ISRAEL JOURNAL OF PLANT SCIENCES, 64 (2017) 3-4 http://dx.doi.org/10.1080/07929978.2016.1246147

Comparisons of the High-affinity Nitrate Transporter Complex AtNRT2.1/AtNAR2.1 and the nidulans AnNRTA: structure function considerations

Zorica Kotura, Shiela E. Unklesb and Anthony D. M. Glassa aDepartment of Botany, University of British Columbia, Vancouver, BC, Canada; bSchool of Biology, University of St Andrews, St Andrews, UK

ABSTRACT ARTICLE HISTORY The high-affinity nitrate transporter of green plants is composed of two polypeptides, NRT2.1 and Received 10 July 2016 NAR2.1, while in fungi it appears that nitrate influx is mediated by NRT2 alone. Another difference Accepted 13 September 2016 between plants and fungi is that the central (cytoplasmic) loop of the 12 membrane spanning KEYWORDS regions of NRT is quite large in fungi, consisting of 91 amino acid residues, compared with the Nitrate uptake; plants; fungi; relatively short (21 amino acid residues) plant NRT2.1. Here we examine potential amino acid NRT2.1; NAR2.1 residues involved in the plant NRT2.1:NAR2.1 association by of conserved amino acids in Arabidopsis thaliana AtNRT2.1. Only the replacement of leucine 85 by glutamine disrupted the association between AtNRT2.1 and AtNAR2.1, as examined using the yeast two-hybrid system. Further, to investigate the nitrate-transporting function of AtNRT2.1 in a context free of other members of the NRT2 family, we expressed AtNRT2.1 in Aspergillus nidulans. In the fungal context the plant NRT alone was capable of restoring nitrate transport to a nitrate transport defective mutant, but only when the AtNRT2.1 central loop was replaced by its fungal counterpart.

Introduction (Cassman et al. 1998; Raun & Johnston 1999). In a review of nitrogen use efficiency (Glass 2003) it was After carbon, hydrogen, and oxygen, nitrogen is the stressed that several physiological characteristics of macronutrient required by plants in the greatest nitrate and ammonium uptake by plant roots lead amount, and healthy plants typically contain up to inevitably to the low efficiency of fertilizer capture by 3%–4% N by dry weight. Hence, in many non-agricul- crop plants and facilitate the losses described. These tural soils, where available nitrogen (N) is finite, N is include (1) N efflux (futile cycling between root and commonly the nutrient which most limits plant extracellular media), (2) down-regulation of the growth. To circumvent this limitation and sustain high uptake of both nitrate and ammonium as plant N rises crop yields under agricultural conditions, global towards its optimum (Rawat et al. 1999; Vidmar et al. annual N fertilizer application has grown steadily over 2000), (3) ammonium inhibition of nitrate uptake the last century to approximately 1011 kg, and FAO when both N forms are present (Lee & Drew 1989; (2015) has forecast that between 2014 and 2018 Kronzucker et al. 1999), (4) the diurnal “shutdown” of demand will increase at the rate of 1.8% per annum. N uptake at night (Clement et al. 1978), and (5) the Of the various forms of N fertilizer available, urea now low demand for N early in the season when plant size represents the most widely used. In soils, urea and is small, and low soil temperatures reduce rates of N other N forms (e.g. anhydrous ammonia and ammo- uptake (Siddiqi et al. 1990; Marschner 2012). Paradoxi- nium sulfate) are rapidly converted to ammonium and cally, then, at a time when fertilizer N is available in nitrate. Unfortunately, fertilizer losses due to volatiliza- excess, plant demand is at its lowest. Considering the tion, denitrification, and leaching (especially of nitrate) above, it is hardly surprising that fertilizer losses are so are substantial and represent a major concern high. If there are to be any future prospects for engi- from both economic and environmental perspectives neering better N use efficiency, perhaps through

CONTACT Anthony D. M. Glass [email protected] Supplemental data for this article (Supplemental Information SI1 and SI2) can be accessed here. This paper has been contributed in honor of Professor Uzi Kafkafi. © Koninklijke Brill NV, Leiden, 2017

