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Over 70% land plants live in mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, and maintenance of symbiosis requires transcriptional and post-transcriptional regulation. The former has been widely studied, whereas the latter mediated by symbiotic microRNAs (miRNAs) remains obscure, especially in woody plants. Here, we performed high-throughput sequencing of the perennial woody citrus plant Poncirus trifoliata and identified 3750 differentially expressed genes (DEGs) and 42 miRNAs (DEmiRs) upon AM fungal colonization. By analyzing cis-regulatory elements in the promoters of the DEGs, we predicted 329 key AM transcription factors (TFs). A miRNA-mRNA regulatory network was then constructed by integrating these data. Several candidate miRNA families of P. trifoliata were identified whose members target known symbiotic genes, such as miR167h-AMT2;3 and miR156e-EXO70I, or key TFs, such as miR164d-NAC and miR477a-GRAS, thus are involved in AM symbiotic processes of fungal colonization, arbuscule development, nutrient exchange and phytohormone signaling. Finally, analysis of selected miRNA family revealed that a miR159b conserved in mycorrhizal plant species and a Poncirus-specific miR477a regulate AM symbiosis. The role of miR477a was likely to target GRAS family gene RAD1 in citrus plants. Our results not only revealed that miRNA-mRNA network analysis, especially miRNA-TF analysis, is effective in identifying miRNA family regulating AM symbiosis, but also shed light on miRNA-mediated post-transcriptional regulation of AM symbiosis in woody citrus plants.

Laser microdissection (LMD) is a microscopy technique that, through the collection of specific cell-type populations from sections of heterogeneous tissues, allows the subsequent extraction of nucleic acids as well as primary and secondary metabolites. In plants, LMD was widely used to study cell-specific gene expression during symbiotic interactions with other organisms, including mycorrhizal fungi. In particular, LMD was extensively used to study cell-specificity in gene expression profiles in arbuscular mycorrhizal (AM) and orchid mycorrhizal (ORM) interactions. These earlier studies were mainly focused on the identification of functional markers in plant cells containing intracellular fungal structures, i.e. arbuscules, the typical structures in AM, and coils, typical of ORM. Several plant and fungal genes coding for nutrient transporters were identified in these cells thanks to LMD, suggesting that symbiotic nutrient exchange is cell specific. In the absence of a stable transformation protocol for the expression of tagged genes in the mycorrhizal fungal partner, LMD protocols represent a useful tool to study fungal gene expression in specific cell-type populations inside symbiotic plant tissues., Italian Journal of Mycology, V. 50 (2021)

Plant-microorganism interactions at the rhizosphere level have a major impact on plant growth and plant tolerance and/or resistance to biotic and abiotic stresses. Of particular importance for forestry and agricultural systems is the cooperative and mutualistic interaction between plant roots and arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycotina, since about 80% of terrestrial plant species can form AM symbiosis. The interaction is tightly regulated by both partners at the cellular, molecular and genetic levels, and it is highly dependent on environmental and biological variables. Recent studies have shown how fungal signals and their corresponding host plant receptor-mediated signalling regulate AM symbiosis. Host-generated symbiotic responses have been characterized and the molecular mechanisms enabling the regulation of fungal colonization and symbiosis functionality have been investigated. This review summarizes these and other recent relevant findings focusing on the molecular players and the signalling that regulate AM symbiosis. Future progress and knowledge about the underlying mechanisms for AM symbiosis regulation will be useful to facilitate agro-biotechnological procedures to improve AM colonization and/or efficiency., This study was supported by grant (PID2020-115336GB-I00) funded by Spanish MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union”.

This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA&ndash, CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.

This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA–CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.

Laser microdissection (LMD) technology has been widely applied to plant tissues, offering novel information on the role of different cell-type populations during plant-microbe interactions. In this chapter, protocols to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations from arbuscular mycorrhizal (AM) roots are described in detail, starting from the biological material preparation to gene expression analyses by RT-PCR and RT-qPCR.

