The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment.

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.

Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation.

Characterization of an Aux/IAA cDNA upregulated in Pinus pinaster roots in response to colonization by the ectomycorrhizal fungus Hebeloma cylindrosporum.

• In an attempt to determine whether fungal auxin affects host plant gene expression during mycorrhizal formation, an auxin upregulated cDNA, Pp-iaa88, was isolated by differential screening of a cDNA library made from auxin-treated Pinus pinaster roots. • Pp-iaa88 codes for a polypeptide that shares extensive homology to auxin-inducible Aux/IAA proteins, which are supposed to act as transcription factors. Cycloheximide did not inhibit auxin-induced mRNA accumulation, indicating that Pp-iaa88 upregulation is a primary (direct) auxin response. • The level of Pp-iaa88 transcripts in roots increased following inoculation with either an indoleacetic acid-overproducing mutant or a wild-type strain of the ectomycorrhizal fungus Hebeloma cylindrosporum. With both strains, mRNA accumulation was detectable as soon as fungal hyphae reached the root and it increased during differentiation of symbiotic structures. The kinetics of Pp-iaa88 transcript accumulation was closely connected with the dynamics of symbiosis establishment and was more rapid with the mutant than with the wild-type strain. • As a putative transcription factor expressed at the very early stages of symbiosis establishment, Pp-iaa88 could play a key role in mycorrhizal formation.