Downloaded from Brill.com09/28/2021 02:02:36PM via free access 22 Z. KOTUR ET AL. molecular , much more must be understood may be converted from its monomeric (high-affinity) regarding the structure and regulation of plant nitrate form to a dimeric (low-affinity) form according to the and ammonium transport systems. The present paper state of phosphorylation of Thr 101. sequences focuses on recent findings regarding the structure of for members of the NRT2 and NRT1 families are unre- the Arabidopsis thaliana high-affinity nitrate trans- lated. Since these earlier discoveries, encoding porter and that of its fungal counterpart in Aspergillus HATS and LATS have been identified in many species nidulans from the laboratory of the senior author and including barley (Trueman et al. 1996), Nicotiana plum- his collaborators. baginofolia (Quesada et al. 1997; Krapp et al. 1998), Physiological studies of nitrate uptake by plant soybean (Amarasinghe at al. 1998), tomato (Ono et al. 14 ¡ roots using measurements of net nitrate ( NO3 ) 2000), rice (Araki & Hasegawa 2006; Miller et al. 2007; depletion from external solution, or by measuring Cai et al. 2008), wheat (Zhao et al. 2004; Cai et al. 15 ¡ 13 ¡ NO3 or NO3 accumulation in roots and shoots 2007; Yin et al. 2007), Physcomitrella patens (Tsujimoto after brief exposure to these tracers, demonstrate et al. 2007), and Lotus japonica (Criscuolo et al. 2012). ¡ that in higher plants there are two classes of nitrate In C. reinhardtii it was demonstrated that NO3 transporter responsible for nitrate accumulation transport activity by the high-affinity transporter (Glass 2009). High-affinity Transport Systems (HATS) CrNRT2.1 required the expression of a second, struc- ¡ operate at low external NO3 concentrations, and dis- turally unrelated gene, namely CrNAR2 (Galvan et al. play Michaelis–Menten kinetics, saturating at 1996; Galvan & Fernandez 2001). Mutants disrupted in »200 mM (Glass & Siddiqi 1995). These are the “work- CrNAR2 failed to absorb nitrate despite the presence horse” transporters that are largely responsible for of a WT CrNRT2.1. This genetic evidence of a require- root nitrate uptake. By contrast, Low-affinity Transport ment for NAR2 was confirmed using the Xenopus Systems (LATS) make significant contribution to total oocyte system, in which nitrate transport was only nitrate uptake only at elevated concentrations (Siddiqi observed when both CrNRT2.1 and CrNAR2 cDNA was ¡ et al. 1990). Moreover, NO3 influx through LATS fails co-injected (Zhou et al. 2000). Again using the Xeno- ¡ to saturate even at 50 mM external NO3 concentra- pus expression system, Tong et al. (2005) provided evi- tion (Siddiqi et al. 1990). Because of the presence of a dence that in barley too, functional expression of the strong electrical potential (up to 300 mV) across the barley NRT2.1 (HvNRT2.1) required coexpression of a ¡ root plasma membrane, NO3 uptake through both barley NAR2-like mRNA. In A. thaliana,asinChlamydo- HATS and LATS represents active (energy-requiring) monas, nitrate influx was reduced to a few percent of transport processes, fueled by the proton motive force WT values in mutants disrupted in the NAR2.1 gene (Glass et al. 1992). (Okamoto et al. 2006), and these mutants grew Beginning in the 1990s, genes encoding the HATS extremely poorly when grown on micromolar concen- were first identified in the Aspergillus nidulans trations of nitrate. Consistent with the proposed role (Unkles et al. 1991) and soon afterwards in the single- for NRT1.1, at high nitrate concentrations, the same celled green alga Chlamydomonas reinhardtii (Galvan mutants grew normally on 2.5 mM nitrate (Okamoto et al. 1996), and in the higher plants barley (Trueman et al. 2006). Subsequently, nitrate uptake by several et al. 1996) and Arabidopsis thaliana (Zhuo et al. 1999). other plants has been demonstrated to depend upon Genes encoding the high-affinity transporters from all expression of both the NRT2.1 and NAR2.1 homo- of these species belong to a family named NRT2 (an logues. These include rice (Araki & Hasegawa 2006; abbreviation for Nitrate Transporter). In Arabidopsis Cai et al. 2008; Yan et al. 2011), wheat (Triticum aesti- there are seven different NRT2 genes, of which NRT2.1 vum) (Cai et al. 2007), and the moss Physcomitrella pat- has been shown to be largely responsible for nitrate ens (Tsujimoto et al. 2007). Indeed, using the Y2H and uptake from low concentration external sources the Xenopus systems, Kotur and Glass demonstrated (Filleur et al. 2001; Okamoto et al. 2003; Li et al. 2007). that all of the Arabidopsis NRT2s (except for NRT2.7) A gene encoding the LATS was cloned from A. thali- require interaction with NAR2.1 for nitrate transport ana (Tsay et al. 1993) and was initially designated (Kotur et al. 2012). CHL1, subsequently NRT1.1, and is presently desig- A partial explanation of the apparently obligate nated NPF6.3. Sun et al. (2014) have reported on the requirement for NAR2.1 expression was provided by crystal structure of NRT1.1, suggesting that the latter Yong et al. (2010), who used blue native gel

