The ectomycorrhizal fungus Laccaria bicolor stimulates lateral root formation in poplar and Arabidopsis through auxin transport and signaling

Abstract

The early phase of the interaction between tree roots and ectomycorrhizal fungi, prior to symbiosis establishment, is accompanied by a stimulation of lateral root (LR) development. We aimed to identify gene networks that regulate LR development during the early signal exchanges between poplar (Populus tremula x Populus alba) and the ectomycorrhizal fungus Laccaria bicolor with a focus on auxin transport and signaling pathways. Our data demonstrated that increased LR development in poplar and Arabidopsis (Arabidopsis thaliana) interacting with L. bicolor is not dependent on the ability of the plant to form ectomycorrhizae. LR stimulation paralleled an increase in auxin accumulation at root apices. Blocking plant polar auxin transport with 1-naphthylphthalamic acid inhibited LR development and auxin accumulation. An oligoarray-based transcript profile of poplar roots exposed to molecules released by L. bicolor revealed the differential expression of 2,945 genes, including several components of polar auxin transport (PtaPIN and PtaAUX genes), auxin conjugation (PtaGH3 genes), and auxin signaling (PtaIAA genes). Transcripts of PtaPIN9, the homolog of Arabidopsis AtPIN2, and several PtaIAAs accumulated specifically during the early interaction phase. Expression of these rapidly induced genes was repressed by 1-naphthylphthalamic acid. Accordingly, LR stimulation upon contact with L. bicolor in Arabidopsis transgenic plants defective in homologs of these genes was decreased or absent. Furthermore, in Arabidopsis pin2, the root apical auxin increase during contact with the fungus was modified. We propose a model in which fungus-induced auxin accumulation at the root apex stimulates LR formation through a mechanism involving PtaPIN9-dependent auxin redistribution together with PtaIAA-based auxin signaling.

Figure 1.

Time course of colonization of poplar roots by L. bicolor in an in vitro sandwich culture system. A and B, L. bicolor and poplar precultures, respectively. C to E, Root development of poplar at 3 DODI (C), 10 DODI (D), and 30 DODI (E). Note the increasing LR number starting at 10 DODI. Root swelling at the LR basis started at 10 DODI, and LR arrest was observed at 30 DODI. F, L. bicolor hyphae from precultures after UVitex staining. G, Transverse root section after propidium iodide UVitex dual staining (green, UVitex; magenta, propidium iodide). H to J, Dual fluorescence-stained transverse root sections at 3 DODI (H), 10 DODI (I), and 30 DODI (J). Co, Cortex; Ep, epidermis; HN, Hartig net; M, mantle; RC, root cap cells; RH, root hair. Note hyphae attachment at 3 DODI and mantle and Hartig net development from 10 to 30 DODI. In H, single, magenta-colored cells surrounding the epidermis are detached root cap cells. Images shown are representative from a series of three experiments. Bars = 10 μm.

Figure 2.

Time course of early LR development during the interaction of L. bicolor with mycorrhizal poplar and nonmycorrhizal Arabidopsis. A and B, Time course of LR development in poplar in response to L. bicolor (A) and in Arabidopsis (B). Beginning at 4 d (A) and 2 d (B) of poplar and Arabidopsis-L. bicolor interaction, a significant (Student's t test P < 0.05) LR stimulation was observed. LR stimulation was similar in indirect and direct contact in poplar. C, Effect of the polar auxin transport inhibitor NPA on LR increase in poplar (10 μm NPA) after 8 DOII and in Arabidopsis (5 μm NPA) after 6 DOII. LR ratio of plants in indirect contact versus control plants without fungus in the presence and absence of NPA is shown. LR stimulation was significantly (Student's t test P < 0.01) reduced by the NPA treatment. In each experiment and each condition, 10 to 15 individual poplar plants or 15 Arabidopsis seedlings were observed. Error bars indicate se. If not visible, they were smaller than the symbol at the data point.

Figure 3.

