The Study On Genetic Diversity Of Citrus Bark Cracking Viroid And Its Antagonistic Mechanism Against Citrus Exocortis Viroid

Viroids are single-stranded,circular,small RNA molecules that are not able to encode any protein.They infect plant cells by mechanical means and cause serious diseases in some important crops.Up to date,citrus plants have been shown to be natural hosts for at least eight types of viroids including Citrus exocortis viroid（CEVd）,Citrus bent leaf viroid（CBLVd）,Hop stunt viroid（HSVd）,Citrus dwarfing viroid（CDVd）,Citrus bark cracking viroid（CBCVd）,Citrus viroid V（CVd-V）,Citrus viroid VI（CVd-VI）and Citrus viroid VII（CVd-VII）.Specific strains of CEVd cause pronounced cleavage symptoms on trifoliate orange（Poncirus trifoliata）and citranges（Citrus sinensis × P.trifoliate）,which are widely used as rootstocks and can rapidly proliferate in commercial orchards by mechanical means such as grafting.CBCVd is considered as a chimeric recombinant of CEVd and HSVd.There are a large number of citrus species and citrus relatives infected by CBCVd without obvious symptoms.Previous reports confirmed that CBCVd had cross protection against CEVd on trifoliate orange and their combined infection could reduce the symptoms of CEVd on sensitive rootstocks,but the molecular mechanism has been unknown yet.The “Arizona 861-S1” of “Etrog” citron（C.medica）is widely used as an indicator plant for citrus viroids and provides a platform for studying the interaction mechanism between citrus viroids.The studies of interaction between viroids and hosts are mostly concentrated on herbaceous hosts,while the natural infection reports of viroids are more common in woody plants such as citrus.Therefore,studies on interaction between woody plants and viroids are practical.In this study,the identification of citrus viroids and the construction of infectious clones were performed.The genetic diversity of CBCVd,a potential antagonistic resource for citrus crackle disease,was analyzed in this study.Changes of the citron plants in responding to viroid infection were analyzed by next generation sequencing,and the molecular mechanism of cross protection between CEVd and CBCVd was analyzed.Ⅰ.The analysis of genetic diversity（1）Identification of CEVd and CBCVd sources and their genetic diversityIn order to clarify the differences between citrus viroids in different regions and construct infectious clones,the viroid resources preserved in our laboratory and their genetic diversity were analyzed.We analyzed citrus samples from Pakistan by transcriptome sequencing and obtained the full genome sequences of CEVd,CBLVd,HSVd,CBCVd,CDVd,and CVd-V,and they differed little from the reported sequences.The Pakistani CEVd sequence（Contig-4665）was identical to the one isolated from “Meishan 9” in China.We provisionally called the newly discovered Pakistani CBCVd variants “CBCVd-LSS” for their low sequence similarity（80.9%–88.9%）with the CBCVd RefSeq sequence（NC₀03539）.Molecular characterization of CBCVd from citrus in Pakistan and China were also reported in our work.The length of CBCVd from China ranged from 282 to 286 nucleotides,while that of the one from Pakistan ranged from 273 to 277 nucleotides.Our results indicated that the CBCVd sequences from Pakistan and China were significantly different with respect to genome and secondary structure and Pakistan might be one of the independent geographical origins of CBCVd worldwide.Due to the high sequence homology with the reference sequence,CEVd isolated from “Meishan 9” and CBCVd isolated from “Akemi” were selected to construct the infectious clones.（2）Construction of infectious clones of CEVd and CBCVd and the genetic diversity of their progeny populationsIn this study,we described a method to rapidly prepare the cDNA dimers of CEVd by one-step RT-PCR and the results showed that transcripts from the dimeric cDNA products were infectious to viroid-free hosts.Moreover,this method for rapid preparing cDNA dimers was also applicable to CBCVd.This method reduced the cloning steps,and the viroid RNAs obtained in this study could spread in the plant tissues.These results indicated that it was feasible to prepare the cDNA dimers of CEVd and CBCVd by this method.In addition,the analysis of the progeny populations of CEVd and CBCVd in citron showed that the CBCVd progeny population had a more complex population structure and higher variability levels,as well as haplotype diversity and nucleotide diversity.The progeny populations had complex variant sequences.In particular,we found a different sequence with the frequency close to the original inoculation sequence in CBCVd populations.The results showed that the viroids formed complex genetic diversity to adapt to the host after inoculating with a single sequence.