Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

STUDY QUESTION: What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? SUMMARY ANSWER: Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. WHAT IS KNOWN ALREADY: The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. PARTICIPANTS/MATERIALS, SETTING, METHODS: (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. STUDY FUNDING/COMPETING INTEREST(S): This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Profiling oocytes with neural networks from images and mechanical data.

The success rate of assisted reproductive technologies could be greatly improved by selectively choosing egg cells (oocytes) with the greatest chance of fertilization. The goal of mechanical profiling is, thus, to improve predictive oocyte selection by isolating the mechanical properties of oocytes and correlating them to their reproductive potential. The restrictions on experimental platforms, however - including minimal invasiveness and practicality in laboratory implementation - greatly limits the data that can be acquired from a single oocyte. In this study, we perform indentation studies on human oocytes and characterize the mechanical properties of the zona pellucida, the outer layer of the oocyte. We obtain excellent fitting with our physical model when indenting with a flat surface and clearly illustrate localized shear-thinning behavior of the zona pellucida, which has not been previously reported. We conclude by outlining a promising methodology for isolating the mechanical properties of the cytoplasm using neural networks and optical images taken during indentation.

Impact of the COVID-19 pandemic on fertility preservation activities in France: A survey by the Groupe de Recherche et d'Etude sur la Conservation Ovarienne et Testiculaire (GRECOT; group for research and studies on ovarian and testicular preservation).

BACKGROUND: To study the repercussions of the COVID-19 pandemic for fertility preservation activities in France. BASIC PROCEDURES: A questionnaire was sent to all the fertility preservation centres, requesting, for fertility preservation techniques (gamete and gonadal tissue preservation), the number of patients managed before, during and after the lockdown, and the number of patients who were not able to have access to these techniques and thus suffered definitive losses of fertility, during the lockdown period in spring 2020. MAIN FINDINGS: Fertility preservation activities in France did not cease entirely during the lockdown, but a 42.6% decrease in activity was observed. After lockdown, the levels of sperm, testicular and ovarian tissue cryopreservation returned to pre-lockdown levels (95.2%). The restoration of activity was partial only for oocyte freezing, which reached a level 56.8% that before lockdown. In total, 45 patients (8.35%) lost all chance of fertility preservation during the lockdown period. PRINCIPAL CONCLUSIONS: In France, fertility preservation activities were significantly affected by the lockdown in spring 2020 linked to the COVID-19 pandemic.

Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients.

BACKGROUND: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. RESULTS: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. CONCLUSIONS: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Regulation of mating genes during arbuscular mycorrhizal isolate co-existence-where is the evidence?

A recent study published by Mateus et al. [1] claimed that 18 "mating-related" genes are differentially expressed in the model arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis when genetically distinct fungal strains co-colonize a host plant. To clarify the level of evidence for this interesting conclusion, we first aimed to validate the functional annotation of these 18 R. irregularis genes using orthology predictions. These analyses revealed that, although sequence relationship exists, only 2 of the claimed 18 R. irregularis mating genes are potential orthologues to validated fungal mating genes. We also investigated the RNA-seq data from Mateus et al. [1] using classical RNA-seq methods and statistics. This analysis found that the over-expression during strain co-existence was not significant at the typical cut-off of the R. irregularis strains DAOM197198 and B1 in plants. Overall, we do not find convincing evidence that the genes involved have functions in mating, or that they are reproducibly up or down regulated during co-existence in plants.

The genome of Geosiphon pyriformis reveals ancestral traits linked to the emergence of the arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) (subphylum Glomeromycotina)1 are among the most prominent symbionts and form the Arbuscular Mycorrhizal symbiosis (AMS) with over 70% of known land plants.2,3 AMS allows plants to efficiently acquire poorly soluble soil nutrients4 and AMF to receive photosynthetically fixed carbohydrates. This plant-fungus symbiosis dates back more than 400 million years5 and is thought to be one of the key innovations that allowed the colonization of lands by plants.6 Genomic and genetic analyses of diverse plant species started to reveal the molecular mechanisms that allowed the evolution of this symbiosis on the host side, but how and when AMS abilities emerged in AMF remain elusive. Comparative phylogenomics could be used to understand the evolution of AMS.7,8 However, the availability of genome data covering basal AMF phylogenetic nodes (Archaeosporales, Paraglomerales) is presently based on fragmentary protein coding datasets.9Geosiphon pyriformis (Archaeosporales) is the only fungus known to produce endosymbiosis with nitrogen-fixing cyanobacteria (Nostoc punctiforme) presumably representing the ancestral AMF state.10-12 Unlike other AMF, it forms long fungal cells ("bladders") that enclose cyanobacteria. Once in the bladder, the cyanobacteria are photosynthetically active and fix nitrogen, receiving inorganic nutrients and water from the fungus. Arguably, G. pyriformis represents an ideal candidate to investigate the origin of AMS and the emergence of a unique endosymbiosis. Here, we aimed to advance knowledge in these questions by sequencing the genome of G. pyriformis, using a re-discovered isolate.

