A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase

Abstract

Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase.

Keywords: Rhizophagus irregularis; arbuscular mycorrhizal fungi; dipeptide transporter; organic nitrogen; symbiosis.

Figure 1

Phylogenetic position of RiPTR2 protein sequence in comparison with homologous sequences from selected species representative of the major fungal phyla (Table S1). The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model as implemented in MEGA5. The percentage of trees higher than 75% in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Figure 2

RiPTR2 complements growth defects of ptr2Δ, dal5Δ or ptr2Δ dal5Δ yeast transporter mutants on dipeptides. Serial dilutions (1:10–1:100–1:1,000–1:10,000 from left to right) of cultured strains transformed with RiPTR2 or the empty vector (EV) were plated on YCB solid medium containing H₂0 (negative control), NH⁺₄ (positive control), 0.25 mM Ala-Tyr, Tyr-Ala or Ala-Leu. Plates were incubated at 30°C for 4 days.

Figure 3

Relative expression of RiPTR2 assessed by qRT-PCR in intraradical mycelium (IRM) and extraradical mycelium (ERM) from mycorrhizal roots of M. truncatula grown in the sandwich system (A) and mycorrhizal roots of C. intybus grown in monoaxenic culture (B). Data for each condition are presented as mean ± standard deviation. Different letters indicate statistically significant difference (p < 0.05, ANOVA).

Figure 4

Relative expression of MtPT4 (A) and RiPTR2 (B) assessed by qRT-PCR in a time course experiment of root colonization at 7, 14, 28 and 60 days post-inoculation (dpi). Data for each condition are presented as mean ± standard deviation. Different letters indicate statistically significant difference (p < 0.05, ANOVA). (C) Gel electrophoresis of RT-PCR products obtained from two independent samples (1, 2) of RNA from laser-microdissected arbuscule-containing cells using primers specific for MtPT4 or RiPTR2. No RNA sample (-); M: 100 bp (Invitrogen).

Figure 5

Relative expression of RiPTR2 (A) assessed by qRT-PCR in mycorrhizal roots exposed for 24 h to 10 mM Ala-Leu, 1 mM nitrate or no N. MtPT4 expression was used as marker of mycorrhization (B). Data for each condition are presented as mean ± standard deviation. Different letters indicate statistically significant difference (p < 0.05, ANOVA).

Figure 6

Relative expression of RiPTR2 assessed by qRT-PCR in the extraradical mycelium developed in ROC exposed for 24 h to 10 mM Ala-Leu or 3.2 mM nitrate or no N. Data for each condition are presented as mean ± standard deviation. Different letters indicate statistically significant difference (p < 0.05, ANOVA).

Figure 7

Total fresh weight (A) and shoot to root ratio (B) of M. truncatula plants grown in the sandwich system inoculated (Myc) or not (C) and watered with either 2.5 mM Ala-Leu and 0.25 mM nitrate (Ala-Leu + nitrate) or 0.5 mM nitrate (LA). Data for each condition are presented as mean ± standard deviation. Different letters indicate statistically significant difference (p < 0.05, ANOVA).

Figure 8

Relative expression of RiPTR2 (A) assessed by qRT-PCR in mycorrhizal roots grown in the presence of 0.5 mM nitrate (LA) or 2.5 mM Ala-Leu and 0.25 mM nitrate (Ala-Leu + nitrate). MtPT4 expression was used as marker of mycorrhization (B). Data for each condition are presented as mean ± standard deviation. Different letters indicate statistically significant difference (p < 0.05, ANOVA).