The colonization of a host plant root by arbuscular mycorrhizal (AM) fungi is a progressive process, characterized by asynchronous hyphal growth in intercellular and intracellular spaces, leading to the coexistence of diverse intraradical structures, such as hyphae, coils, arbuscules, and vesicles. In addition, the relative abundance of intercellular and intracellular fungal structures is highly dependent on root anatomy and the combination of plant and fungal species. Lastly, more than one fungal species may colonize the same root, adding a further level of complexity. For all these reasons, detailed imaging of a large number of samples is often necessary to fully assess the developmental processes and functionality of AM symbiosis. To this aim, the use of rapid and efficient staining methods that can be used routinely is crucial.We herein present a simple protocol to obtain high detail images of both overall intraradical fungal colonization pattern and fine morphology, in AM root sections of Lotus japonicus. The procedure is based on tissue clearing, fluorescent staining of fungal cell walls with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), and the combined counterstaining of plant cell walls with propidium iodide (PI). The resulting images can be acquired using traditional or confocal fluorescence microscopes and used for qualitative and quantitative analyses of fungal colonization, of particular interest for the comparison of mycorrhizal phenotypes between different experimental conditions or genetic backgrounds.

Climate changes include the intensification of drought in many parts of the world, increasing its frequency, severity and duration. However, under natural conditions, environmental stresses do not occur alone, and, in addition, more stressed plants may become more susceptible to attacks by pests and pathogens. Studies on the impact of the arbuscular mycorrhizal (AM) symbiosis on tomato response to water deficit showed that several drought-responsive genes are differentially regulated in AM-colonized tomato plants (roots and leaves) during water deficit. To date, global changes in mycorrhizal tomato root transcripts under water stress conditions have not been yet investigated. Here, changes in root transcriptome in the presence of an AM fungus, with or without water stress (WS) application, have been evaluated in a commercial tomato cultivar already investigated for the water stress response during AM symbiosis. Since root-knot nematodes (RKNs, Meloidogyne incognita) are obligate endoparasites and cause severe yield losses in tomato, the impact of the AM fungal colonization on RKN infection at 7 days post-inoculation was also evaluated. Results offer new information about the response to AM symbiosis, highlighting a functional redundancy for several tomato gene families, as well as on the tomato and fungal genes involved in WS response during symbiosis, underlying the role of the AM fungus. Changes in the expression of tomato genes related to nematode infection during AM symbiosis highlight a role of AM colonization in triggering defense responses against RKN in tomato. Overall, new datasets on the tomato response to an abiotic and biotic stress during AM symbiosis have been obtained, providing useful data for further researches.

The arbuscular mycorrhizal (AM) symbiosis between terrestrial plants and AM fungi is regulated by plant hormones. For most of these, a role has been clearly assigned in this mutualistic interaction; however, there are still contradictory reports for cytokinin (CK). Here, pea plants, the wild type (WT) cv. Sparkle and its mutant E151 (Pssym15), were inoculated with the AM fungus Rhizophagus irregularis. E151 has previously been characterized as possessing high CK levels in non-mycorrhizal (myc-) roots and exhibiting high number of fungal structures in mycorrhizal (myc+) roots. Myc- and myc+ plants were treated 7, 9, and 11 days after inoculation (DAI) with synthetic compounds known to alter CK status. WT plants were treated with a synthetic CK [6-benzylaminopurine (BAP)] or the CK degradation inhibitor INCYDE, whereas E151 plants were treated with the CK receptor antagonist PI-55. At 13 DAI, plant CK content was analyzed by mass spectrometry. The effects of the synthetic compounds on AM colonization were assessed at 28 (WT) or 35 (E151) DAI via a modified magnified intersections method. The only noticeable difference seen between myc- and myc+ plants in terms of CK content was in the levels of nucleotides (NTs). Whereas WT plants responded to fungi by lowering their NT levels, E151 plants did not. Since NTs are thought to be converted into active CK forms, this result suggests that active CKs were synthesized more effectively in WT than in E151. In general, myc+ and myc- WT plants responded similarly to INCYDE by lowering significantly their NT levels and increasing slightly their active CK levels; these responses were less obvious in BAP-treated WT plants. In contrast, the response of E151 plants to PI-55 depended on the plant mycorrhizal status. Whereas treated myc- plants exhibited high NT and low active CK levels, treated myc+ plants displayed low levels of both NTs and active CKs. Moreover, treated WT plants were more colonized than treated E151 plants. We concluded that CKs have a stimulatory role in AM colonization because increased active CK levels were paralleled with increased AM colonization while decreased CK levels corresponded to reduced AM colonization.

