Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

Abstract

Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (K m = 4.7 µM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate-(or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.

Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

“…In Arabidopsis, 10 lM chlorate produced sensitivity, as expected for the high-affinity phase of CHL1, while the chl1-5 mutant was tolerant (Liu et al 1999). This capacity to transport chlorate has not been observed for either Ynt1 or T. borchii high-affinity nitrate transporter TbNRT2 expressed in H. polymorpha (Machin et al 2004;Montanini et al 2006). CHL1 functional expression was consistently observed in about 30% of the transformants analysed.…”

“…In this way, Nia2 tobacco nitrate reductase has been successfully produced in this yeast (Perdomo et al 2002). Likewise, a putative nitrate transporter from the mycorrhizal ascomycete Tuber borchii was characterized as a high-affinity nitrate transport system (Montanini et al 2006). However, plant nitrate transporters have so far not been functionally expressed in any yeast.…”

Gene expression profiling of the nitrogen starvation stress response in the mycorrhizal ascomycete Tuber borchii

“…Most of them display the same kind of N status responsiveness previously documented in non-mycorrhizal fungi (Magasanik and Kaiser, 2002;Marzluf, 1997). The main exceptions are the lack of Nmediated repression for NADP-glutamate dehydrogenase in Tuber borchii (Montanini et al, 2003) and the nitrate-independent upregulation of nitrate assimilation components in both Hebeloma cylindrosporum (Jargeat et al, 2000(Jargeat et al, , 2003 and T. borchii (Montanini et al, 2006). Additional genes coding for N metabolism components have been uncovered in the course of microarray studies aimed at exploring gene expression variations accompanying mycorrhiza (Morel et al, 2005;Podila et al, 2002;Wright et al, 2005) or fruitbody formation (Lacourt et al, 2002).…”

“…Table 1 Arrayed cDNAs coding for proteins directly involved in N metabolism a GenBank annotation of the best reciprocal hits retrieved from BLASTX analysis (E 6 10 ¡5 ). b Experimentally validated function: TbAMT1 (Montanini et al, 2002); TbGS (Montanini et al, 2003); TbGDH ; TbNrt2 (Montanini et al, 2006). c Functional classiWcation according to Gene ontology, 'biological process' category (Ashburner et al, 2000).…”

Down-regulation of Eukaryotic Nitrate Transporter by Nitrogen-dependent Ubiquitinylation

“…1A). In all these transporters, Ynt1, NrtA, NrtB, TbNrt2, (32) and NIT10, this domain contains regions resembling PEST sequences, and in the case of NIT10 the PESTfind algorithm (33) defines a proper PEST sequence. In the PEST-like sequences of Ynt1 and fungal nitrate transporters the numerous Lys, Ser and Thr residues are remarkable, which could constitute ubiquitinylation and phosphorylation sites.…”