Downloaded from Brill.com09/28/2021 02:02:36PM via free access ISRAEL JOURNAL OF PLANT SCIENCES 23 electrophoresis (BNGE) and anti-NRT2.1 and anti- AtNRT2.1 mutants altered in conserved amino acids NAR2.1 antibodies to identify a 150-kDa plasma mem- and tested for their capacity to associate with brane complex from the roots of A. thaliana. AtNAR2.1 in the yeast two-hybrid system (Yong et al. This complex was absent from NRT2.1 mutants as well 2010). The rationale for this approach was to attempt as from NAR2.1 mutants, but was restored when these to disrupt the normal association between NRT2.1 mutants were transformed with corresponding and NAR2.1 by mutating conserved amino acids in AtNRT2.1 or AtNAR2.1 cDNAs. This 150-kDa complex AtNRT2.1. This might identify amino acids involved in was dissociated to yield AtNRT2.1 and AtNAR2.1 subu- the association. While this might have been under- nits when following BNGE, SDS–PAGE was employed taken in planta, by transforming mutants lacking WT for the second-dimension electrophoresis (Yong et al. NRT2.1 with mutant NRT2.1and examining for disrup- 2010). Based upon the molecular masses of AtNRT2.1 tion of nitrate uptake and/or for absence of the 150- and AtNAR2.1 (48 and 26 kDa, respectively), it was kDa complex, these assays would require lengthy proposed that the functional A. thaliana high-affinity transformations and procedures that could require a nitrate transporter is made up of two subunits each of year to generate testable plants. By contrast, the yeast AtNRT2.1 and AtNAR2.1 (Yong et al. 2010). Interest- two-hybrid (Y2H) assay for association among mem- ingly, although mRNA encoding AtNRT2.1 is present brane polypeptides can be completed within a few in the NAR2.1 mutants, the corresponding protein is days. Hence the latter system was employed to iden- absent (Wirth et al. 2007; Yong et al. 2010). Thus, it tify disruptions of the WT NRT2.1/NAR2.1 association. may be that unless the NAR2.1 polypeptide is avail- The second approach was to attempt to express able to generate the 150-kDa NAR2.1/NRT2.1 complex, AtNRT2.1/NAR2.1in A. nidulans. Because of the rapid the NRT2.1 protein is degraded. growth of the fungus, we sought to generate suffi- Unlike the plant high-affinity transporters (includ- cient quantities of the complex for further biophysical ing that of the green alga C. reinhardtii), the corre- analysis as well as to assay mutated versions in hours sponding fungal transporter (named NRTA) appears rather than months. to function without the necessity of a second (NAR2.1-type) polypeptide. This conclusion is arrived Materials and methods at through two lines of evidence. Firstly, when the fun- Fungal strains gal gene is expressed in Xenopus oocytes, nitrate transport was demonstrated when NRTA cDNA alone Aspergillus nidulans strains used in this study, wild- was injected into oocytes (Zhou et al. 2000). Secondly, type biA1, and the double deletion mutant BNGE of A nidulans plasma membrane (PM) polypepti- nrtA747nrtB110 (disrupted in both nrtA and nrtB des revealed no higher molecular weight complex genes, were described earlier by Unkles et al. (2001). comparable to that of A. thaliana, but only a mono- meric 46-kDa NRTA (Unkles, unpublished). Another Aspergillus nidulans transformation difference between the plant NRT2.1 and the fungal Details of the selection strategy and transformation NRTA concerns the length of the hydrophilic cyto- procedure for A. nidulans were described previously plasmic loop between transmembrane regions (TMR) (Riach & Kinghorn 1996). For direct selection on mini- 6 and 7. NRT polypeptides of both plants and fungi mal medium containing nitrate as the sole nitrogen traverse the PM lipid bilayer 12 times. In A. thaliana source, strain T110 (nrtA747 nrtB110) was used as the loop that extends into the cytoplasm from recipient, and for indirect selection on the basis of between TMR 6 and 7 is quite short (21 amino acids) arginine prototrophy, strain JK1060 (nrtA747 nrtB110 compared to the fungal counterpart (91 amino acids argB2) was the recipient (Unkles et al. 2001). Detailed in A. nidulans). Up to the present time there appears description of A. nidulans protoplast transformation to be no explanation for the differences between protocol can be found in Kotur (2013). fungi and plants with respect to loop length, or of the requirement for NAR2 in plants. Generation of fungal expression constructs In order to further examine the relationship between NRT2.1 and NAR2.1, we have employed two A plasmid for nitrate inducible expression of AtNRT2.1 approaches. We have generated a large number of was generated by amplification of the coding region

