Comparative Transcriptome Analysis Of Citrus Am Symbiosis And Functional Characterization Of Am-host Plant Sugar Efflux Transporter SWEET1b

Citrus have rare root hairs and are largely dependent on symbiotic arbuscular mycorrhizal fungi to uptake water and mineral nutrient.Therefore,studies on the molecular mechanism of citrus-arbuscular mycorrhizal symbiosis would provide theoretical basis towards practical application.In this study,we investigated AMF colonization-induced transcriptomic changes in roots of citrus rootstock Poncirus trifoliata(L.)Raf,and then assessed the conservation level in AMF-induced transcriptional reprogramming between P.trifoliata and four herbaceous model plants.Further reverse-genetic analyses identified core genes involved in AM symbiosis in model plant M.truncatula.AM fungi are strictly biotrophic and thus depend on organic carbon provided by their host plants to support its growth.It is known that the AM fungi could obtain carbon in the form of lipids and sugars.Though the molecular mechanism of lipids transfer from host to AM fungi was stated,the mechanism by which sugars are transported to the fungi is unclear.Here,we identified and functional characterized one candidate gene SWEET1 b which could export sugars from host plant cells into AM fungi.The main results are showed as following:1.Transcriptome analysis of Poncirus trifoliata identified AM responsive genesTotal RNA was extracted from roots of the most common citrus rootstock Poncirus trifoliata(L.)Raf inoculated or non-inoculated with AM fungi Glmous versiforme and were subjected to RNA-seq analysis.We identified 282 differentially expressed genes(DEGs)in response to AMF colonization,with 245 genes up-regulated and 37down-regulated.The DEGs included orthologs of 21 genes with characterized roles in AMS and 83 genes that are considered to be conserved in AM-host plants.Further q RT-PCR analysis showed 36 genes indeed displayed induced expression in Gv-colonized root samples compared with non-inoculated controls,which well confirmed the RNA-seq data.Our transcriptome analysis provided a set of reliable AM-responsive genes,as well the known genes showed induced expression in Poncirus trifoliata probably play a conserved role in AM symbiosis.2.Comparative transcriptome analysis of Poncirus trifoliata identified a core set of genes involved in AM symbiosisTo further investigate the extent to which the transcriptional reprogramming upon AMS is conserved in the five flowering plant species(P.trifoliata,M.truncatula,L.japonicus,S.lycopersicum,and O.sativa),we performed a wider comparative transcriptome analysis.The results showed 75.9% of all up-regulated P.trifoliata genes contained orthologous genes also induced expression from at least one another species.Among this,153 P.trifoliata genes that were members of orthogroups containing genes were induced by AMF from at least two other AM-host plants.The results strengthen the hypothesis that the ancient symbiotic pathway triggers a conserved expression program in the evolutionarily diverse woody plant P.trifoliata and four herbaceous model plants.The153 gene set reflected a core genetic program that was induced independent of plant and fungal identity.We therefore consider this core set of genes as likely key candidates to play important roles in AM symbiosis,at least within angiosperms.3.Reverse genetic analysis of one AMF-specifically induced FatG gene demonstrated its role in mycorrhizal symbiosisOne of the core set gene Ptr FatG(Cs1g21320)only exists in AM-host plants,and it encods a 3-keto-acyl-ACP reductase involved in fatty acids synthesis.Promoter-GUS analysis showed the Ptr FatG promoter only have activity in arbuscule-containing cells.Regarding that its putative ortholog in M.truncatula(Mt FatG;Medtr4g097510)was highly induced in arbuscule-containing cells,we generated transgenic M.truncatula roots expressing an RNAi construct targeting Mt FatG.Our observation that silencing of the Mt FatG in M.truncatula roots resulted in impaired arbuscule development suggests a conserved role for FatG in the fatty acid synthesis regulon to provide lipids to the fungus.4.Functional characterization of one candidate gene SWEET1 b involved in the sugar efflux from host plant to AM fungiOur previous study showed one citrus SWEET gene is strongly induced in mycorrhization,therefore,we hypothesize that specific SWEET proteins may be crucial players in the supply of sugars to AM fungi.Based on this,we conducted a further study using the mycorrhizal model plant M.truncatula.We made use of laser microdissection combined with RNA-seq analysis,and found one of the 26 M.truncatula SWEETs were prominent expressed in arbuscule containing cells.Further q RT-PCR analysis confirmed MtSWEET1 b is induced in mycorrhizal roots.Promoter-GUS analysis showed strong GUS signal present in arbuscule cells.Native promoter drived SWEET1b-GFP fusion protein was localized to peri-arbuscular membrane(PAM).The results indicate that the MtSWEET1 b is well positioned to transport sugars across the PAM to the fungus.Phylogenetic analyses showed that MtSWEET1 b belongs to the At SWEET1 clade.Heterologous expression of MtSWEET1 b in a yeast hexose deficiency mutant showed that it could transport glucose.Two MtSWEET1 b mutants carrying Y57 A or G58 D substitutions lost the glucose transport activity,indicating that the two residues are important for glucose transport in MtSWEET1 b.To investigate the importance of MtSWEET1 b in the AM symbiosis,we overexpressed the Y57 A and G58 D mutant MtSWEET1 b versions using the arbusucle-specific Mt PT4 promoter.Results showed that the dominant-negative interference of MtSWEET1 b function impaired the maintenance of arbuscules.Our results indicate that the symbiotic MtSWEET1 b plays an important role in glucose delivery at arbuscules to AM fungi,as well for arbuscule development.In summary,this study investigated the citrus common rootstock P.trifoliata AM-reponsive gene;Comparative transcriptome analysis revealed the conserved transcriptional program of woody plant P.trifoliata and four herbaceous model plants(M.truncatula,L.japonicus,S.lycopersicum,and O.sativa)in arbuscular mycorrhizal symbiosis,and identified a core set of AM co-responsive genes.It highlights that comparative transcriptome is a new and powerful approach to study the molecular mechanism of plant-arbuscular mycorrhizal symbiosis.We found a novel fatty acids synthesis regulon FatG,which only exist in AM-host plants,specifically expressed in arbuscule-containing cells and is required for arbuscule development,thus our results further suggest that lipids are one of the important carbon source of AM fungi.This study provides the first insight into the molecular machinery of the sugar supply from host plants to AM fungi.Our results showed that plants employ a peri-arbuscular membrane localized MtSWEET1 b to deliver glucose into AM fungi to sustain symbiosis.These data extend our understanding about the mechanism of AM-host plants carbon efflux into AM fungi during AM symbiosis.