Arbuscular mycorrhizal (AM) symbiosis alleviates drought stress in plants. However, the intimate mechanisms involved, as well as its effect on the production of signalling molecules associated with the host plant–AM fungus interaction remains largely unknown. In the present work, the effects of drought on lettuce and tomato plant performance and hormone levels were investigated in non-AM and AM plants. Three different water regimes were applied, and their effects were analysed over time. AMplants showed an improved growth rate and efficiency of photosystem II than non-AM plants under drought from very early stages of plant colonization. The levels of the phytohormone abscisic acid, as well as the expression of the correspondingmarker genes, were influenced by drought stress in non-AMandAMplants. The levels of strigolactones and the expression of corresponding marker genes were affected by both AM symbiosis and drought. The results suggest that AM symbiosis alleviates drought stress by altering the hormonal profiles and affecting plant physiology in the host plant. In addition, a correlation between AM root colonization, strigolactone levels and drought severity is shown, suggesting that under these unfavourable conditions, plants might increase strigolactone production in order to promote symbiosis establishment to cope with the stress.

Secondary metabolites (SMs) have high economic impact thanks to their exploitability in chemical, pharmaceutical and cosmetic industries. Trigonella foenum-graecum has an importance due to the production of bioactive compounds with pharmaceutical values. Among them, the alkaloid trigonelline is known for its role in the treatment of different human diseases. SM accumulation is influenced by environmental factors but is modulated by the application of exogenous compounds. Ethephon, a precursor of the phytohormone ethylene, was already used to influence SM accumulation. Our work is aimed at evaluating the accumulation of trigonelline and the phytohormone abscisic acid (ABA) when three factors were combined: i) two levels of water regimes (well-watered and water deficit), ii) ethephon treatments and iii) inoculation with an arbuscular mycorrhizal (AM)-based inoculum also leading to nodulation. The content of trigonelline and ABA was significantly affected by symbioses, showing high accumulation in AM-colonized plants irrespective of the water regimes applied. In terms of trigonelline accumulation with respect to ethephon treatments, while symbiotic plants showed a dose-dependent trend, non-symbiotic plants showed a significantly difference only when 550 ppm of ethephon was applied. In conclusion, our work provides new information on the effects of both ethephon and symbioses on plant growth and accumulation of valuable compounds, such as trigonelline, in fenugreek.

AM symbiosis did not strongly affect Arundo donax performances under salt stress, although differences in the plants inoculated with two different fungi were recorded. The mechanisms at the basis of the improved tolerance to abiotic stresses by arbuscular mycorrhizal (AM) fungi have been investigated mainly focusing on food crops. In this work, the potential impact of AM symbiosis on the performance of a bioenergy crop, Arundo donax, under saline conditions was considered. Specifically, we tried to understand whether AM symbiosis helps this fast-growing plant, often widespread in marginal soils, withstand salt. A combined approach, involving eco-physiological, morphometric and biochemical measurements, was used and the effects of two different AM fungal species (Funneliformis mosseae and Rhizophagus irregularis) were compared. Results indicate that potted A. donax plants do not suffer permanent damage induced by salt stress, but photosynthesis and growth are considerably reduced. Since A. donax is a high-yield biomass crop, reduction of biomass might be a serious agronomical problem in saline conditions. At least under the presently experienced growth conditions, and plant–AM combinations, the negative effect of salt on plant performance was not rescued by AM fungal colonization. However, some changes in plant metabolisms were observed following AM-inoculation, including a significant increase in proline accumulation and a trend toward higher isoprene emission and higher H2O2, especially in plants colonized by R. irregularis. This suggests that AM fungal symbiosis influences plant metabolism, and plant–AM fungus combination is an important factor for improving plant performance and productivity, in presence or absence of stress conditions.

