Characterization Of Root Microbiome And Identification Of Mirnas Involved In Arbuscular Mycorrhizal Symbiosis In Citrus

Citrus roots have rare root hairs and thus heavily depend on forming symbiosis with arbuscular mycorrhizal fungi(AMF)for absorbing nutrients from soil.Except for AMF,other plant associated microbes can promote plant growth,and increase the resistance of plant to the biotic and abiotic stress,such as pathogen infection,drought,cold,high salinity and heavy mental.Thus,the study of citrus root microbial community structure,especially the AMF community structure,as well as its functions and regulation are of great significance in citrus production.In this study,high-throughput sequencing was used to uncover the arbuscular mycorrhizal fungi community structure and identify mi RNAs in response to mycorrhizal symbiosis in citrus,and the overexpression and Short tandem target mimic(STTM)were utilized to validate the functions of key mi RNAs.This study would provide the basis for researches on the molecular mechanisms of mycorrhizal symbiosis.Additionally,the microbiome and metagenome techniques were applied to identify the microbial community structure and function associated with citrus roots,as well as their variations under Huanglongbing infection.The main results were listed as follows:1.Identification of arbuscular mycorrhizal fungi community colonized citrus roots,and evaluation of its major influencing factorsThe citrus seedlings and grafted combinations were sampled from 8 citrus producing areas.By using 454-pyrosequencing of AMF 18 S r RNA gene fragment,we investigated the genetic diversity of AMF colonized citrus roots and evaluated the impact of habitats and rootstock & scion genotypes on the AMF community structure.Over 740000 effective sequences were obtained from 77 citrus root samples,corresponding to 1028 AMF OTU.These AMF sequences were assigned to 75 AMF virtual taxa,of which 66 belong to Glomus,highlighting an absolute dominance of this AMF genus in symbiosis with citrus roots.The citrus AMF community structure was significantly affected by habitats and host genotypes,and habitats was the most important influencing factor.Interestingly,our data suggested that the genotype of the scion exerted a greater impact on the AMF community structure than that of the rootstock where the physical root-AMF association occurs.This result indicated that scion might regulate the mycorrhizal symbiosis through transport of metabolites or other long-distance regulation.2.Identification and functional analysis of mycorrhizal symbiosis responsive mi RNAs in citrusIn this study,a commonly used citrus rootstock,Poncirus trifoliata was utilized as materials,and small RNA sequencing was performed to identify the AMF responsive mi RNAs in citrus roots.A total of 148 known mi RNAs and 15 novel mi RNAs were identified in the roots,among which 20 mi RNAs and mi RNA*s were induced or repressed by mycorrhizal colonization.The 20 differentially expressed mi RNAs include two previously reported arbuscular mycorrhizal symbiosis(AMS)related mi RNAs(mi R171 b and mi R167h)and AMS responsive mi RNAs(mi R473,mi R1446b/c and mi R477a/c)that could target AMS-related genes,including GRAS family RAD1 gene and RFCb gene.To investigate the role of these mi RNAs,mi R477 a was selected for constructing the over-expression vector and STTM vector,and then transformed into poncirus.The positive lines were obtained for checking phenotypes next.In addition,we found the homolog mi RNA of mi R477 a in medicago(mi R5037a/b/c),and the expression level of mi R5037a/b/c was also induced by AMS through q RT-PCR.Due to the fact that mi R5037a/b/c have 3 precusors and 1 mature mi RNA,3 over-expression vectors and 1 STTM vector were constructed and transformed to the medicago with hair root transformation.By checking the phenotypes,we found the mycorrhizal colonization rate and arbuscular abundance were slightly increased in the mi R5037 over-expression lines(no significant difference),while a significant reduce of mycorrhizal colonization rate and arbuscular abundance in 5037 STTM lines were observed,which might indicate that mi R5037a/b/c may play a role in AMS.3.Identification of the microbial community structures of citrus root,as well as their response to HLB infectionThe healthy and HLB infected citrus roots(Newhall sweet orange grafted on Poncirus trifoliata)and rhizosphere soil were sampled from the same orchard in the Institute of Citrus Science Research of Ganzhou,and the high-throughput sequencing of bacteria 16 S r RNA specific fragments was performed to identify the bacterial community structures of citrus root endophytic compartment(EC)and rhizosphere,as well as the influence of HLB infection on the citrus root bacterial communities.A total of1010116 effective reads were obtained from sequencing of 16 citrus root and rhizosphere samples,and a total of 2464 bacterial OTU were identified.Among them,Proteobacteria,which accounted for 37.42% of the total OTU number and 58.25% of the total reads number,was the most abundant phylum in the bacterial communities of both citrus root EC and rhizosphere,indicating the dominance of Proteobacteria in citrus root microbiome.HLB infection affected the citrus root bacterial community structures by altering the relative abundance of partial bacterial species,while no significant difference was observed in the total richness and diversity of citrus root bacterial communities.Interestingly,the abundance of Amycolatopsis(a disease resistance related bacteria genus)and Sphingopyxis(an aromatic compounds degradation related bacteria genus)were increased during HLB infection,indicating a possible involvement of these two bacteria genera in response to HLB infection.4.Identification of the community structure and function of citrus rhizosphere microbiome based on metagenomic sequencingThe healthy and HLB infected citrus(Newhall sweet orange grafted on Poncirus trifoliata)rhizosphere soils were sampled from the same orchard in the Institute of Citrus Science Research of Ganzhou,and the metagenomic sequencing was used in this study to identify the community structure and function of citrus rhizosphere microbiome,as well as their variations under HLB infection.A total of 102474 genes were obtained from metagenomic sequencing,and 43 phyla,1167 genera and 3571 species were annotated from the citrus rhizosphere microbiome,indicating a high diversity of the citrus rhizosphere microbiome and a high accuracy of metagenome approach at genus level and species level.Bacterial genes were accounting for 99.59% of the total annotated genes in the citrus rhizosphere metagenome,of which Proteobacteria was the most abundant phylum.The main functions of citrus rhizosphere microbiome were “Amino acid transport and metabolism”,“Energy production and conversion”,“Carbohydrate tansport and metabolism”,“Cell wall/membrane/envelope biogenesis”,“Transcription”,“Signal transduction mechanisms” and so on.More than 74% of the differentially expressed genes identified in citrus rhizosphere metagenome during HLB infection were involved in metabolism related pathways,and all the most abundant three pathways were metabolism related pathways(including Metabolic pathways,Microbial metabolism in diverse environments and Biosynthesis of secondary metabolites),which indicated that HLB infection played a significant impact on the metabolism functions of the citrus rhizosphere microbial community.The abundance of key enzymes for degrading carbohydrate,including maltase,?-glucosaccharase,Glucose-6-phosphate isomerase,enolase and pyruvate kinase,were significantly decreased in the citrus rhizosphere metagenome under HLB infection,while the abundance of enzymes for degrading aromatic compounds,aldehyde dehydrogenase and 4-oxalocrotonate tautomerase,were significantly increased.This result suggested that the plugging phloem caused by HLB infection would be able to decrease the root exudates of citrus plants,and thus leads to the decreased abundance of bacteria which used the root exudates as the main carbon source.In addition,the depletion of carbohydrate in HLB infected citrus roots would lead to the root death,and thus resulted in the increase of aromatic compounds,like recalcitrant lignin.As a result,the abundance of bacteria genera which could degrade aromatic compounds,including Comamonas,Ralstonia,Sphingopyxis and Sphingobacterium,were significantly increased.We hypothesized that HLB infection might affect the microbial community structure through altering the root exudates and the situation of the rhizosphere.