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      Functional analysis of the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants

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          Low availability of nitrogen (N), mainly nitrate in aerobic soils, is a primary limiting factor for crop production. Most terrestrial plants live in symbiosis with arbuscular mycorrhizal (AM) fungi to increase nutrient uptake, including N, from soil. Research on the AM symbiosis field has focused almost exclusively on ammonium as the form of N transferred to the plants, and there has been no direct evidence of N transfer as nitrate thus far. Here, we report that mycorrhizal rice could receive more than 40% of its N via the mycorrhizal pathway and that the AM-specific nitrate transporter OsNPF4.5 accounted for approximately 45% of the mycorrhizal nitrate uptake. Our work suggests the presence of a mycorrhizal route for nitrate uptake in plants.

          Abstract

          Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus Rhizophagus irregularis remarkably promoted rice ( Oryza sativa) growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO 3 supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene OsNPF4.5 in rice roots, and its orthologs ZmNPF4.5 in Zea mays and SbNPF4.5 in Sorghum bicolor. OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO 3 transport activity when expressed in Xenopus laevis oocytes. Moreover, knockout of OsNPF4.5 resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO 3 was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO 3 acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.

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          Plant nitrogen assimilation and use efficiency.

          Crop productivity relies heavily on nitrogen (N) fertilization. Production and application of N fertilizers consume huge amounts of energy, and excess is detrimental to the environment; therefore, increasing plant N use efficiency (NUE) is essential for the development of sustainable agriculture. Plant NUE is inherently complex, as each step-including N uptake, translocation, assimilation, and remobilization-is governed by multiple interacting genetic and environmental factors. The limiting factors in plant metabolism for maximizing NUE are different at high and low N supplies, indicating great potential for improving the NUE of current cultivars, which were bred in well-fertilized soil. Decreasing environmental losses and increasing the productivity of crop-acquired N requires the coordination of carbohydrate and N metabolism to give high yields. Increasing both the grain and N harvest index to drive N acquisition and utilization are important approaches for breeding future high-NUE cultivars.
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            Targeted mutagenesis in rice using CRISPR-Cas system