Grapevine, cultivated for both fruit and beverage production, represents one of the most economically important fruit crops worldwide. With the aim of better understanding how grape roots respond to beneficial microbes, a transcriptome sequencing experiment has been performed to evaluate the impact of a single arbuscular mycorrhizal (AM) fungal species (Funneliformis mosseae) versus a mixed inoculum containing a bacterial and fungal consortium, including different AM species, on Richter 110 rootstock. Results showed that the impact of a single AM fungus and of a complex microbial inoculum on the grapevine transcriptome differed. After 3 months, roots exclusively were colonized after the F. mosseae treatment and several AM marker genes were found to be upregulated. The mixed inoculum led only to traces of colonization by AM fungi, but elicited an important transcriptional regulation. Additionally, the expression of genes belonging to categories such as nutrient transport, transcription factors, and cell wall-related genes was significantly altered in both treatments, but the exact genes affected differed in the two conditions. These findings advance our understanding about the impact of soil beneficial microbes on the root system of a woody plant, also offering the basis for novel approaches in grapevine cultivation.

Arbuscular mycorrhizal (AM) symbiosis alleviates drought stress in plants. However, the intimate mechanisms involved, as well as its effect on the production of signalling molecules associated with the host plant–AM fungus interaction remains largely unknown. In the present work, the effects of drought on lettuce and tomato plant performance and hormone levels were investigated in non-AM and AM plants. Three different water regimes were applied, and their effects were analysed over time. AMplants showed an improved growth rate and efficiency of photosystem II than non-AM plants under drought from very early stages of plant colonization. The levels of the phytohormone abscisic acid, as well as the expression of the correspondingmarker genes, were influenced by drought stress in non-AMandAMplants. The levels of strigolactones and the expression of corresponding marker genes were affected by both AM symbiosis and drought. The results suggest that AM symbiosis alleviates drought stress by altering the hormonal profiles and affecting plant physiology in the host plant. In addition, a correlation between AM root colonization, strigolactone levels and drought severity is shown, suggesting that under these unfavourable conditions, plants might increase strigolactone production in order to promote symbiosis establishment to cope with the stress.

Plants are able to discriminate and respond to structurally related chitooligosaccharide (CO) signals from pathogenic and symbiotic fungi. In model plants Arabidopsis thaliana and Oryza sativa LysM-receptor like kinases (LysM-RLK) AtCERK1 and OsCERK1 (chitin elicitor receptor kinase 1) were shown to be involved in response to CO signals. Based on phylogenetic analysis, the pea Pisum sativum L. LysM-RLK PsLYK9 was chosen as a possible candidate given its role on the CERK1-like receptor. The knockdown regulation of the PsLyk9 gene by RNA interference led to increased susceptibility to fungal pathogen Fusarium culmorum. Transcript levels of PsPAL2, PsPR10 defense-response genes were significantly reduced in PsLyk9 RNAi roots. PsLYK9’s involvement in recognizing short-chain COs as most numerous signals of arbuscular mycorrhizal (AM) fungi, was also evaluated. In transgenic roots with PsLyk9 knockdown treated with short-chain CO5, downregulation of AM symbiosis marker genes (PsDELLA3, PsNSP2, PsDWARF27) was observed. These results clearly indicate that PsLYK9 appears to be involved in the perception of COs and subsequent signal transduction in pea roots. It allows us to conclude that PsLYK9 is the most likely CERK1-like receptor in pea to be involved in the control of plant immunity and AM symbiosis formation.

The arbuscular mycorrhizal (AM) symbiosis is considered a natural instrument to improve plant health and productivity since mycorrhizal plants often show higher tolerance to abiotic and biotic stresses. However, the impact of the AM symbiosis on infection by viral pathogens is still largely uncertain and little explored. In the present study, tomato plants were grown under controlled conditions and inoculated with the AM fungus Funneliformis mosseae. Once the mycorrhizal colonization had developed, plants were inoculated with the Tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus causing one of the most serious viral diseases of tomatoes in Mediterranean areas. Biological conditions consisted of control plants (C), TYLCSV-infected plants (V), mycorrhizal plants (M), and TYLCSV-infected mycorrhizal plants (MV). At the time of analysis, the level of mycorrhiza development and the expression profiles of mycorrhiza-responsive selected genes were not significantly modified by virus infection, thus indicating that the AM symbiosis was unaffected by the presence and spread of the virus. Viral symptoms were milder, and both shoot and root concentrations of viral DNA were lower in MV plants than in V plants. Overall F. mosseae colonization appears to exert a beneficial effect on tomato plants in attenuating the disease caused by TYLCSV.

