| Citrus Huanglongbing(HLB),associated with‘Candidatus Liberibacter asiaticus'(CLas),is the most devastating disease in citrus.CLas is a gram-negative bacteria living exclusively in the sieve of phloem.HLB has caused serious yield losses in citrus-planting regions all over the world,and there is no effective control method.Leaf mottle yellowing is a typical symptom of citrus huanglongbing.It is generally believed that the pathogenic bacteria cause the blockage of leaf phloem,hinder the transport of photosynthetic products,lead to the transitional accumulation of chloroplast starch and the destruction of chloroplast,and finally produce mottle yellowing.At the same time,the carbohydrate transport in the affected citrus was blocked,and the carbohydrate shortage in the roots caused the epidermis of the diseased roots to fall off and rot.However,it has been suggested that the colonization of the roots of huanglongbing is earlier than that of the leaves,and the root damage is not caused by the lack of carbohydrates caused by the overground phloem blockage,but by the destruction of huanglongbing in the roots.Conversely,the stability of the root system is closely related to the disease resistance of the above-ground parts.For example,the stability of the root system affects the absorption and transportation of mineral elements such as N,Fe,Mg,Zn,which are involved in the synthesis of chlorophyll,and the reduction of any element will lead to yellowing symptoms.This vicious cycle between the overground and underground parts of the affected tree eventually leads to its deat.Obviously,how the citrus root and leaf respond to the infection of huanglongbing cooperatively,whether the root or the leaf plays the leading role remains to be further studied.In addition,the pathogenic mechanism of huanglongbing in citrus root and leaves is still unclear.Therefore,in order to parse the huanglongbing with citrus roots and leaves of mechanism,this study use susceptible cultivars citrus sinensis csbeck as experimental materials,and the leaf-disc grafting method was used to transmission the pathogenic bacteria,starch content in root and leaf change,corpus callosum anatomic structure of sedimentary characteristics,organization change is analyzed,further,proteome sequencing of roots and leaves using TMT labeling technology,after the pathogen infect the host and pathogen in the protein group level variation characteristics and molecular mechanism interactions.Through the above research,it is expected to elucidate the internal mechanism of the interaction between huanglongbing with citrus roots and leaves,and provide theoretical reference for the exploration of the key resistance genes for resistance breeding of citrus.The principal research results are as follows: 1?Comparison of the spreading characteristics of pathogens in roots and leaves of wanjingchengWithin 10 months of transmission,the contents of pathogenic bacteria in the leaves and veins showed an obvious trend of increasing,while the contents of pathogenic bacteria in the roots showed an obvious trend of increasing from the 2nd month to the 6th month,but decreased at the 10 th month.At the 2nd and 6th months,the content of pathogen in root was slightly higher than that in leaf vein,and at the 10 th month,the content of pathogen in leaf vein was significantly higher than that in root.2?Comparison of response characteristics of wanjingcheng root and leaf after infectionIn the 2nd month after grafting,there was no obvious difference between the infected plants and the control plants.In the sixth and tenth month,the roots of the infected plants rotted seriously and the leaves showed signs of yellowing.The contents of starch and callose gradually accumulated.In the tenth month after the onset of the disease,the phloem of the vein was thickened,the epidermal cells were stained deeply,and a large number of starch grains were deposited.3?Sequencing analysis of root and leaf proteomes of susceptible wanjingchengA total of 7,779 host proteins were identified in this study,6,759 of which contained quantitative information.Then among the proteins quantified,43 proteins in the vein are up-regulated and 85 proteins are down-regulated.In the root,183 proteins were up-regulated and 97 proteins were down-regulated.At the same time,38 pathogenic proteins were identified,among which 6 were differentially expressed.4?Variation characteristics of host proteome in response to CLas infectionThe proteome data showed that at second months after infection,the number of proteins in root responding to huanglongbing was greater than that in vein.The KEGG enrichment results showed that there were more proteins in vein enriched in the sugar metabolism pathway,and more proteins in root enriched in the synthesis of secondary metabolites and the anabolism pathway of phenylpropanoids.The results of subcellular localization annotation showed that there were more proteins located in chloroplast.According to the results of differentially expressed genes,a large number of peroxidase,chitinase and other pathogenesis related proteins were identified.The results showed that in the early stage of Huanglongbing infection of citrus,vein mainly responded to CLas infection through sugar metabolism related pathways,while root mainly responded to CLas infection through secondary metabolism related pathways.5?Characteristics of proteome changes in pathogens of CLasThe GO annotation results of the proteins in the pathogen showed that the molecular functions of more pathogen proteins were transporters and nucleic acidrelated functional proteins.The results of protein network interaction analysis showed that these proteins were closely related to cell growth and development and protein folding.Among the six significantly expressed pathogen proteins identified,2 were significantly up-regulated and 4 were significantly down-regulated.These proteins are mainly closely related to cell proliferation and apoptosis,energy metabolism and transport of substances across the membrane.The results suggest that CLas mainly maintains the proliferation and spread of pathogenic cells in citrus by regulating the transport of substances across the membrane and energy metabolism.6?Quantitative analysis of differential protein gene expression induced by CLasThe expression characteristics of differential protein genes infected by Las were analyzed by q-PCR.Among 66 analyzed host differential protein genes,the gene expression level of 23 up-regulated proteins were up-regulated,9 up-regulated proteins were down-regulated,6 down-regulated proteins were up-regulated,and 28 downregulated proteins were down-regulated.Amang the pathogenic bacteria proteins,the gene expression of 13 upregulated proteins were up-regulated,15 down-regulated proteins were up-regulated.7?Preliminary study on the response mechanism of Zn metabolism in the interaction between citrus and huanglongbingThe host protein is the ethanol dehydrogenase gene CsADH1,which is analyzed by bioinformatics,hormone and zinc ion treatment,the results showed that the protein contained a zinc binding site,and the results of hormone and zinc ion treatment showed that SA,MeSA,IAA,ABA and zinc ion could induce the expression of this gene.In roots,CsADH1 was significantly more affected by hormones and zinc ions than in leaves.It is suggested that CsADH1 plays an important role in the response to adversity in citrus roots.Identification of pathogen proteins is ABC transporter,bioinformatics analysis showed that the protein contains a malE(permease)conservative domain structure,can transfer membrane phospholipid molecules inside and outside,it is essential to maintain osmotic pressure inside and outside the membrane and asymmetry inside and outside the membrane and regulate the transport of Zn and other substances across the membrane.The results suggest that the host CsADH1 and pathogen LasmalE gene may play an important role in root Zn ion metabolism in response to pathogen infectionTo sum up,in the early stage of the interaction between HLB and citrus,the host vein and root regulate sugar metabolism and secondary metabolism-related pathways in response to Las infection,to coordinate the dynamics balance of substance and energy metabolism between source and sink during pathogen infection,and Las maintains the proliferation and spread of pathogenic cells in citrus by regulating the transmembrane transport of substances and energy metabolism,suggesting that coordinating the dynamic balance of substances and energy between pathogens and hosts may be effective in reducing Las harm and it is essential to improvecitrus tolerance to HLB. |
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