            Dear Editor, Genome editing of model organisms is essential for gene function analysis and is thus critical for human health and agricultural production. The current technologies used for genome editing include ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcription activator-like effector nucleases), etc. 1 . These technologies can generate double stranded breaks (DSBs) to either disrupt gene function through generation of premature stop codons by non-homologous end joining (NHEJ) pathway, or to facilitate gene targeting through homologous recombination (HR) with an incoming template. Recently, a new technology for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems, has been developed 2 . CRISPR/Cas systems are adaptive defense systems in prokaryotic organisms to fight against alien nucleic acids 3 . The spacer sequences acquired from foreign DNA are positioned between host repeats, and transcribed together as CRISPR RNA (crRNA). In the type II CRISPR system, a single nuclease Cas9, guided by a dual-crRNA:tracrRNA, is sufficient to cleave cognate DNA homologous to the spacer 2 . Efficient cleavage also requires the presence of protospacer adjacent motif (PAM) 5′-NGG-3′ following the spacer sequence. The dual-crRNA:tracrRNA has been further streamlined to a single RNA chimera, called sgRNA (single guide RNA) 2 . Compared with protein-guided technologies, CRISPR/Cas system is much easier to implement, as only short guide RNAs need to be customized to target the genes of interest. Up to now, the CRISPR/Cas system has been successfully applied to efficient genome editing in many eukaryotic organisms including human 1 , mice 4 , zebra fish 5 , fly 6 , worm 7 , and yeast 8 . However, the application of CRISPR/Cas system in plants has not been reported. Rice (Oryza sativa L.) is a major staple crop in the grass family (Poaceae), feeding half of the world's population. Rice is also used as a model monocot plant for biological studies because it has a relatively small genome compared to other cereal crops and is easy to be manipulated genetically. We demonstrate in this study that the CRISPR/Cas technology can achieve efficient targeted mutagenesis in transgenic rice. Our work paves the way for large-scale genome editing in rice, which is important for quality improvement and yield increase of rice. To accommodate the CRISPR/Cas system to Agrobacterium-mediated plant transformation, we designed Gateway™ binary T-DNA vectors for co-expression of CAS9 and guide RNA (either sgRNA or dual-crRNA:tracrRNA, see Figure 1A). Gene-specific spacer sequence was cloned into entry vectors for expression of guide RNA (Supplementary information, Figure S1 and Data S1), which was then cloned into destination vectors containing the CAS9 expression cassette. To reconstitute Cas9 ribonucleoprotein complex in the nucleus, Cas9 was attached with a nuclear localization signal, and guide RNAs were driven by pol III type promoter of U3 snRNA. CAS9 coding sequence was codon-optimized for expression in rice (Supplementary information, Figure S2), and was driven by the maize Ubiquitin (Ubi) promoter. To facilitate Cas9 binding and R-loop formation, we chose the sgRNA with the secondary structure containing a dangling spacer, an extended hairpin region and a long 3′ end (Figure 1B). To test whether the CRISPR/Cas system can be applied in plant cells, we first investigated whether the system can generate DSB in rice callus using the DGU.US as a reporter (Supplementary information, Figure S3). GUS activity can be restored through single strand annealing (SSA) upon DNA cleavage between the repeat regions in the DGU.US reporter 9 (Figure 1C). We designed both sgRNA and dual-crRNA:tracrRNA to target the reporter. The result showed that strong GUS staining spots were detected in rice calli after the CRISPR/Cas system and the reporter were co-transformed through particle bombardment, whereas no GUS signal was observed in those transformed with the reporter alone (Figure 1D). Both sgRNA and dual-crRNA:tracrRNA were effective in this assay, indicating that pre-crRNA can be properly processed in rice (plant) cells. To further test whether the CRISPR/Cas technology can be used to specifically disrupt an endogenous gene in rice, we designed sgRNAs and dual-crRNA:tracrRNAs targeting either the second exon of the CHLOROPHYLL A OXYGENASE 1 (CAO1) gene or the third exon of the LAZY1 gene, and transformed them into Kitaake, a japonica rice variety with short life cycle 10 . The seedlings of the loss-of-function mutant cao1 show a pale green leaf phenotype due to defective synthesis of Chlorophyll b (Chl b), which is easily observed at an early developmental stage, whereas the la1 mutant, loss-of-function mutant of LAZY1 gene, exhibited a pronounced tiller-spreading phenotype that can be observed after tillering stage 11 . We obtained 30 independent transgenic lines for sgRNA construct and 45 lines for dual-crRNA:tracrRNA construct for the CAO1 gene. We found that some transgenic plants displayed pale green leaf blades (Figure 1E), and we conducted genotyping analysis on these lines using gene-specific primers. The result showed that either loss of peak/gain of peak or overlapping peak around the target site was observed in the sequencing chromatograms (Figure 1F and Supplementary information, Figure S4), which confirmed mutations in the CAO1 gene. The occurrence of indels at 3-4-bp upstream of PAM is consistent with the location of Cas9 cleavage site. We randomly selected transgenic lines with biallelic mutation, and measured the contents of Chl a and Chl b 12 . The result showed that Chl b content was reduced to a marginal level, consistent with the cao1 phenotype (Figure 1G). We obtained only 12 independent transgenic lines for sgRNA construct for LAZY1 gene. Sequencing analysis showed that 11 out of 12 lines bear mutations in the specific region of LAZY1 gene, confirming the disruption of LAZY1 gene (Figure 1H and Supplementary information, Figure S5). Appearance of tiller-spreading phenotypes in 6 homogenous mutation lines further supported the conclusion (Figure 1I). Notably, in the case of sgRNA, about 83.3% and 91.6% of the independent lines of the T1 transgenic rice beared mutations in CAO1 and LAZY1, respectively, among which 4 lines (13.3%) for CAO1 and 6 lines (50%) for LAZY1 beared biallelic mutations (Supplementary information, Table S1). These results indicate that transgenic rice with mutated gene of interest can be easily generated in the T1 generation by using CRISPR/Cas technology, and that the high efficiency of targeted mutagenesis can be achieved in different genes. This high efficiency is possibly due to the unique feature of the CRISPR/Cas system, i.e., different from ZFN or TALEN, Cas9-mediated cleavage tolerates DNA methylation 13 . This gives the CRISPR/Cas technology leverage for genome editing in plants with high GC content in the genome such as rice. It seems that sgRNA constructs generate targeted mutagenesis more efficiently than dual-crRNA:tracrRNA constructs (Supplementary information, Table S1), the mechanism of which needs to be investigated in the future. To facilitate selection of spacers to target other rice genes, we also used criteria reported recently, which exclude potential mismatches of the first 8 nucleotides of the 20-nt spacer region and leaky activity on 5′-NAG-3′ PAM 8 , and designed more than 3.6 million spacers (Supplementary information, Data S2). We showed that transgenic rice with mutations in specific genes could be generated through the CRISPR/Cas technology in a straightforward manner. Our strategy for vector construction is modular, efficient, and expandable for multiplex gene editing. Because dual expression of pre-crRNA:tracrRNA can be correctly processed in the plant cells although the mutagenesis efficiency is relatively low in the two genes that we tested (Figure 1 and Supplementary information, Table S1), the entry vectors that we designed has the potential to host two sgRNAs or multiple spacers, in an array of crRNA. The facile genome editing at specific sites in rice will speed up functional characterization of rice genes, especially for those genes with family members, and will greatly promote the effort to improve rice quality and yield through agricultural biotechnology. The compiled list of the specific spacers for each gene in rice genome will also help to accelerate the application of the CRISPR/Cas system in rice. Further work will be focused on whether the CRISPR/Cas system can be applied successfully to other cereals with bigger and more complicated genomes such as maize and wheat. Meanwhile, further modification of the CRISPR/Cas system, e.g., removal of the components of the CRISPR/Cas system after the target genes are mutated, will promote the application of this new technology in agriculture.
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              Nitrogen transfer in the arbuscular mycorrhizal symbiosis.