Obligate symbiotic fungi that form arbuscular mycorrhizae (AMF; belonging to the Glomeromycota phylum) are some of the most important soil microorganisms. AMFs facilitate mineral nutrient uptake from the soil, in exchange for plant-assimilated carbon, and promote water-stress tolerance and resistance to certain diseases. AMFs colonize the root by producing inter- and intracellular hyphae. When the fungus penetrates the inner cortical cells, it produces a complex ramified structure called arbuscule, which is considered the preferential site for nutrient exchange. Direct DNA extraction from the whole root and sequencing of ribosomal gene regions are commonly carried out to investigate intraradical AMF communities. Nevertheless, this protocol cannot discriminate between the AMFs that actively produce arbuscules and those that do not. To solve this issue, the authors have characterized the AMF community of arbusculated cells through a laser microdissection (LMD) approach, combined with sequencing-based taxa identification. The results were then compared with the AMF community that was found from whole root DNA extraction. The AMF communities originating from the LMD samples and the whole root samples differed remarkably. Five taxa were involved in the production of arbuscules, while two taxa were retrieved inside the root but not in the arbusculated cells. Unexpectedly, one taxon was found in the arbusculated cells, but its detection was not possible when extracting from the whole root. Thus, the LMD technique can be considered a powerful tool to obtain more precise knowledge on the symbiotically active intraradical AMF community.

Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers, whose exploitation could represent a promising avenue for the development in the future of a more sustainable next-generation agriculture. The best understood function in symbiosis is an improvement in plant mineral nutrient acquisition, as exchange for carbon compounds derived from the photosynthetic process: this can enhance host growth and tolerance to environmental stresses, such as water stress (WS). However, physiological and molecular mechanisms occurring in arbuscular mycorrhiza-colonized plants and directly involved in the mitigation of WS effects need to be further investigated. The main goal of this work is to verify the potential impact of AM symbiosis on the plant response to WS. To this aim, the effect of two AM fungi (Funneliformis mosseae and Rhizophagus intraradices) on tomato (Solanum lycopersicum) under the WS condition was studied. A combined approach, involving ecophysiological, morphometric, biochemical, and molecular analyses, has been used to highlight the mechanisms involved in plant response to WS during AM symbiosis. Gene expression analyses focused on a set of target genes putatively involved in the plant response to drought, and in parallel, we considered the expression changes induced by the imposed stress on a group of fungal genes playing a key role in the water-transport process. Taken together, the results show that AM symbiosis positively affects the tolerance to WS in tomato, with a different plant response depending on the AM fungi species involved.

As one of the most important food source for humans, rice has a relevant social and economic role worldwide. Understanding rice responses to the interaction with arbuscular mycorrhizal (AM) fungi has gained increasing importance in order to enhance plant yield. While it is known that AM fungi do not preferentially colonize flooded roots, the mechanisms which control this phenomenon remain largely unknown. The goal of our work is to elucidate, through morphological and molecular approaches, the factors which control colonization dynamics of rice roots by AM fungi in different water regimes such as flooding and dry conditions. Using controlled growth systems, two sets of mycorrhizal and control rice plants were grown in both water regimes, considering also the transfer from water to dry and viceversa. Roots from all biological conditions were sampled at different time points, starting from 7 dpi (days after inoculation with Glomus intraradices) to 35 dpi. We evaluated both morphological and molecular parameters, such as root branching (the ratio between the number of large lateral roots and the crown ones), the mycorrhizal colonization level, and the expression of plant and fungal functional marker genes. As expected, mycorrhizal colonization decreased with flooding, while differences on root anatomy between dry and flooding conditions started from 7 dpi, leading to a more important branching under dry conditions. In mycorrhizal plants, root branching was significantly higher as a consequence of the fungal presence added to the effect of the dry soil. In addition, the root apparatus of plants shifted from the water condition to the dry rapidly adapted to the new condition revealing a high organ plasticity. Quantitative real time PCRs performed on plant and fungal P and N transporters revealed that symbiosis functionality was directly linked to the root anatomy and the success of mycorrhizal colonization. However, even if reduced, the expression of fungal P and N transporters was maintained also in the flooding condition. In conclusion, our experiments demonstrate that the water regime influences the fungal colonization, and therefore the expression of symbiosis marker genes, first modifying root morphology.