Lineage-Specific Genes and Cryptic Sex: Parallels and Differences between Arbuscular Mycorrhizal Fungi and Fungal Pathogens.

Arbuscular mycorrhizal fungi (AMF) live as obligate root symbionts on almost all land plants. They have long been regarded as ancient asexuals that have propagated clonally for millions of years. However, genomic studies in Rhizophagus irregularis and other AMF revealed many features indicative of sex. Surprisingly, comparative genomics of conspecific isolates of R. irregularis revealed an unexpected interstrain diversity, suggesting that AMF carry a high number of lineage-specific (LS) genes. Intriguingly, cryptic sex and LS genomic regions have previously been reported in a number of fungal pathogens of plants and humans. Here, we discuss these genomic similarities and highlight their potential relevance for AMF adaptation to the environment and for symbiotic functioning.

Characterization of the microbiome associated with in situ earthen materials.

BACKGROUND: The current increase in public awareness of environmental risks is giving rise to a growth of interest in the microbiological safety of buildings. In particular, microbial proliferation on construction materials can be responsible for the degradation of indoor air quality that can increase health-risk to occupants. Raw earth materials are still widely used throughout the world and, in some cases, are linked to heritage habitats, as in the southwest of France. Moreover, these building materials are currently the subject of renewed interest for ecological and economic reasons. However, the microbial status of earthen materials raises major concerns: could the microbiome associated with such natural materials cause disease in building occupants? Very few analyses have been performed on the microbial communities present on these supports. Characterizing the raw earth material microbiome is also important for a better evaluation and understanding of the susceptibility of such materials to microbial development. This study presents the distribution of in situ bacterial and fungal communities on different raw earth materials used in construction. Various buildings were sampled in France and the microbial communities present were characterized by amplicon high-throughput sequencing (bacterial 16S rRNA gene and fungal ITS1 region). Bacterial culture isolates were identified at the species level by MALDI-TOF mass spectrometry. RESULTS: The major fungal and bacterial genera identified were mainly associated with conventional outdoor and indoor environmental communities, and no specific harmful bacterial species were detected on earthen materials. However, contrary to expectations, few human-associated genera were detected in dwellings. We found lower microbial alpha-diversity in earthen material than is usually found in soil, suggesting a loss of diversity during the use of these materials in buildings. Interestingly enough, the main features influencing microbial communities were building history and room use, rather than material composition. CONCLUSIONS: These results constitute a first in-depth analysis of microbial communities present on earthen materials in situ and may be considered as a first referential to investigate microbial communities on such materials according to environmental conditions and their potential health impact. The bacterial and fungal flora detected were similar to those found in conventional habitats and are thought to be mainly impacted by specific events in the building's life, such as water damage.

Development of Ovarian Tissue Autograft to Restore Ovarian Function: Protocol for a French Multicenter Cohort Study.

BACKGROUND: Sterility is a major late effect of radiotherapy and chemotherapy treatments. Iatrogenic sterility is often permanent and greatly impacts long-term quality of life. Ovarian tissue cryopreservation (OTC) performed before gonadotoxic treatments with subsequent autograft is a method of fertility preservation available for girls and women. Its application in prepubertal girls is of particular value as it is the only possible approach in this patient group. In addition, it does not require a delay in cancer therapy and no ovarian stimulation is needed. OBJECTIVE: The primary aim of this protocol is to help increase the implementation of ovarian tissue autografting in France. Knowledge is still lacking regarding the efficacy of ovarian transplantation in restoring ovarian function and regarding the safety of this procedure, especially the risk of cancer cell reseeding in certain types of cancer. A secondary aim of this study is to generate data to improve our understanding of these two essential aspects. METHODS: The DATOR (Development of Ovarian Tissue Autograft in Order to Restore Ovarian Function) study is ongoing in 17 university hospitals. The DATOR protocol includes the autograft of ovarian cortex fragments. Candidates are identified from an observational prospective cohort (called the Prospective Cohort of Patients Candidates for Ovarian Tissue Autograft [PERIDATOR]) of patients who have undergone OTC. Enrollment in the study is initiated at the patient's request and must be validated by the center's multidisciplinary team and by the study steering committee. The DATOR study begins with a total medical checkup. Ovarian tissue qualification and residual disease detection, if required, are performed. RESULTS: The study is ongoing. Currently, 38 patients have provided informed consent and have been entered into the DATOR study. Graft has been performed for 34 of these patients. An interim analysis was conducted on the first 25 patients for whom the period of at least 1 year posttransplantation was achieved. Out of these 25 patients, 11 women succeeded in becoming pregnant (pregnancy rate=44% [11/25]; delivery rate=40% [10/25]). Among these, 6 women conceived twice, and 1 pregnancy led to a miscarriage. CONCLUSIONS: Our preliminary analysis appears to be coherent with the accumulating body of evidence indicating the potential utility of ovarian tissue autograft for patients with premature ovarian failure. All these elements justify the pursuit of our study. TRIAL REGISTRATION: ClinicalTrials.gov NCT02846064; https://clinicaltrials.gov/ct2/show/NCT02846064. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/12944.