              Most land plants are symbiotic with arbuscular mycorrhizal fungi (AMF), which take up mineral nutrients from the soil and exchange them with plants for photosynthetically fixed carbon. This exchange is a significant factor in global nutrient cycles as well as in the ecology, evolution and physiology of plants. Despite its importance as a nutrient, very little is known about how AMF take up nitrogen and transfer it to their host plants. Here we report the results of stable isotope labelling experiments showing that inorganic nitrogen taken up by the fungus outside the roots is incorporated into amino acids, translocated from the extraradical to the intraradical mycelium as arginine, but transferred to the plant without carbon. Consistent with this mechanism, the genes of primary nitrogen assimilation are preferentially expressed in the extraradical tissues, whereas genes associated with arginine breakdown are more highly expressed in the intraradical mycelium. Strong changes in the expression of these genes in response to nitrogen availability and form also support the operation of this novel metabolic pathway in the arbuscular mycorrhizal symbiosis.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                14 July 2020
                25 June 2020
                25 June 2020
                : 117
                : 28
                : 16649-16659
                Affiliations
                [1] aState Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University , 210095 Nanjing, China;
                [2] bKey Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University , 210095 Nanjing, China;
                [3] cInstitute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Sciences, Texas Tech University , Lubbock, TX 79409;
                [4] dLaboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica Avanzada del Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional , 36500 Irapuato, Mexico
                Author notes
                2To whom correspondence may be addressed. Email: chenaq8@ 123456njau.edu.cn , ghxu@ 123456njau.edu.cn , or luis.herrera-estrella@ 123456ttu.edu .

                Contributed by Luis Rafael Herrera-Estrella, May 13, 2020 (sent for review January 17, 2020; reviewed by Alain Gojon, Maria J. Harrison, and Ertao Wang)

                Author contributions: A.C., L.R.H.-E., and G.X. designed research; S.W., K.X., X.Y., Z.L., J.C., D.Z., Y.R., C.Y., L.W., H.F., and D.L.L.-A. performed research; A.C. and G.X. contributed new reagents/analytic tools; S.W., A.C., D.L.L.-A., L.R.H.-E., and G.X. analyzed data; and S.W., A.C., D.L.L.-A., L.R.H.-E., and G.X. wrote the paper.

                Reviewers: A.G., Institut National de la Recherche Agronomique Montpellier; M.J.H., Cornell University; and E.W., Chinese Academy of Sciences.

                1S.W. and A.C. contributed equally to this work.

                Author information
                https://orcid.org/0000-0002-1679-4226
                https://orcid.org/0000-0001-7389-3143
                https://orcid.org/0000-0001-7936-3856
                https://orcid.org/0000-0002-3283-2392
                Article
                202000926
                10.1073/pnas.2000926117
                7368293
                32586957
                cd3e2249-22de-4f3a-a97d-2391de324a21
                Copyright © 2020 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 11
                Funding
                Funded by: National Key Research and Developement Program/China
                Award ID: 2016YFD0100700
                Award Recipient : Aiqun Chen Award Recipient : Kun Xie Award Recipient : Xiaofeng Yang Award Recipient : Zhenzhen Luo Award Recipient : Jiadong Chen Award Recipient : Dechao Zeng Award Recipient : Yuhan Ren Award Recipient : Congfan Yang Award Recipient : Lingxiao Wang Award Recipient : Huimin Feng Award Recipient : Damar Lizbeth López-Arredondo Award Recipient : Luis Rafael Herrera-Estrella Award Recipient : Guohua Xu
                Funded by: National Science Foundation of China
                Award ID: 31572188
                Award Recipient : Aiqun Chen Award Recipient : Kun Xie Award Recipient : Xiaofeng Yang Award Recipient : Zhenzhen Luo Award Recipient : Jiadong Chen Award Recipient : Dechao Zeng Award Recipient : Yuhan Ren Award Recipient : Congfan Yang Award Recipient : Lingxiao Wang Award Recipient : Huimin Feng Award Recipient : Damar Lizbeth López-Arredondo Award Recipient : Luis Rafael Herrera-Estrella Award Recipient : Guohua Xu
                Funded by: National Science Foundation of China
                Award ID: 31372121
                Award Recipient : Aiqun Chen Award Recipient : Kun Xie Award Recipient : Xiaofeng Yang Award Recipient : Zhenzhen Luo Award Recipient : Jiadong Chen Award Recipient : Dechao Zeng Award Recipient : Yuhan Ren Award Recipient : Congfan Yang Award Recipient : Lingxiao Wang Award Recipient : Huimin Feng Award Recipient : Damar Lizbeth López-Arredondo Award Recipient : Luis Rafael Herrera-Estrella Award Recipient : Guohua Xu
                Categories
                Biological Sciences
                Plant Biology

                arbuscular mycorrhiza,rna sequencing,nitrate transporter,nitrogen uptake,osnpf4.5

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