Downloaded from Brill.com09/28/2021 02:02:36PM via free access 24 Z. KOTUR ET AL. from Arabidopsis root cDNA to create a fragment with cDNA of AtNRT2.1 gene was cloned into pDL2Nx prey EcoRI ends, which was cloned into vector pMUT vector using BamHI and EcoRI restriction sites, in (Unkles et al. 2005) such that the coding region was frame with N-Ubiqutin. Detailed protocol of Y2H under the control of the A. nidulans nrtA promoter screening is available in Kotur et al. (2012). Site- and terminator. Plasmids for nitrate-inducible coex- directed mutagenesis of pDL2NxNRT2.1 was done pression of the AtNRT2.1 and AtNAR2.1 were gener- using a QuikChange II site-directed mutagenesis kit ated in the twin reporter vector pTRAN3-1 (Punt et al. (Agilent Technologies, TX, USA). Sequences of oligo- 1991) such that the AtNRT2.1 coding region was under nucleotide primers used to clone NRT2.1 and the control of the niiA promoter and the AtNAR2.1 AtNAR2.1, as well as primers used for site-directed under the control of the niaD promoter in plasmid mutagenesis, are shown in Tables 4, 5 and 6. All Y2H pATP. Plasmid pATP2 contained the genes in tests were done in two different strains of yeast, DSY1 exchanged order with respect to the promoters. For and NMY51 (Dualsystems Biotech AG), and included expression of the chimeric protein, the coding region multiple markers for detection of interaction of the of AtNRT2.1 with EcoRI ends was cloned into pUC8. tested (-histidine and -adenine growth, as EcoRV and XhoI sites were introduced by polymerase well as b-galactosidase activity). Conserved NRT2.1 chain reaction (PCR) overlap extension (Warrens et al. amino acids were chosen for based on 1997) in the regions encoding the N- and C-terminal MUSCLE (Edgar 2004) multiple protein sequence ends, respectively, of the predicted loop 6/7 between alignment of 110 NRT2 sequences (dicots, monocots, transmembrane domains 6 and 7. The DNA encoding and C. reinhardtii; see Supplemental Material). Rules A. nidulans loop 6/7 was amplified by PCR with EcoRV for amino acid (aa) changes were: charged aa (positive and XhoI ends and cloned in replacement of the and negative) to polar neutral, hydrophobic to polar AtNRT2.1 loop 6/7 region. The EcoRI fragment was (uncharged), polar (uncharged) to small hydrophobic, cloned into pMUT such that the recombinant coding possible phosphorylation sites to neutral polar. region was under the control of the A. nidulans nrtA promoter and terminator to give plasmid AtNRT2.1- Results and discussion AnLoop. Expression of the resulting protein would result Y2H testing of AtNRT2.1 mutants in a chimera composed of AtNRT2.1 N-terminal region up to and including residue D248 and the C-terminal Based on multiple sequence alignment of NRT2.1 pro- region from F268 (inclusive), encompassing 91 residues teins from different plant species (see Supplemental of the predicted NRTA loop 6/7 from residues P223 to Information SI1), conserved amino acids of AtNRT2.1 S313 inclusive. In addition, the final cloning introduced were selected, and using site-directed mutagenesis, a sequence encoding a V5 epitope tag fusion to the C- changed into different amino acids as shown in terminus of the protein. PCR-amplified regions of all Tables 1–3. Positions of amino acids were estimated plasmids were verified by DNA sequencing of the based on NRT2.1 topology prediction HMMTOP server amplified region and covering the cloning sites. by Tusnady and Simon (1998) (Supplemental Informa- tion SI2). A single change in the hydrophobic regions 13 NO3¡ influx in A. nidulans of NRT2.1 resulted in disruption of the interaction between NAR2.1 and NRT2.1, and that was the muta- Growth of strains and assay of 13N-nitrate influx at the tion of leucine at position 85 to glutamine (L895Q), standard concentration range for high-affinity trans- localized in the first putative TMR. None of the other port (10–250 mM) were as detailed in Unkles et al. 44 mutations of the predicted hydrophobic trans- (2004). membrane regions of NRT2.1 (Table 1) succeeded in disrupting the association between NRT2.1 and Membrane yeast-two-hybrid screening for NAR2.1. Likewise, none of the 14 mutations of amino interaction of AtNRT2.1 gene with AtNAR2 as bait acids in the predicted central hydrophilic loop region Membrane Y2H screening for interactions of AtNRT2.1 of NRT2.1 (Table 2) or the 21 mutations of amino acids with AtNAR2.1 was done using the DUAL membrane in the small extracellular hydrophilic loop regions of kit from Dualsystems Biotech AG (Switzerland). cDNA NRT2.1 (Table 3) disrupted the interaction in the Y2H of AtNAR2.1 was cloned into Y2H bait vector pTMBV4. assays.

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Table 1. Screening for interactions of mutated NRT2.1 with Table 2. Screening for interactions of mutated NRT2.1 with AtNAR2.1 as bait in the yeast two-hybrid system. Mutations of AtNAR2.1 in the yeast two-hybrid system. Mutations of amino amino acids are in the predicted hydrophobic transmembrane acids are in the predicted central hydrophilic loop region of regions of NRT2.1. Protein topology predicted by HMMTOP NRT2.1. Protein topology predicted by HMMTOP (Tusnady & (Tusnady & Simon 1998; Tusnady & Simon 2001) Simon 1998; Tusnady & Simon 2001). Growth Assay Growth Assay Amino acid change -HIS-LEU-TRP b-galactosidase Amino acid change -HIS-LEU-TRP b-galactosidase 1 F72Q CC C 1 G246A CC C 2 L85Q ––2 D248N CC C 3 P87G CC C 3 P250V CC C 4 I89Q CC C 4 D251N CC C 5 G101L CC C 5 G252A CC C 6 G110L CC C 6 G261A CC 7 M118N CC C 7 D266N CC C 8 G162L CC C 8 K265N CC C 9 V169N CC C 9 K270N CC C 10 W174Q CC10 D248N-D251N CC C 11 G193L CC C 11 D248N-D266N CC C 12 G195L CC C 12 D251N-D266N CC C 13 G198A-G199A-G200A CC C 13 K265N-K270N CC C 14 P207G CC C 14 D265N-D266N-K270N CC C 15 A227N CC C 16 P231G CC C 17 Y279F CC C fi 18 R280Q CC C position 85 to glutamine (L85Q), localized in the rst 19 Y288F CC C putative transmembrane region, disrupted the 20 G289L CC C 21 A317N CC C NRT2.1/NAR2.1 association suggests that (apart from 22 G318L CC C L85) none of the amino acid substitutions we gener- 23 G325L CC C 24 N328A CC C ated disrupted the capacity of the mutant AtNRT2.1 to 25 R332Q CC C associate with AtNAR2.1. This observation is perplex- 26 Q359A CC C 27 S386A CC C ing, unless there are sufficient other remaining 28 G394L CC C 29 G409L CC C AtNRT2.1 WT residues to participate in the AtNRT2.1: 30 S412A CC C AtNAR2.1 association. Hence, we might conclude that 31 G416L CC C 32 G418A-G419A CC C no one particular pair of residues is critical to the asso- 33 G422L CC C ciation, but rather AtNRT2.1 and AtNAR2.1 are held by 34 T426V CC C 35 Q427A CC C multiple non-covalent associations. Disrupting one 36 M445N CC 37 G446L CC C 38 C366A CC C 39 F159Q CC C Table 3. Screening for interactions of mutated NRT2.1 with 40 G119L CC C AtNAR2.1 in the yeast two-hybrid system. Mutations of amino 41 A103N CC C acids are in the small extracellular hydrophilic loop regions of 42 N278A CC C NRT2.1. Protein topology predicted by HMMTOP (Tusnady & 43 F431Q CC C  44 A391N CC C Simon 1998; Tusnady & Simon 2001). 45 G293L CC C Growth Assay Amino acid change -HIS-LEU-TRP b-galactosidase 1 R90N CC 2 E91N CC Given that the association between AtNRT2.1 and 3 L93Q CC AtNAR2.1 is so critical for nitrate transport and, 4 L95Q CC 5 R158N CC indeed, for the apparent protection of AtNRT2.1 from 6 G217A CC degradation, it was hypothesized that at least some of 7 A223N CC 8 W224N CC the conserved residues among AtNRT2.1 might be 9 R225N CC involved in the linkage(s) responsible for the associa- 10 E295N CC 11 N300A CC tion of AtNRT2.1 and AtNAR2.1. A priori, we considered 12 Y305Q CC CC that if, for example, a salt bridge (negative acidic to 13 D308N 14 F310G CC positive basic R-group association) was important in 15 G370A CC 16 T374N CC the non-covalent linkages between AtNRT2.1 and 17 G441A CC AtNAR2.1, then replacement of an acidic R-group by, 18 R90N - E91N CC 19 L93Q - L95Q CC for example, a basic residue might disrupt this linkage. 20 W224N - R225N CC The finding that only the mutation of leucine at 21 E295N - D308N CC

