Functional Characterization Of A Putative UDP-Glucosyl Transferase Gene SlUGT132 In The Arbuscular Mycorrhizal Symbiosis

Arbuscular mycorrhizal(AM)symbiosis that is formed by AM fungi belonging to Glomeromycotina and the roots of most land plants is one of the most widespread and oldest mutualistic association in nature.The primary benefit for both partners is the acquisition of nutrients:the fungi improve the supply of water and mineral nutrients,mainly P,to the host plants,whereas,in exchange,plants transfer up to 20%of their fixed carbohydrate to the fungi.AM fungi also provide non-nutritional benefits to their host,such as improving the resistance of plants against several abiotic(drought,salinity)and biotic(pathogen infections)stresses.As a typical endosymbiosis,AM interaction requires the differentiation of novel symbiotic interfaces within the root cells,and a continuous exchange of signals between the two partners.In the AM symbiosis,the fungal hyphae penetrate the root epidermis,grow through the intercellular spaces of the root and subsequently invade cortical cells,developing highly-branched tree-like structures called arbuscules.Development of AM symbiosis is controlled by an intricate procedure involving a number of genes from both the symbionts.Glycosylation is an important secondary metabolism reaction that can transfer one or multiple molecules of sugar to a wide range of acceptor molecules such as lipids,proteins,hormones,and oligosaccharides.The catalytic reaction is mediated by a large group of enzymes termed glycosyltransferases.These enzymes participate in diverse biological processes including hormone homeostasis,flower and fruit pigmentation,biosynthesis of plant cell walls and defense responses.Flavonoids are a group of secondary metabolites derived from the phenylpropanoid pathway,and their biosynthesis usually involves the glycosylation modification in the later steps.Diverse functions,including vegetative growth,reproduction process,disease defense and pathogen resistance,have been proposed for flavonoids in diverse plant species.It has been documented that low concentrations of flavonoids could trigger developmental responses of the AM fungi,such as stimulating spore germination and priming hyphal growth and branching,indicating that flavonoids are implicated in the development of AM symbiosis.However,limited information regarding whether the presence of glycosylation-mediated modification of flavonoids in the modulation of AM symbiosis is available thus far.In our previous study,through extensively hunting the released transcriptome data of tomato mycorrhizal and nonmycorrhizal roots,we identified a putative UDP-glucosyl transferase gene,referred as SLUGT132,that was strongly induced in response to AM symbiosis.Bioinformatic analysis of the putative functional domains in SLUGT132 protein further led to the prediction that SLUGT132 could catalyze the glycosylation reaction with flavonoids as potential substrates.Thus we proposed that SlUGT132 might be able to regulate the establishment of AM symbiosis through glycosylated modification of certain flavonoids.To test this,in the present study,we performed detailed investigations,including tissue-specific expression patterns,in-vitro enzymatic activity assay,functional analysis of the transgenic plants with knock-out or enhanced expression activity,to dissect the potential roles of SlUGT132 in AM symbiosis.The main findings were listed as follows:1.Though gnome-wide hunting of the released tomato genomic sequences,a total of 175 genes(named as SlUGT1-175,respectively,according to their chromosomal localizations)encoding putative UDP-glucosyl transferase were identified from tomato.These UGT genes could be classified into six subfamilies,and SlUGT132,that was shown to be strongly induced in the mycorrhizal roots,is a member of the V subfamily.Quantitative expression analysis revealed that except the SlUGT132,none of the members of the V subfamily was upregulated in response to AM symbiosis.SlUGT132 was also shown to be highly expressed in fruits.2.The glycosylation enzymatic activity of SlUGT132 was evaluated in vitro.SlUGT132 was heterologously expressed in E.coli,and the purified protein was used for glycosylation reaction by using UDP as the donor and kaempferol,apigenin and quercetin as the putative