,SAUR20,SAUR32,SAUR40,PR1 a,PRB1 TGA10 and Sn RK2.3 are key candidate genes responsible for cherry fruit staleness.In conclusion,this study has laid the foundation for understanding phytoplasma diseases,the molecular mechanisms of cherry fruit staleness and better understanding of fruit development,as well as identifying potential candidate genes associated with cherry fruit staleness that can be used further by researchers in related fields.