International audience; Molecular pathways governing the life cycle of arbuscular mycorrhizal (AM) fungi and their symbiotic interactions with root tissues are not yet fully understood. Most studies fo fungal responses to host plants have targeted developmental stages before root contact (germinating spores), or after root colonization (intraradical mycelium). We are focusing on the early cell events of appressoria contact with the root surface which are essential to the successful outcome of the AM symbiosis. Recent monitoring of Glomus intraradices gene expression at this stage has revealed differential fungal responses to roots of host and non-host (Myc- mutants) M. truncatula (Seddas et al. submitted), suggesting a fine regulation of fungal genes by the host plant. Transcription factors are central regulators of gene expression which control many physiological and developmental processes but little is known of these elements in AM fungi. We have identified seven sequences in G. intraradices with similarity to genes encoding transcription factors in other fungi, by homology searches in databases. Transcript profiling of the corresponding genes in appressoria formed on roots of wild-type or Myc- mutans of M. truncatula points to their differential expression when symbiosis-related plant genes are inactivated, suggesting that the encoded transcription factors could be implicated in regulating early events leading to root colonization. Characterisation of the corresponding genes and analysis of transcription factor function is on-going in order to obtain new insight into their role in symbiosis establishment.

The establishment of a symbiotic interaction between plant roots and arbuscular mycorrhizal (AM) fungi requires both partners to undergo significant morphological and physiological modifications which eventually lead to reciprocal beneficial effects. Extensive changes in gene expression profiles recently have been described in transcriptomic studies that have analyzed the whole mycorrhizal root. However, because root colonization by AM fungi involves different cell types, a cell-specific gene expression pattern is likely to occur. We have applied the laser microdissection (LMD) technology to investigate expression profiles of both plant and fungal genes in Lycopersicon esculentum roots colonized by Glomus mosseae. A protocol to harvest arbuscule-containing cells from paraffin sections of mycorrhizal roots has been developed using a Leica AS LMD system. RNA of satisfactory quantity and quality has been extracted for molecular analysis. Transcripts for plant phosphate transporters (LePTs), selected as molecular markers for a functional symbiosis, have been detected by reverse-transcriptase polymerase chain reaction assays and associated to distinct cell types, leading to novel insights into the distribution of LePT mRNAs. In fact, the transcripts of the five phosphate transporters (PTs) have been detected contemporaneously in the same arbusculated cell population, unlike from the neighboring noncolonized cells. In addition, fungal H(+)ATPase (GmHA5) and phosphate transporter (GmosPT) mRNAs were found exclusively in arbusculated cells. The discovery that five plant and one fungal PT genes are consistently expressed inside the arbusculated cells provides a new scenario for plant-fungus nutrient exchanges.

The hypothesis of the present study was that bacterial communities would differentiate under Eucalyptus camaldulensis and that an enhancement of arbuscular mycorrhizal (AM) density would minimize this exotic plant species effect. Treatments consisted of control plants, preplanting fertilizer application and AM inoculation. After 4 months of culture in autoclaved soil, E. camaldulensis seedlings were either harvested for growth measurement or transferred into containers filled with the same soil but not sterilized. Other containers were kept without E. camaldulensis seedlings. After 12 months, effects of fertilizer amendment and AM inoculation were measured on the growth of Eucalyptus seedlings and on soil microbial communities. The results clearly show that this plant species significantly modified the soil bacterial community. Both community structure (assessed by denaturing gradient gel electrophoresis profiles) and function (assessed by substrate-induced respiration responses including soil catabolic evenness) were significantly affected. Such changes in the bacterial structure and function were accompanied by disturbances in the composition of the herbaceous plant species layer. These results highlight the role of AM symbiosis in the processes involved in soil bio-functioning and plant coexistence and in afforestation programmes with exotic tree species that target preservation of native plant diversity.