Modification of the auxin gradient in P. tremula × P. tremuloides and Arabidopsis upon interaction with L. bicolor. A, Left, distribution of average, background-corrected GFP fluorescence intensity in the 18 optical sections of the Z-stack through the LR apices of P. tremula × P. tremuloides DR5:GFP plants in control conditions or at 3 DOII. Right, quantification of the signal intensity change as percentage of fluorescence in control root apices. Total intensity (sum of 18 sections) as well as maximum intensity (one section, around Z-stack section 9–11) increased significantly. Quantification from at least seven root apices per condition is shown (** Student's t test P < 0.05). B to E, P. tremula × P. tremuloides DR5:GFP LR apex under control conditions (B and C) and at 3 DOII (D and E). F and G, Arabidopsis DR5:GUS in control conditions (F) and at 3 DOII (G). Note the signal in provasculature at 3 DOII (arrows in G). Examples shown are out of more than 30 biological replicates. H and I, Arabidopsis pin2 DR5:GUS in control conditions (H) and at 3 DOII (I). A strong auxin accumulation at the root apex in control plants is observed, and the presence of the fungus enlarged this signal upwards. Examples shown are out of 15 biological replicates.

Figure 4.

Expression profiles of auxin-related target genes in poplar roots at 1, 3, and 10 DOII as well as 30 DODI (horizontal axis). Log₂-transformed relative expression compared with control roots is shown. A, PtaIAA19.3 transcripts were significantly up-regulated with an accumulation maximum at 3 DOII. PtaIAA28.1 was down-regulated but bottomed out at the same time point. With the exception of PtaIAA33.2, these members of this gene family were only induced during the early phase of contact. B, PtaGH3 genes were slowly up-regulated only at 10 DOII. C, PtaAUX6 was early induced at 3 DOII, whereas PtaAUX3 was only induced in the late phase (30 DODI). D, PtaPIN transcript profiles differed from one another. PtaPIN9 and PtaPIN12 were induced early (3 DOII) but PtaPIN9 levels decreased in the late phase (30 DODI). * Student's t test P < 0.05; ** Student's t test P < 0.01.

Figure 5.

Influence of the polar auxin transport inhibitor NPA on gene expression after 3 DOII (white bars) or 10 DOII (gray bars) of poplar with L. bicolor. Ratios of the transcript levels in roots in the presence versus the absence of NPA during indirect interaction with L. bicolor are presented. The graph is separated into three parts. The left part presents genes whose fungal induction was significantly abolished by NPA (in order of magnitude referring to 3 DOII). A drastic reduction in PtaIAA19.3, PtaPIN9, and PtaIAA33.2 transcripts by the presence of NPA at 3 DOII was observed. The middle part presents genes whose fungal induction was significantly increased by the presence of NPA. Note the high up-regulation of PtaPIN12 and PtaGH3-1. The right part refers to genes whose expression was not statistically affected by NPA. * Student's t test P < 0.05; ** Student's t test P < 0.01.

Figure 6.

LR development in Arabidopsis auxin mutants in the presence of L. bicolor after 8 DOII. A, Ratio of LR number that developed in plants in contact with L. bicolor versus control plants without fungus. Significant LR increases are marked by asterisks (** Student's t test P < 0.01). Ratios in mutants that differed significantly from ectoype Columbia (Col0) are represented as white bars. LR development was stimulated by L. bicolor in Columbia, pin3, and aux1 to a similar extent and significantly less in pin2 and pin2,3,4,7. The quadruple auxin receptor mutant tir1afb1,2,3 and the IAA14 gain-of-function mutant slr1 were completely insensitive to L. bicolor in terms of LR stimulation. B, Root development of control plants and plants after 8 DOII. Note the absence of LRs in the slr1 and tir1afb1,2,3 negative control plants and at 8 DOII. For each treatment and mutant line, 10 to 15 biological replicates were analyzed.

Figure 7.

Hypothetical model of the molecular mechanism underlying fungus-induced LR development in poplar. A, PtaPIN9 directed basipetal polar auxin transport (arrows) before contact with the fungus. The auxin maximum around the quiescent center (QC) is indicated by gray shading in the meristematic zone (MZ). Pericycle founder cells (single gray box) are primed for LRI in the elongation zone (EZ), and LRs are initiated through subsequent cell divisions (multiple gray boxes) in the differentiation zone (DZ). B, The presence of the fungus stimulates auxin accumulation at the root apex (gray shading) through an unknown mechanism. The increased auxin level and/or other fungal signals stimulate PtaPIN9 expression. PtaPIN9 protein enhances basipetal auxin transport (thick arrows), which then primes more pericycle cells for LRI. LRI occurs through a PtaIAA19.3-dependent signaling mechanism in the differentiation zone. Whether fungal signals act directly on PtaIAA19.3 expression needs to be analyzed. C, Cortex; E, epidermis; H, hyphae; P, pericycle.