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Table 4. Sequences of oligonucelotides used for site-directed mutagenesis of NRT2.1 amino acids in the predicated small hydrophilic loop regions. NRT2.1 amino acid change Primer name Primer sequence (5' to 3') R90N c268a_g269a_g270t_ 5'-tttggtgaggttgagattctcattgatgatagggacaagtggtgc-3' 5'-gcaccacttgtccctatcatcaatgagaatctcaacctcaccaaa-3 E91N g271a_g273t_ 5'-gtttggtgaggttgagattattccggatgatagggacaagt-3' 5'-acttgtccctatcatccggaataatctcaacctcaccaaac-3' L93Q t278a_c279g_ 5'-gtttggtgaggttgagattattccggatgatagggacaagt-3' 5'-acttgtccctatcatccggaataatctcaacctcaccaaac-3' L95Q t284a_c285g_ 5'-tgtcttgtttggtgaggttctgattctcccggatgatagg-3' 5'-cctatcatccgggagaatcagaacctcaccaaacaagaca-3' R158N g473a_g474t_ 5'-ttccaatgtcttgtttggtctggttgagattctcccggat-3' 5'-atccgggagaatctcaaccagaccaaacaagacattggaa-3' G217A g650c_ 5'-tgaaggctgtggaagcgcagcgcctaatg-3' 5'-cattaggcgctgcgcttccacagccttca-3' A223N g667a_c668a_ 5'-gaaggcgatcctccagttcgtgaaggctgtggaa-3' 5'-ttccacagccttcacgaactggaggatcgccttc-3' W224N t670a_g671a_g672t_ 5'-gtacaaagaaggcgatcctattggccgtgaaggctgtggaa-3' 5'-ttccacagccttcacggccaataggatcgccttctttgtac-3' R225N g674a_g675t_ 5'-ggtacaaagaaggcgatattccaggccgtgaaggct-3' 5'-agccttcacggcctggaatatcgccttctttgtacc-3' E295N g883a_g885t_ 5'-attatcagtgctcaaattaactcccatggagtatccgtagagaaga-3' 5'-tcttctctacggatactccatgggagttaatttgagcactgataat-3' N300A a898g_a899c_ 5'-agtactcggcgataacagcatcagtgctcaactcaactccc-3' 5'-gggagttgagttgagcactgatgctgttatcgccgagtact-3' Y305Q t913c_c915g_ 5'-caagtgaaacctgtcaaagaactgctcggcgataacattatcagt-3' 5'-actgataatgttatcgccgagcagttctttgacaggtttcacttg-3' D308N g922a_ 5'-caagtgaaacctgttaaagaagtactcggcgataacattatc-3' 5'-gataatgttatcgccgagtacttctttaacaggtttcacttg-3' F310G t928g_t929g_ 5'-cgagtacttctttgacaggggtcacttgaagctccacaca-3' 5'-tgtgtggagcttcaagtgacccctgtcaaagaagtactcg-3' G370A g1109c_ 5'-gtgttggcgcgggcgagccacacac-3' 5'-gtgtgtggctcgcccgcgccaacac-3' T374N c1121a_ 5'-gcagttacaaggttgttggcgcggccga-3' 5'-tcggccgcgccaacaaccttgtaactgc-3' G441A g1322c_ 5'-ctcccatccacgttagcgcttgttcagttgtgaag-3' 5'-cttcacaactgaacaagcgctaacgtggatgggag-3' R90N & E91N c268a_g269a_g270t_ g271a_g273t_ 5'-atgtcttgtttggtgaggttgagattattattgatgatagggacaagtggtgcagctg 5'-cagctgcaccacttgtccctatcatcaataataatctcaacctcaccaaacaagacat L93Q & L95Q t278a_c279g_t284a_ c285g_ 5'-gtttccaatgtcttgtttggtctggttctgattctcccggatgatagggac-3' 5'-gtccctatcatccgggagaatcagaaccagaccaaacaagacattggaaac-3’ W224N & R225N t670a_g671a_g672t _g674a_g675t_ 5'-accgggtacaaagaaggcgatattattggccgtgaaggctgtggaacc-3' 5'-ggttccacagccttcacggccaataatatcgccttctttgtacccggt-3’ such pair would leave intact other amino acid:amino made use of the double deletion mutant nrtA747 acid associations. Alternatively (apart from L85), all of nrtB110 (disrupted in both nrtA and nrtB genes) that is the conserved residues we substituted may be incapable of growth on nitrate as the sole source of N involved, not in the association between AtNRT2.1 (Unkles et al. 2001). Transformants were incapable of and AtNAR2.1, but as critical for nitrate transport per growth on nitrate as the sole source of N, indicating se. The latter hypothesis might be tested by trans- that neither AtNRT2.1 alone nor in combination with forming a NRT2.1-null mutant using our amino acid AtNAR2.1 was able to rescue the fungal double dele- substitution mutations. The resulting lines could then tion mutant. Next, the AtNRT2.1 central cytoplasmic be tested for nitrate uptake directly as well as examin- loop (between TMR 6 and 7) was replaced by the ing PM preparations for the presence of the 150-kDa much larger A. nidulans loop to generate AtNRT2.1– complex. Interestingly, Leu 85 is also present in the AnLoop. The nrtA747 nrtB110 line was transformed Aspergillus NRTA. Therefore, it may be that this con- with this modified AtNRT12.1–AnLoop. Resulting A. served amino acid serves a function in nitrate trans- nidulans transformants were found to grow success- port as well as in the association with NAR2.1. fully on nitrate as the sole N source, and Western blot analysis using appropriate antibody confirmed that the chimeric protein AtNRT2.1–AnLoop was expressed Expression of AtNRT2.1 in A. nidulans in the membranes of A. nidulans. We had earlier fl In order to attempt to express AtNRT2.1, either alone (Unkles et al. 2001) examined nitrate in ux in WT and or together with AtNAR2.1, in Aspergillus nidulans,we the double deletion mutants and demonstrated the