Deciphering the phylogeny of violets based on multiplexed genetic and metabolomic approaches.

Molecular phylogenetics based on nucleotide sequence comparisons has profoundly influenced plant taxonomy. A comprehensive chemotaxonomical approach based on GC-MS and UHPLC-HRMS profiling was evaluated for its ability to characterize a large collection of plants all in the violet family Violaceae (n = 111) and thus decipher the taxonomy. A thorough identification of violets is challenging due to their natural hybridization and phenotypic variability. Phylogenetic inference performed on ribosomal internal transcribed spacer sequences using maximum likelihood and neighbor-joining distance methods allowed the clear identification of 58% of the collection. Metabolomic approaches with multivariate data analysis were performed on SPME/GC-MS chromatograms of volatile compounds emitted by fresh mature flowers and on UHPLC-HRMS/MS leaf extracts for non-volatile compounds. Interestingly, molecular and biochemical approaches provided separate classifications while highlighting several common clusters. The profiling of secondary metabolites was proved most suitable for the classification of hundreds of extracts. The combination of phylogenetic and chemotaxonomic approaches, allowed the classification of 96% of the entire collection. A correlation network revealed specific chemotaxonomic biomarkers, in particular flavonoids, coumarins and cyclotides. Overall, our pioneering approach could be useful to solve misclassification issues within collections of close plant species.

Correction to: Identification of new signalling peptides through a genome-wide survey of 250 fungal secretomes.

Following the publication of this article [1] the authors noted that the image in Fig. 1 was incorrect.

Comparative genomics of Rhizophagus irregularis, R. cerebriforme, R. diaphanus and Gigaspora rosea highlights specific genetic features in Glomeromycotina.

Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.

Identification of new signalling peptides through a genome-wide survey of 250 fungal secretomes.

BACKGROUND: Many small peptides regulate eukaryotic cell biology. In fungi, some of these peptides are produced after KEX2 protease activity on proteins displaying repetitions of identical or nearly identical motifs. Following this endoprotease activity, peptides are released in the extracellular space. This type of protein maturation is involved in the production of the α-type sexual pheromone in Ascomycota. In other cases, this processing allows the production of secreted peptides regulating fungal cell wall structure or acting as mycotoxins. In this work, we report for the first time a genome-wide search of KEX2-processed repeat proteins that we call KEPs. We screened the secreted proteins of 250 fungal species to compare their KEP repertoires with regard to their lifestyle, morphology or lineage. RESULTS: Our analysis points out that nearly all fungi display putative KEPs, suggesting an ancestral origin common to all opisthokonts. As expected, our pipeline identifies mycotoxins but also α-type sexual pheromones in Ascomycota that have not been explored so far, and unravels KEP-derived secreted peptides of unknown functions. Some species display an expansion of this class of proteins. Interestingly, we identified conserved KEPs in pathogenic fungi, suggesting a role in virulence. We also identified KEPs in Basidiomycota with striking similarities to Ascomycota α-type sexual pheromones, suggesting they may also play alternative roles in unknown signalling processes. CONCLUSIONS: We identified putative, new, unexpected secreted peptides that fall into different functional categories: mycotoxins, hormones, sexual pheromones, or effectors that promote colonization during host-microbe interactions. This wide survey will open new avenues in the field of small-secreted peptides in fungi that are critical regulators of their intimate biology and modulators of their interaction with the environment.

