Abstract： 【Objective】 Ectomycorrhizae is an important trait that enables chestnut trees to obtain limited soil nutrients, such as nitrogen. The molecular mechanism involved in nitrogen absorption and utilization by ectomycorrhizae has not yet been clarified in chestnut. In this study, the highly induced nitrate transporter gene CmNRT3 of Chinese chestnut in response to ectomycorrhizal symbiosis was identified and further its spatial expression pattern and protein function were analyzed.【Methods】The plant materials used were Chinese chestnut 'Jingshuhong', which produced healthy, pest-free fruits, and Medicago truncatula ecotype A17 and Nicotiana benthamiana, which were propagated in our laboratory. The ectomycorrhizal fungus selected was Scleroderma citrinum, obtained by propagation on P20 medium. The arbuscular mycorrhizal fungus used was spores of Rhizophagus irregularis, provided by thearbuscular mycorrhizal fungi germplasm resource center of Beijing Academy of Agriculture and Forestry Sciences. △ynt-Leu- yeast lacking of nitrate transportation ability was provided by Yuan Lixing's research group (China Agricultural University), and cultured in YNB yeast medium. To investigate which chestnut NRTs were involved in ectomycorrhizal symbiosis, the excavation and expression analysis of Chinese chestnut NRT gene family in both control and ectomycorrhizal roots were conducted. The CmNRTs by using protein sequence BLAST of Arabidopsis and rice NRTs against the chestnut genome were identified, and MEGA7.0.14 software for gene sequence alignment and evolutionary analysis was employed. The highly induced CmNRT3 among NRT gene family in chestnut ectomycorrhizal roots was confirmed using quantitative real-time PCR (BIO-RAD, USA). Primers used were designed by Primer 3 (v.0.4.0) (http://bioinfo.ut.ee/primer3-0.4.0/). Primer sequences of CmNRT3 were CmNRT3-F: 5'- GTCTAGCTGTAACTTGTTATGGA-3', CmNRT3-R: 5'-CTGGCAAACTCTGGTTTAGA-3'. Primer sequences of CmACTIN as a reference gene were CmACTIN-F: 5'-GTGGCGGTTCAACCATGTTC-3', CmACTIN-R: 5'- GGATGGACCACTCTCATCGT-3'. Further, a 1.6 kb promoter of CmNRT3 was cloned, and the CmNRT3 promoter-GUS construct was generated and transgenic roots expressing this reporter by performing hairy root transformation were obtained. Additionally, this construct was introduced into Medicago through hairy root transformation as well, and then the transgenic roots were inoculated with R. irregularis. The transgenic roots underwent GUS staining, plastic embedding, and sectioning with a microtome (Leica). The sections were counterstained with ruthenium red and observed under a light microscope (Leica 5500). In this way the spatial expression pattern of CmNRT3 was studied in chestnut control roots, and heterozygously analyzed in Medicago arbuscular mycorrhizal roots. To further identify the CmNRT3’s subcellular localization, 35S::CmNRT3::GFP and CmNRT3pro::CmNRT3::GFP fusion constructs were generated and transiently expressed in N. benthamiana leaves and Medicago roots, respectively. Here, plasma membrane-tagged protein pm-rb CD3-1008 expression vectors were used as a marker, and a laser confocal microscope (Leica STELLARIS 5) was used for protein expression observation. To test whether this CmNRT3 function as a nitrate transporter, CmNRT3-pDR-F1-GW vector by using pENTR-TOPO-CmNRT3 vector (with stop codon) through LR recombination reaction was generated. Subsequently, yeast functional complementation experiments by expressing the aforementioned vector in △ynt-Leu- yeast were conducted. 【Results】60 putative nitrate transporter (NRT) gene family members in chestnut genome were characterized, and divided into three subfamilies, NRT1, NRT2 and NRT3. In NRT3 subfamily, Cm06G00423 was the only member and therefore named as CmNRT3. Based on transcriptome data analyses of Chinese chestnut control and ectomycorrhizal roots, CmNRT3 was induced 8 folds in 2 months old ectomycorrhizal roots. Meanwhile, through quantitative real-time PCR the expression level of CmNRT3 gene was up-regulated 3.13-fold in 1 month old ECM, compared with control roots. A 1.6 kb CmNRT3 promoter-driven GUS signal was mainly detected in the epidermis and weakly in the cortex in chestnut and Medicago control roots. However, the GUS signal was predominantly present in arbuscule containing cells upon arbuscular mycorrhizal symbiosis in Medicago roots. The subcellular localization analysis by using 35S::CmNRT3::GFP construct indicated that CmNRT3 was localized on the cell membrane, marked by the plasma membrane pm-rb CD3-1008 reporter in N. benthamiana leaves. In Medicago arbuscular mycorrhizal roots expressing CmNRT3pro::CmNRT3::GFP fusion vectors, CmNRT3 protein was mainly localized in arbuscule containing cells and specifically on the peri-arbuscular membrane, an interface between plants and arbuscular mycorrhizal fungi. Through yeast complementation assay, the nitrate transportation defective yeast expressing CmNRT3 failed to recover the growth in Leu-deficient medium, in contrast, the growth could be restored by adding Leu in the medium. 【Conclusion】 This analysis indicated that CmNRT3 is a unique gene within NRT3 subfamily in Chinese chestnut, indicating this gene has a specific function. Expression of CmNRT3 was up-regulated in both early and late stages of ectomycorrhizal symbiosis, suggesting it is required during the whole process of symbiotic interaction. Promoter-GUS assay showed that its expression pattern was conserved in different plant species. However, upon arbuscular mycorrhizal signal, its expression pattern was altered, indicating a specific transcriptional regulation during fungal symbiosis. Whether this was also the case during ectomycorrhizal symbiosis need to be further clarified. When heterogeneously expressed either in tabacco leaves or in Medicago arbuscular mycorrhizal roots, the CmNRT3-protein fusions were localized on membranes. To be specific the former in the plasma membrane, but the later in arbuscular plasma membrane, was in line with an alteration in gene expression pattern. Although sequence analysis predicted its nitrate transportation potential, CmNRT3 did not display nitrate absorption or transportation ability in the yeast complementation experiment. It was well possible that CmNRT3 functioned as a membrane co-transporter that could be investigated by protein-protein interactions. This study underscored the role of ectomycorrhiza in promoting nitrogen absorption and utilization in Chinese chestnut.
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