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Table 5. Sequences of oligonucelotides used for site-directed mutagenesis of NRT2.1 amino acids in the predicated central hydrophilic loop region. NRT2.1 amino acid change Primer name Primer sequence (5' to 3') G246A g737c_ 5'-catctggcagatcttgagctagattgagcaccaag-3' 5'-cttggtgctcaatctagctcaagatctgccagatg-3' D248N g742a_ 5'-catctggcagattttgacctagattgagcaccaag-3' 5'-cttggtgctcaatctaggtcaaaatctgccagatg-3' P250V c748g_c749t_ 5'-ggtagctcgatttccatctaccagatcttgacctagattg-3' 5'-caatctaggtcaagatctggtagatggaaatcgagctacc-3' D251N g751a_ 5'-ggtagctcgatttccatttggcagatcttgaccta-3' 5'-taggtcaagatctgccaaatggaaatcgagctacc-3' G252A g755c_ 5'-ccaaggtagctcgatttgcatctggcagatcttga-3' 5'-tcaagatctgccagatgcaaatcgagctaccttgg-3' G261A g782c_ 5'-gtctttggcaacttctgccgctttctccaaggt-3' 5'-accttggagaaagcggcagaagttgccaaagac-3' D266N g796a_ 5'-gaatctttccgaatttgtttttggcaacttctcccgctttc-3' 5'-gaaagcgggagaagttgccaaaaacaaattcggaaagattc-3' K265N a795t_ 5'-tctttccgaatttgtcattggcaacttctcccgc-3' 5'-gcgggagaagttgccaatgacaaattcggaaaga-3' K270N g810t_ 5'-gccaaagacaaattcggaaatattctgtggtatgccgtta-3' 5'-taacggcataccacagaatatttccgaatttgtctttggc-3' D248N & D251N g742a_g751a_ 5'-cttggtgctcaatctaggtcaaaatctgccaaatggaaatcgag-3' 5'-ctcgatttccatttggcagattttgacctagattgagcaccaag-3' K265N & K270N a795t_g810t_ 5’ gcgggagaagttgccaatgacaaattcggaaatattctgtggtatgccgtta-3’ 5’ taacggcataccacagaatatttccgaatttgtcattggcaacttctcccgc-3’ K265N & D266N & K270N 5’ gcgggagaagttgccaataacaaattcggaaatattctgtggtatgccgtta-3’

