Study of Nitrogen Assimilation in Ectomycorrhiza by RNAi-based Gene Silencing: The Role of the Laccaria Bicolor Nitrate Transporter

Abstract

In soil N is present in inorganic forms as ammonium (NH4+) and nitrate (NO3-), and as organic compounds such as amino acids, peptides and proteins. However, availability of N appears to be a limiting factor for forest tree growth in most temperate and boreal ecosystems where a significant pool of N is present in complex organic compounds. Ectomycorrhizal fungi play a major role in soil N cycling by their utilization of both inorganic and organic forms. Nutrients, including N, are mobilized from soil by fungal mycelia and transferred to the host plant. On the other hand, the host feeds the fungal symbiont with carbon compounds derived from photosynthesis. Despite the widely accepted importance of ectomycorrhizas on forest tree nutrition and N cycling, the control of basic N metabolism in ectomycorrhizal fungi is still poorly studied. Therefore, resolving how ectomycorrhizal fungi regulate their N adquisition will contribute to a better understanding of forest tree nutrition. Laccaria bicolor is an ectomycorrhizal basidiomycete which forms symbiosis with several boreal and temperate forest trees such as birch, pine and poplar. Ectomycorrhizal fungi such as L. bicolor compete with saprotrophs and plants for the limited pool of nutrients. Ectomycorrhizal fungi are well known to utilize complex organic N sources especially abundant in acid boreal and temperate forest soils but mineral N is efficiently utilized as well. Ectomycorrhizal fungi appear to be well adapted to low mineral N concentrations and efficiently assimilate inorganic N as NH4+, NO3-, the first generally being the preferred N source. While NH4+ is the dominant mineral N form in un­disturbed boreal and temperate forest soils the true availability of NO3- for ectomycorrhizal fungi in their habitat is not well established. The majority of studied ectomycorrhizal fungi shows a conserved capacity to grow on NO3- as the sole N source and some of them have even been reported to prefer NO3- over NH4+. This suggests that NO3- may play a more significant role as N source in forest ecosystems as traditionally believed. Laccaria, like many other ectomycorrhizal fungi, clearly prefers NH4+ but it also grows well on NO3-. Its genome encodes for one copy of each NO3- assimilation genes: the transporter Lbnrt, the NO3--reductase Lbnr and the NO2--reductase Lbnir. The genes are organized in the so-called fungal high-affinity N-assimilation cluster (fHANT-AC). We found massive effects on the formation of ECM root tips of poplar when N-assimilation in L. bicolor was impaired by silencing of a selected NO3--metabolism gene, the nitrate reductase encoding gene Lbnr. Alteration of the symbiotic capacity was specific to the RNAi-induced reduction in N assimilation of the fungal partner. Silencing of Lbnr strongly impaired mycorrhiza formation on NO3- but not on NH4+. The participation of NO3- -utilization genes in the symbiotic interaction of Laccaria is hence clear. We have extended the nitrate utilization RNAi-based gene silencing studies of the ECM basidiomycete Laccaria bicolor by altering the expression of LbNrt, the sole nitrate transporter encoding gene of the fungus. Here we report the first nutrient transporter mutants for mycorrhizal fungi. Silencing of LbNrt results in fungal strains with minimal detectable LbNrt transcript levels, significantly reduced growth capacity on nitrate and altered symbiotic interaction with poplar. Transporter silencing also produces co-downregulation of the whole fHANT-AC. Moreover, this effect on the nitrate utilization pathway seems to be independent of extracellular nitrate or the nitrogen status of the fungus. Our results show a novel and key nitrate uptake-independent regulatory role for a eukaryotic nitrate transporter. The possible cellular mechanisms underneath this regulation are discussed in the light of the current knowledge on NRT2-type nitrate transporters.