Fungal hypaphorine reduces growth and induces cytosolic calcium increase in root hairs of Eucalyptus globulus.

Root hairs are tubular cells resulting from a tip-localized growth in which calcium ions play a key role. Hypaphorine, an indole alkaloid secreted by the fungus Pisolithus microcarpus during the formation of ectomycorrhizae with the host plant Eucalyptus globulus, inhibits root hair tip growth. Hypaphorine-induced inhibition is linked to a transient depolarization of the plasma membrane and a reorganization of the actin and microtubule cytoskeletons. Here we investigated the activity of hypaphorine on calcium distribution in E. globulus root hairs with the ratiometric fluorochrome calcium indicator Indo-1. In 85% of actively growing root hairs, a significant but modest calcium gradient between the apex and the base was observed due to an elevated cytoplasmic calcium concentration at the apical tip. Following exposure to 1 mM hypaphorine, the apical and basal cytoplasmic Ca(2+) concentration increased in 70 and 77% of the hairs, respectively, 10 min after treatment. This led to a reduced calcium gradient in 81% of the cells. The hypothetical links between calcium concentration elevation, regulation of actin cytoskeleton dynamics, and root hair growth inhibition in response to hypaphorine treatment are discussed.

Fungal auxin antagonist hypaphorine competitively inhibits indole-3-acetic acid-dependent superoxide generation by horseradish peroxidase.

Plant peroxidases (EC 1.11.1.7) including horseradish peroxidase (HRP-C), but not the nonplant peroxidases, are known to be highly specific indole-3-acetic acid (IAA) oxygenases which oxidize IAA in the absence of H2O2, and superoxide anion radicals (O2*-) are produced as by-products. Hypaphorine, a putative auxin antagonist isolated from ectomycorrhizal fungi, inhibited the IAA-dependent generation of O2*- by HRP-C, which occurs in the absence of H2O2. Hypaphorine has no effect on the nonspecific heme-catalyzed O2*- generation induced by high concentration of ethanol. It is probable that the inhibitory effect of hypaphorine on O2*- generation is highly specific to the IAA-dependent reaction. The mode of inhibition of the IAA-dependent O2*--generating reaction by hypaphorine was analyzed with a double-reciprocal plot and determined to be competitive inhibition, indicating that hypaphorine competes with IAA by binding to the putative IAA binding site on HRP-C. This implies the importance of structural similarity between hypaphorine and IAA. This work presented the first evidence for antagonism between IAA and a structurally related fungal alkaloid on binding to a purified protein which shares some structural similarity with auxin-binding proteins.

Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid.

Hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation rate of root hairs. At inhibitory concentrations (100 microM), hypaphorine induced a transitory swelling of root hair tips of Eucalyptus globulus Labill. ssp. bicostata. When the polar tip growth resumed, a characteristic deformation was still visible on elongating hairs. At higher hypaphorine concentrations (500 microM and greater), root hair elongation stopped, only 15 min after application. However, root hair initiation from trichoblasts was not affected by hypaphorine. Hypaphorine activity could not be mimicked by related molecules such as indole-3-acetic acid (IAA) or tryptophan. While IAA had no activity on root hair elongation, IAA was able to restore the tip growth of root hairs following inhibition by hypaphorine. These results suggest that hypaphorine and endogenous IAA counteract in controlling root hair elongation. During ectomycorrhiza development, the absence of root hairs might be due in part to fungal release of molecules, such as hypaphorine, that inhibit the elongation of root hairs.

Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings.

Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified.

The expression of a symbiosis-regulated gene in eucalypt roots is regulated by auxins and hypaphorine, the tryptophan betaine of the ectomycorrhizal basidiomycete Pisolithus tinctorius.

A full-length cDNA coding for a symbiosis-regulated transcript, EgHypar, was isolated by differential screening from a Eucalyptus globulus bicostata--Pisolithus tinctorius ectomycorrhiza. The sequence of this clone revealed a protein with an estimated molecular mass of 25.5 kDa that exhibited a high degree of homology (66%) with plant auxin-induced glutathione-S-transferases. Expression of the EgHypar gene in seedlings was confined largely in roots and it is drastically increased by ectomycorrhiza development. The concentration of EgHypar transcripts was similarly up-regulated in roots incubated in media supplemented with P. tinctorius cell-free extracts, indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid or hypaphorine (tryptophan betaine), the major indolic compound secreted by P. tinctorius. The latter fungal alkaloid concomitantly induced a decrease in root hair elongation in eucalypt seedlings. Up-regulation of EgHypar expression by auxins and fungal metabolites suggests that this symbiosis-regulated gene could be involved in the morphological changes taking place in plants roots upon symbiosis development. To our knowledge, these results provide the first molecular evidence that gene expression of the host plant is altered by molecules produced by the ectomycorrhizal mycobiont.

Chitinase and peroxidase activities are induced in eucalyptus roots according to aggressiveness of Australian ectomycorrhizal strains of Pisolithus sp.

Peroxidase and chitinase activities were measured in roots of Eucalyptus globulus spp bicostata Kirkp. during colonization by Pisolithus sp. isolated from under Eucalyptus. Ten fungal isolates, ranging from poor to good root colonizers, were selected to represent a range of ectomycorrhizal aggressivity. The induction of chitinases and peroxidases was strongly related to the aggressiveness of the fungal strain. Only good colonizers, that is strains which rapidly form differentiated ectomycorrhizas, induced a strong response in the plant. Therefore, it can be concluded that these enzymes are not responsible for poor root colonization by the less aggressive strains. The chitinase response of Eucalyptus roots to contact with fungal extracts differed only slightly between weakly and strongly aggressive strains. This suggests that a major component of differential induction observed in vivo is the consequence of root colonization, tissue penetration and the ability to deliver elicitors to the plain print to and during rant colonization.

ECTOMYCORRHIZAL VS ENDOMYCORRHIZAL FUNGI WITHIN THE SAME ROOT SYSTEM.

Vesicular-arbuscular (VA) mycorrhizal fungi and ectomycorrhizal fungi were observed together in the same root system, and even within the same root apices, of seedlings of Eucalyptus dumosa A. Cunn. ex Schau. On a population basis, there was a succession of two overlapping mycorrhizal epidemics-VA mycorrhizas followed by ectomycorrhizas. This succession was interpreted as follows. Early build-up of VA mycorrhizas was related to initial higher inoculum potential of that fungus and to rapidity of colonization of individual roots. The ectomycorrhizal fungus was more successful later in achieving secondary infections by hyphal spread and in colonization of lateral root branches. The ectomycorrhizal fungus had no difficulty infecting pre-existing VA mycorrhizas, but the ectomycorrhizal sheath provided a barrier to subsequent VA mycorrhizal infections. While the proportion of VA mycorrhizas to ectomycorrhizas changed between two and five months, the overall proportion of mycorrhizal roots remained constant, suggesting that there could be competition between the different fungi for limiting substrate.