Table 6. Sequences of oligonucelotides used for site-directed mutagenesis of NRT2.1 amino acids in the predicated transmembrane regions. NRT2.1 amino acid change Primer name Primer sequence (5' to 3') F72Q t214c_t215a_c216g_ 5'-agacaaaacatgtggactgagagatccacgagagatggaacgttct-3' 5'-agaacgttccatctctcgtggatctctcagtccacatgttttgtct-3' L85Q t254a_t255g_ 5'-tcgcagctgcaccacaggtccctatcatccgg-3' 5'-ccggatgatagggacctgtggtgcagctgcga-3' P87G c259g_c260g_ 5'-gattctcccggatgataccgacaagtggtgcagctg-3' 5'-cagctgcaccacttgtcggtatcatccgggagaatc-3' I89Q a265c_t266a_c267g_ 5'-gaggttgagattctcccgctggatagggacaagtggtgc-3' 5'-gcaccacttgtccctatccagcgggagaatctcaacctc-3' G101L g301t_g302t_ 5'-caactccggcgtttaaaatgtcttgtttggtgaggttgaga-3' 5'-tctcaacctcaccaaacaagacattttaaacgccggagttg-3' A103N g307a_c308a_ 5'-agagacagaggcaactccgttgtttccaatgtcttgtttg-3' 5'-caaacaagacattggaaacaacggagttgcctctgtctct-3' R115Q a343c_g344a_ 5'-tcccatcacgagctgagagaagatactcccagagacaga-3' 5'-tctgtctctgggagtatcttctctcagctcgtgatggga-3' M118N t353a_g354t_ 5'-acacacggctccattcacgagcctagagaagatactc-3' 5'-gagtatcttctctaggctcgtgaatggagccgtgtgt-3' G119L g355t_g356t_ 5'-caaaagatcacacacggctaacatcacgagcctagagaag-3' 5'-cttctctaggctcgtgatgttagccgtgtgtgatcttttg-3' F159Q t475c_t476a_c477g_ 5'-gccaggcaaaaaccaatcatctgcctcaccgttatgaagcctg-3' 5'-caggcttcataacggtgaggcagatgattggtttttgcctggc-3' G162L g484t_g485t_t486a_ 5'-acacaaacgtcgccaggcaaaataaaatcatgaacctcaccgttatg-3' 5'-cataacggtgaggttcatgattttattttgcctggcgacgtttgtgt-3' V169N g505a_t506a_g507t_ 5'-gctcatccagtattgacaagaattaaacgtcgccaggcaaaaaccaat-3' 5'-attggtttttgcctggcgacgtttaattcttgtcaatactggatgagc-3' W174Q t520c_g521a_ 5'-gacgtttgtgtcttgtcaataccagatgagcactatgttcaacagt-3' 5'-actgttgaacatagtgctcatctggtattgacaagacacaaacgtc-3' G193L g577t_g578t_ 5'-cacccatgtttccccataaggctgctgtcccattca-3' 5'-tgaatgggacagcagccttatggggaaacatgggtg-3' G195L g583t_g584t_ 5'-ccgccacccatgtttaaccatccggctgctgt-3' 5'-acagcagccggatggttaaacatgggtggcgg-3' G198–199–200A g593c_g596c_g599c_ 5'-agcaactgcgttatggcggcagccatgtttccccatc-3' 5'-gatggggaaacatggctgccgccataacgcagttgct-3' P207G c619g_c620g_ 5'-ctaatgatttcatacacaatgcccatgagcaactgcgttatgcc-3' 5'-ggcataacgcagttgctcatgggcattgtgtatgaaatcattag-3' A227N g679a_c680a_ 5'-accgggtacaaagaagttgatcctccaggccgtg-3' 5'-cacggcctggaggatcaacttctttgtacccggt-3' P231G c691g_c692g_ 5'-gcaaccaaccgcctacaaagaaggcgatcctcc-3' 5'-ggaggatcgccttctttgtaggcggttggttgc-3' (continued)