substrates.The results showed that SlUGT132 has the ability to catalyze the glycosylation reaction for both kaempferol and apigenin,but not quercetin.3.To obtain a more detailed view of SlUGT132 expression in tomato,the promoter of SlUGT132 was fused to the GUS reporter gene,and introduced into tomato plants via Agrobacterium tumefaciens-mediated transformation.Histochemical staining showed that the GUS staining could be detected strongly in AM fungal-colonized roots of transgenic tomato plants,but no GUS staining could be detected in nonmycorrhizal roots.Consistent with the results obtained from quantitative expression analysis,strong GUS staining could also be detected in tomato fruits.Promoter deletion assay identified a putative cis-element,MYCU1,that might be responsible for the AM-induced expression in the SlUGT132 promoter.4.To determine the subcellular localization of SlUGT132,the coding sequence of SlUGT132 was fused in-frame with the 5' end of the GFP reporter gene under the control of either the 35S cauliflower mosaic virus promoter(35S::SlUGT132-GFP)or its native promoter(pUGT::SlUGT132-GFP).The two constructs were transformed into the N.Benthamiana leaf epidermal cells and tomato,respectively.The SlUGT132-GFP fusion protein expressed in the N.benthamiana epidermal cells showed a distinct localization signal in both cytoplasm and nucleus,while in the tomato mycorrhizal roots colonized by R.irregularis,the SlUGT132-GFP fusion protein showed a specific localization in the cells containing hyphae without fully developed arbuscules.5.To determine whether the AM-induced SlUGT132 is required for the maintenance of AM symbiosis,we generated knockout and knockdown mutant lines of SlUGT132 by using the CRISPR-Cas9 and the VIGS(virus-induced gene silence)systems,respectively.The mycorrhizal slugt132 knockout mutants generated from CRISPR-Cas9 showed a significant decrease in total flavonoid content compared with that in the mycorrhizal WT plants,while no significant difference could be detected between the nonmycorrhizal slugt132 mutants and WT plants.Compared to mycorrhizal WT plants,the mycorrhizal slugt132 mutants had significantly higher total root length colonization and arbuscule incidence,but relatively lower average arbuscule size.Consistent with the results obtained from the slugt132 knockout mutants,the slugt132 knockdown lines generated by VIGS also showed a significant increase in both total root length colonization and arbuscule incidence,compared with the corresponding WT plants.These finding strongly suggest that SlUGT132 might have a negative effect on AM symbiosis,probably through modification of flavonoids.6.To gain further insights into the possible role of SlUGT132,we further generated transgenic tomato and rice plants overexpressing SlUGT132 under the control of the cauliflower mosaic virus 35S promoter.Both the transgenic tomato and rice plants overexpressing SlUGT132 showed a significantly decreased biomass,soluble sugar content and yield compared with the corresponding WT plants,irrespective of the presence or absence of AM fungal colonization.Compared to WT plants,both the transgenic tomato and rice plants showed a lower arbuscule abundance when inoculated with the AM fungus Rhizophagus irregularis.However,arbuscule morphology in the SlUGT132-overexpressing plants did not differ significantly with that in the corresponding WT plants.In conclusion,we performed reverse genetic approaches to characterize the function of the tomato AM-induced UDP-glucosyl transferase gene,namely,SlUGT132,in AM symbiosis.SlUGT132 was strongly expressed in the cortical cells containing arbuscules and also in the fruits,and its protein could catalyze glycosylation for both kaempferol and apigenin.Knockout or knockdown of SlUGT132 significantly decreased the content of total flavonoids,but increased the total root length colonization and arbuscule abundance in transgenic plants.Constitutive overexpression of SlUGT132 in either tomato and rice plants resulted in a significant decrease in biomass,fruit/grain productivity,soluble sugar accumulation and arbuscule incidence.These results suggest that SlUGT132 could negatively regulate the development of AM symbiosis,probably through the galactosylated modification of certain kinds of flavonoids,such as kaempferol and apigenin.