Armelle Gollotte

In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections

A histochemical procedure was developed to visualize and estimate the proportion of arbuscular mycorrhizal infections showing fungal alkaline phosphatase activity, as compared to the total amount of fungal tissue (trypan blue staining) and of living mycelium, indicated by succinate dehydrogenase activity. In roots of Allium porrum and Platanus acerifolia, only a small proportion of living intraradical mycelium showed alkaline phosphatase activity during early infection but this increased greatly just before the mycorrhizal growth response of the host plant. Infection revealed by all three stains reached a maximum at 6 weeks after inoculation, after which the level of trypan blue stained infection remained constant but the proportion showing succinate dehydrogenase and alkaline phosphatase activity declined as the infection aged. Alkaline phosphatase activity was absent from virtually all abortive entry point hyphae formed on roots of a resistant myc−, nod− mutant of Pisum sativum, although succinate dehydrogenase activity was detected. Observations suggest that the alkaline phosphatase activity is induced by colonization of host roots and that this fungal enzyme could provide a useful marker for analyzing the symbiotic efficiency of arbuscular mycorrhizal infections.

On the mechanisms of cadmium stress alleviation in Medicago truncatula by arbuscular mycorrhizal symbiosis: A root proteomic study

The arbuscular mycorrhizal (AM) symbiosis belongs to the strategies plants have developed to cope with adverse environmental conditions including contamination by heavy metals such as cadmium (Cd). In the present work, we report on the protective effect conferred by AM symbiosis to the model legume Medicago truncatula grown in presence of Cd, and on the 2‐D‐based proteomic approach further used to compare the proteomes of M. truncatula roots either colonised or not with the AM fungus Glomus intraradices in Cd‐free and Cd‐contaminated substrates. The results indicated that at the proteome level, 9 out of the 15 cadmium‐induced changes in nonmycorrhizal roots were absent or inverse in those Cd‐treated and colonized by G. intraradices, including the G. intraradices‐dependent down‐accumulation of Cd stress‐responsive proteins. Out of the twenty‐six mycorrhiza‐related proteins that were identified, only six displayed changes in abundance upon Cd exposure, suggesting that part of the symbiotic program, which displays low sensitivity to Cd, may be recruited to counteract Cd toxicity through the mycorrhiza‐dependent synthesis of proteins having functions putatively involved in alleviating oxidative damages, including a cyclophilin, a guanine nucleotide‐binding protein, an ubiquitin carboxyl‐terminal hydrolase, a thiazole biosynthetic enzyme, an annexin, a glutathione S‐transferase (GST)‐like protein, and a S‐adenosylmethionine (SAM) synthase.

Gene Expression and Molecular Modifications Associated with Plant Responses to Infection by Arbuscular Mycorrhizal Fungi

Microorganisms that colonize living plant tissues generally show a relatively restricted host range, but the striking feature of arbuscular mycorrhizal fungi is their ability to form a symbiotic association with roots of the large majority of terrestrial plant species. Likewise, the ancestral nature of arbuscular mycorrhiza [1] makes them a remarkable example of reciprocal cellular and physiological compatibility between plant and fungal taxa that must have been established early during land colonization and maintained through evolution. In spite of the widespread occurrence of arbuscular mycorrhiza, analyses into the molecular mechanisms and genetic determinants involved are still in their infancy due to the complexity of symbiont interactions and the incalcitrance of the fungal partner to pure culture. We have adopted two strategies to investigate plant processes regulating symbiotic fungus-root interactions: firstly, by probing tissues for molecular events that may be in common with other plant-microbe associations and, secondly, by characterizing of genes and gene products specific to arbuscular mycorrhiza. In the present paper we discuss recent data and speculate about the plant’s role in establishment of the symbiotic state and in control over fungal development in arbuscular mycorrhiza.

First Identification of Polymorphic Microsatellite Markers in the Burgundy Truffle, Tuber aestivum (Tuberaceae)

Premise of the study: Tuber aestivum, the most common truffle in Europe, plays an important role in the commercial truffle market. For the first time, microsatellite primers were developed to investigate polymorphism within this species. Methods and Results: Using direct shotgun pyrosequencing, 15 polymorphic microsatellites were identified out of the 7784 perfect microsatellites present in the 534620 reads obtained. Tested on 75 samples, these microsatellites were highly polymorphic. The number of alleles varied from four to 15, and the expected heterozygosity ranged from 0.266 to 0.620. A multilocus analysis allowed the identification of 63 genotypes over the 75 samples analyzed. Conclusions: Direct shotgun pyrosequencing is a fast and relatively low-cost technique allowing identification of microsatellites in nonmodel species. The microsatellites developed in this study will be useful in population genetic studies to infer the evolutionary history of this species.

Etude immunocytochimique des interfaces plante-champignon endomycorhizien à arbuscules chez des pois isogéniques myc+ ou résistant à l'endomycorhization (myc−)

Summary Interactions between an arbuscular mycorrhizal fungus and pea cv. Frisson (myc+) or an isogenic mycorrhiza-resistant mutant (myc−) were investigated by immunocytochemistry. Observations of the presence of proteins and polysaccharides accumulating in other symbioses (pectins) or in plant-pathogenic interactions (PR-b1 protein, s-1,3-glucans) indicated a weak activation of defence mechanisms during establishment of symbiosis. In contrast, the myc mutant showed an important defence reaction against the mycorrhizal fungus, suggesting that there are complex interactions between defence genes and symbiosis genes during endomycorrhiza establishment.

A multigene phylogeny demonstrates that Tuber aestivum and Tuber uncinatum are conspecific

For almost 2 centuries it has been disputed whether Tuber aestivum and Tuber uncinatum constitute two different species of truffles. Molecular markers have been applied previously to contribute to resolving this question, coming to different conclusions. In this study, we address this question by analyzing the genetic structure of truffles assigned to either of the two putative species from a geographically broad sampling across Europe. We used an approach involving multigene phylogenies and coalescent analyses of nine regions from five genes. All tests conducted supported the conspecificity of Tuber aestivum and Tuber uncinatum.

SSR-based identification of genetic groups within European populations of Tuber aestivum Vittad

Tuber species are ectomycorrhizal ascomycetes establishing relationships with different host trees and forming hypogeous fruiting bodies known as truffles. Among Tuber species, Tuber aestivum Vittad. has a wide distributional range being found naturally all over Europe. Here, we performed large-scale population genetic analyses in T. aestivum to (i) investigate its genetic diversity at the European scale, (ii) characterize its genetic structure and test for the presence of ecotypes and (iii) shed light into its demographic history. To reach these goals, 230 ascocarps from different populations were genotyped using 15 polymorphic simple sequence repeat markers. We identified 181 multilocus genotypes and four genetic groups which did not show a clear geographical separation; although, one of them was present exclusively in Southeast France, Italy and Spain. Fixation index values between pairs of genetic groups were generally high and ranged from 0.29 to 0.45. A significant deficit of heterozygosity indicated a population expansion instead of a recent population bottleneck, suggesting that T. aestivum is not endangered in Europe, not even in Mediterranean regions. Our study based on a large-scale population genetic analysis suggests that genetically distinct populations and likely ecotypes within T. aestivum are present. In turn, this study paves the way to future investigations aimed at addressing the biological and/or ecological factors that have concurred in shaping the population genetic structure of this species. Present results should also have implications for the truffle market since defining genetic markers are now possible at least for some specific T. aestivum genetic groups.