In silico definition of new ligninolytic peroxidase sub-classes in fungi and putative relation to fungal life style.

Ligninolytic peroxidases are microbial enzymes involved in depolymerisation of lignin, a plant cell wall polymer found in land plants. Among fungi, only Dikarya were found to degrade lignin. The increase of available fungal genomes allows performing an expert annotation of lignin-degrading peroxidase encoding sequences with a particular focus on Class II peroxidases (CII Prx). In addition to the previously described LiP, MnP and VP classes, based on sequence similarity, six new sub-classes have been defined: three found in plant pathogen ascomycetes and three in basidiomycetes. The presence of CII Prxs could be related to fungal life style. Typically, necrotrophic or hemibiotrophic fungi, either ascomycetes or basidiomycetes, possess CII Prxs while symbiotic, endophytic or biotrophic fungi do not. CII Prxs from ascomycetes are rarely subjected to duplications unlike those from basidiomycetes, which can form large recent duplicated families. Even if these CII Prxs classes form two well distinct clusters with divergent gene structures (intron numbers and positions), they share the same key catalytic residues suggesting that they evolved independently from similar ancestral sequences with few or no introns. The lack of CII Prxs encoding sequences in early diverging fungi, together with the absence of duplicated class I peroxidase (CcP) in fungi containing CII Prxs, suggests the potential emergence of an ancestral CII Prx sequence from the duplicated CcP after the separation between ascomycetes and basidiomycetes. As some ascomycetes and basidiomycetes did not possess CII Prx, late gene loss could have occurred.

Imbalanced Regulation of Fungal Nutrient Transports According to Phosphate Availability in a Symbiocosm Formed by Poplar, Sorghum, and Rhizophagus irregularis.

In arbuscular mycorrhizal (AM) symbiosis, key components of nutrient uptake and exchange are specialized transporters that facilitate nutrient transport across membranes. As phosphate is a nutrient and a regulator of nutrient exchanges, we investigated the effect of P availability to extraradical mycelium (ERM) on both plant and fungus transcriptomes and metabolomes in a symbiocosm system. By perturbing nutrient exchanges under the control of P, our objectives were to identify new fungal genes involved in nutrient transports, and to characterize in which extent the fungus differentially modulates its metabolism when interacting with two different plant species. We performed transportome analysis on the ERM and intraradical mycelium of the AM fungus Rhizophagus irregularis associated to Populus trichocarpa and Sorghum bicolor under high and low P availability in ERM, using quantitative RT-PCR and Illumina mRNA-sequencing. We observed that mycorrhizal symbiosis induces expression of specific phosphate and ammonium transporters in both plants. Furthermore, we identified new AM-inducible transporters and showed that a subset of phosphate transporters is regulated independently of symbiotic nutrient exchange. mRNA-Sequencing revealed that the fungal transportome was not similarly regulated in the two host plant species according to P availability. Mirroring this effect, many plant carbohydrate transporters were down-regulated in P. trichocarpa mycorrhizal root tissue. Metabolome analysis revealed further that AM root colonization led to a modification of root primary metabolism under low and high P availability and to a decrease of primary metabolite pools in general. Moreover, the down regulation of the sucrose transporters suggests that the plant limits carbohydrate long distance transport (i.e. from shoot to the mycorrhizal roots). By simultaneous uptake/reuptake of nutrients from the apoplast at the biotrophic interface, plant and fungus are both able to control reciprocal nutrient fluxes.

Arbuscular mycorrhizal fungi: intraspecific diversity and pangenomes.

Contents Summary 1129 I. Introduction 1129 II. Intraspecific phenotypic variation and the plant host 1130 III. High inter-isolate genetic diversity in model AMF 1130 IV. Genome diversity within the model AM fungus Rhizophagus irregularis 1131 V. Pangenomes and the future of AMF ecological genomics 1131 Acknowledgements 1133 References 1133 SUMMARY: Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant symbionts with an intriguing population biology. Conspecific AMF strains can vary substantially at the genetic and phenotypic levels, leading to direct and quantifiable variation in plant growth. Recent studies have shown that high intraspecific diversity is very common in AMF, and not only found in model species. Studies have also revealed how the phenotype of conspecific isolates varies depending on the plant host, highlighting the functional relevance of intraspecific phenotypic plasticity for the AMF ecology and mycorrhizal symbiosis. Recent work has also demonstrated that conspecific isolates of the model AMF Rhizophagus irregularis harbor large and highly variable pangenomes, highlighting the potential role of intraspecific genome diversity for the ecological adaptation of these symbionts.