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Table 6. (Continued ) NRT2.1 amino acid change Primer name Primer sequence (5' to 3') Y279F a836t 5'-cgaagatccaagtcctgaagtttgtaacggcatac-3' 5'-gtatgccgttacaaacttcaggacttggatcttcg-3' R280Q a838c_g839a_ 5'-gaagaacgaagatccaagtctggtagtttgtaacggcatacc-3' 5'-ggtatgccgttacaaactaccagacttggatcttcgttcttc-3' Y288F a863t 5'-atggagtatccgaagagaagaacgaagatccaagtc-3' 5'-gacttggatcttcgttcttctcttcggatactccat-3' G289L g865t_g866t_ 5'-actcaactcccatggagtataagtagagaagaacgaagatcc-3' 5'-ggatcttcgttcttctctacttatactccatgggagttgagt-3' G293L g877t_g878t_ 5'-ttatcagtgctcaactcaactaacatggagtatccgtagagaag-3' 5'-cttctctacggatactccatgttagttgagttgagcactgataa-3' A317N g949a_c950a_a951t_ 5'-gctgctatgagcccatttgtgtggagcttcaagtgaaacctgtc-3' 5'-gacaggtttcacttgaagctccacacaaatgggctcatagcagc-3' G318L g952t_g953t_g954a_ 5'-tccgaaacatgctgctatgagtaatgctgtgtggagcttcaagtg-3' 5'-cacttgaagctccacacagcattactcatagcagcatgtttcgga-3' G325L g973t_g974t_ 5'-caaagaaattggccattaagaaacatgctgctatgagccctg-3' 5'-cagggctcatagcagcatgtttcttaatggccaatttctttg-3' N328A a982g_a983c_ 5'-gctggacgagcaaagaaagcggccattccgaaacatgc-3' 5'-gcatgtttcggaatggccgctttctttgctcgtccagc-3' R332Q g995a_t996g_ 5'-gcgtagcctcctgctggctgagcaaagaaattggcc-3' 5'-ggccaatttctttgctcagccagcaggaggctacgc-3' Q359A c1075g_a1076c_ 5'-ccaccagccgtcgctatgatccacaacgtccacaac-3' 5'-gttgtggacgttgtggatcatagcgacggctggtgg-3' C366A t1096g_g1097c_ 5'-ggccgagccacacagcgaagaggccaccag-3' 5'-ctggtggcctcttcgctgtgtggctcggcc-3' S386A t1156g 5'-cttgtgcccccatagcgaagagcaccatagc-3' 5'-gctatggtgctcttcgctatgggggcacaag-3' A391N g1171a_c1172a_ 5'-gtggctccgcaagcattttgtgcccccatagagaag-3' 5'-cttctctatgggggcacaaaatgcttgcggagccac-3' G394L g1180t_g1181t_ 5'-caattgcaaaggtggctaagcaagcagcttgtgccc-3' 5'-gggcacaagctgcttgcttagccacctttgcaattg-3' G409L g1225t_g1226t_c1227a_ 5'-cggttaaacccgagatgattaatagagctcgccgggagacaaag-3' 5'-ctttgtctcccggcgagctctattaatcatctcgggtttaaccg-3' S412A t1234g_ 5'-cccggttaaacccgcgatgatgcctagagct-3' 5'-agctctaggcatcatcgcgggtttaaccggg-3' G416L g1246t_g1247t_g1248a_ 5'-gatccaaagttccctccagctaaggttaaacccgagatgatgcc-3' 5'-ggcatcatctcgggtttaaccttagctggagggaactttggatc-3' G418–419A g1253c_g1256c_ 5'-cctgatccaaagttcgctgcagccccggttaaac-3' 5'-gtttaaccggggctgcagcgaactttggatcagg-3' G422L g1264t_g1265t_ 5'-gtgagccctgataaaaagttccctccagccccg-3' 5'-cggggctggagggaactttttatcagggctcac-3' T426V a1276g_c1277t_ 5'-cgagaagaagaggagttgtacgagccctgatccaaagttc-3' 5'-gaactttggatcagggctcgtacaactcctcttcttctcg-3 Q427A c1279g_a1280c_ 5'-gaagaagaggagtgctgtgagccctgatccaaagttccc-3' 5'-gggaactttggatcagggctcacagcactcctcttcttc-3' F431Q t1291c_t1292a_c1293g_ 5'-gttgtgaagtgtgaggtcgactggaagaggagttgtgtgagcc-3' 5'-ggctcacacaactcctcttccagtcgacctcacacttcacaac-3' M445N t1334a_g1335t_ 5'-gcgactatcatcactccattccacgttagcccttgttc-3' 5'-gaacaagggctaacgtggaatggagtgatgatagtcgc-3' G446L g1336t_g1337t_ 5'-gcaagcgactatcatcactaacatccacgttagcccttgt-3' 5'-acaagggctaacgtggatgttagtgatgatagtcgcttgc-3'

inability of the double nrtA nrtB mutant to take up Michaelis–Menten curve gave an r2 value of 0.86. – nitrate. The AtNRT2.1 AnLoop was able to comple- These results indicate that NRT2.1 can function as an ment the double mutant phenotype, enabling it to effective nitrate transporter even in the absence of 13 ¡ grow on nitrate as the sole source of N, and NO3 NAR2.1, when its short cytoplasmic loop (between fl in ux measurements of Aspergillus double mutant TMR6 and 7) is replaced by the longer Aspergillus loop. – strain expressing AtNRT2.1 AnLoop gave values that The further implication of this observation is that, not- were close to those of WT. Figure 1 shows data from withstanding the usual in planta requirement for one such experiment that generated Km and Vmax NAR2.1, the existing structure of the 12 transmem- § m § ¡1 values of 14.4 4 M and 789.4 47 nmol mg DW brane sequences of NRT2.1 alone contains all the ¡1 h , respectively. These values are extremely close to required information for effective nitrate transport. To those reported for WT A. nidulans in Unkles et al. eliminate the possibility that growth was due to fi (2001). A direct t of these data to a hyperbolic recombination between the fungal mutant nrta

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In summary, we have demonstrated that only one amino acid of the large number examined (80 in total), namely L85, appears to be critical for the NRT2.1/ NAR2.1 association. We have demonstrated that NRT2.1 alone is responsible for nitrate uptake, but we are as yet unable to identify the precise role of NAR2.1.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This work was supported by the Natural Sciences and Engineering 13 ¡ Figure 1. NO3 influx in Aspergillus nidulans double mutant Research Council of Canada (NSERC) [grant number A0570]. nrtB110 nrtA747 expressing AtNRT2.1–AnLoop. Values are average of n D 5 § SD. The fitted line is a direct Michaelis–Menten fit, with ¡1 ¡1 estimated vales for Km (mM) and Vmax (nmol mg DW h ). References

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