Ⅱ.The analysis of antagonistic mechanism（1）Response mechanism of citron against CEVd infectionThis study employed CEVd infecting citron as a system to study the feedback regulation mechanism using transcriptome and sRNA analyses and the reliability was verified by quantitative PCR.Transcriptome results indicated that CEVd infection caused a wide range of changes in the leaves of citron and it activated gene silencing pathway,triggered basic defense response,and disturbed phytohormone homeostasis and so on.The replication of CEVd in citron induced significant up-regulation of the genes encoding DCL2（Dicer-like 2）,RDR1（RNA-dependent RNA polymerase 1）,AGO2（Argonaute 2）,AGO7 in RNA silencing pathway.The genes encoding CNGCs（Cyclic nucleotide-gated ion channel）,MAPKs（Mitogen-activated protein kinase）,CMLs（Calcium-binding protein）,FLS2（LRR receptor-like serine/threonine-protein kinase FLS2）,and Rboh（Respiratory burst oxidase homolog protein D）involved in the basic defense response were significantly up-regulated.And the genes encoding disease resistance proteins,pathogenicity-related proteins,and HSP70（Heat shock cognate 70 kDa protein）were also up-regulated in response to CEVd infection.The expression of many genes involved in plant hormone signal transduction pathways were regulated,and the dwarfed symptom in citron plants might be related to the inhibition of IAA（auxin）signal transduction pathway after CEVd infection.JA（jasmonic acid）,GA（gibberellin）and BR（brassinolide）signal transduction pathways were changed by CEVd infection.Small RNA（sRNA）sequencing showed that CEVd infection caused extensive changes in citron plants.CEVd infection affected the expression of host non-coding RNA（ncRNA）.It induced the production of 21-nt and 22-nt sRNA,and relatively inhibit the production of 24-nt sRNA.A total of 116 differentially expressed microRNAs（DEmiRNAs）were identified in response to CEVd infection and the target genes of some DEmiRNA were associated with RNA silencing,plant innate immune responses,and plant hormone signaling pathways.These results indicated that sRNAs could participate in the interaction between viroids and their hosts and played roles in the feedback regulation aginst CEVd in citron.These results suggested that RNA silencing mechanism and basic defense mechanism could all respond to CEVd infection and would help us understand the pathogenic mechanism of CEVd.（2）Analysis of molecular mechanism of CBCVd’s cross protection against CEVdThe genomic sequences of CEVd and CBCVd have 80⁹0-nt highly homologous region in the right-hand end.The combined infection of CBCVd and CEVd could attenuate cracking symptoms of sensitive rootstocks.In this study,CBCVd had a significant cross protection against CEVd at the molecular level and the symptoms in citron plants also showed some antagonistic performance during the early days.Similar response mechanisms were observed in the citron plants infected with different viroids using transcriptome and proteome analysis.sRNA sequencing revealed that CEVd/CBCVd infection could induce the accumulation of 21²2-nt sRNA.The sizes of viroid-derived sRNAs（vd-sRNA）were predominantly 21-nt and 22-nt,and sRNAs derived from CEVd and CBCVd were significantly enriched in their homologous region.The 5’ base bias of 21-nt and 22-nt sRNAs derived from positive and negative strands of CEVd and CBCVd were predominantly U and C and these vd-sRNA might have competition for AGO proteins in the gene silencing pathway.The 22-nt sRNAs produced by DCL2 were the most abundant,and the 22-nt sRNAs and RDR1 were associated with silencing amplification.DCL2 and RDR1 were speculated to be crucial in the interaction between viroids and citron plants.These results indicated that the RNA silencing mechanism was involved in the interaction between citrus viroids and their hosts.CBCVd had cross protection against CEVd,and the antagonism mechanism should be related to RNA silencing and the identical sRNAs from their homologous regions.CEVd and CBCVd produced a large number of identical sRNAs in their homologous regions and the important roles of these sRNAs in RNA silencing pathway might help us understand the molecular mechanism of cross protection.