Publications by authors named "Emilie Tisserant"

31 Publications

Genotype-first in a cohort of 95 fetuses with multiple congenital abnormalities: when exome sequencing reveals unexpected fetal phenotype-genotype correlations.

J Med Genet 2021 06 30;58(6):400-413. Epub 2020 Jul 30.

Service d'Imagerie médicale, CHU de Besançon, Besançon, France.

Purpose: Molecular diagnosis based on singleton exome sequencing (sES) is particularly challenging in fetuses with multiple congenital abnormalities (MCA). Indeed, some studies reveal a diagnostic yield of about 20%, far lower than in live birth individuals showing developmental abnormalities (30%), suggesting that standard analyses, based on the correlation between clinical hallmarks described in postnatal syndromic presentations and genotype, may underestimate the impact of the genetic variants identified in fetal analyses.

Methods: We performed sES in 95 fetuses with MCA. Blind to phenotype, we applied a genotype-first approach consisting of combined analyses based on variants annotation and bioinformatics predictions followed by reverse phenotyping. Initially applied to OMIM-morbid genes, analyses were then extended to all genes. We complemented our approach by using reverse phenotyping, variant segregation analysis, bibliographic search and data sharing in order to establish the clinical significance of the prioritised variants.

Results: sES rapidly identified causal variant in 24/95 fetuses (25%), variants of unknown significance in OMIM genes in 8/95 fetuses (8%) and six novel candidate genes in 6/95 fetuses (6%).

Conclusions: This method, based on a genotype-first approach followed by reverse phenotyping, shed light on unexpected fetal phenotype-genotype correlations, emphasising the relevance of prenatal studies to reveal extreme clinical presentations associated with well-known Mendelian disorders.
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http://dx.doi.org/10.1136/jmedgenet-2020-106867DOI Listing
June 2021

De novo SMARCA2 variants clustered outside the helicase domain cause a new recognizable syndrome with intellectual disability and blepharophimosis distinct from Nicolaides-Baraitser syndrome.

Genet Med 2020 11 22;22(11):1838-1850. Epub 2020 Jul 22.

Department of Genetics, Robert Debré Hospital, AP-HP, Paris, France.

Purpose: Nontruncating variants in SMARCA2, encoding a catalytic subunit of SWI/SNF chromatin remodeling complex, cause Nicolaides-Baraitser syndrome (NCBRS), a condition with intellectual disability and multiple congenital anomalies. Other disorders due to SMARCA2 are unknown.

Methods: By next-generation sequencing, we identified candidate variants in SMARCA2 in 20 individuals from 18 families with a syndromic neurodevelopmental disorder not consistent with NCBRS. To stratify variant interpretation, we functionally analyzed SMARCA2 variants in yeasts and performed transcriptomic and genome methylation analyses on blood leukocytes.

Results: Of 20 individuals, 14 showed a recognizable phenotype with recurrent features including epicanthal folds, blepharophimosis, and downturned nasal tip along with variable degree of intellectual disability (or blepharophimosis intellectual disability syndrome [BIS]). In contrast to most NCBRS variants, all SMARCA2 variants associated with BIS are localized outside the helicase domains. Yeast phenotype assays differentiated NCBRS from non-NCBRS SMARCA2 variants. Transcriptomic and DNA methylation signatures differentiated NCBRS from BIS and those with nonspecific phenotype. In the remaining six individuals with nonspecific dysmorphic features, clinical and molecular data did not permit variant reclassification.

Conclusion: We identified a novel recognizable syndrome named BIS associated with clustered de novo SMARCA2 variants outside the helicase domains, phenotypically and molecularly distinct from NCBRS.
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http://dx.doi.org/10.1038/s41436-020-0898-yDOI Listing
November 2020

Second-tier trio exome sequencing after negative solo clinical exome sequencing: an efficient strategy to increase diagnostic yield and decipher molecular bases in undiagnosed developmental disorders.

Hum Genet 2020 Nov 12;139(11):1381-1390. Epub 2020 May 12.

UFR Des Sciences de Santé, INSERM-Université de Bourgogne UMR1231 GAD, FHU-TRANSLAD, Bâtiment B3, 15 avenue du maréchal Delattre de Tassigny, 21000, Dijon, France.

Developmental disorders (DD), characterized by malformations/dysmorphism and/or intellectual disability, affecting around 3% of worldwide population, are mostly linked to genetic anomalies. Despite clinical exome sequencing (cES) centered on genes involved in human genetic disorders, the majority of patients affected by DD remain undiagnosed after solo-cES. Trio-based strategy is expected to facilitate variant selection thanks to rapid parental segregation. We performed a second step trio-ES (not only focusing on genes involved in human disorders) analysis in 70 patients with negative results after solo-cES. All candidate variants were shared with a MatchMaking exchange system to identify additional patients carrying variants in the same genes and with similar phenotype. In 18/70 patients (26%), we confirmed causal implication of nine OMIM-morbid genes and identified nine new strong candidate genes (eight de novo and one compound heterozygous variants). These nine new candidate genes were validated through the identification of patients with similar phenotype and genotype thanks to data sharing. Moreover, 11 genes harbored variants of unknown significance in 10/70 patients (14%). In DD, a second step trio-based ES analysis appears an efficient strategy in diagnostic and translational research to identify highly candidate genes and improve diagnostic yield.
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http://dx.doi.org/10.1007/s00439-020-02178-8DOI Listing
November 2020

Further delineation of the female phenotype with KDM5C disease causing variants: 19 new individuals and review of the literature.

Clin Genet 2020 07 29;98(1):43-55. Epub 2020 May 29.

INSERM UMR1231, Equipe Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France.

X-linked intellectual disability (XLID) is a genetically heterogeneous condition involving more than 100 genes. To date, 35 pathogenic variants have been reported in the lysine specific demethylase 5C (KDM5C) gene. KDM5C variants are one of the major causes of moderate to severe XLID. Affected males present with short stature, distinctive facial features, behavioral disorders, epilepsy, and spasticity. For most of these variants, related female carriers have been reported, but phenotypic descriptions were poor. Here, we present clinical and molecular features of 19 females carrying 10 novel heterozygous variants affecting KDM5C function, including five probands with de novo variants. Four heterozygous females were asymptomatic. All affected individuals presented with learning disabilities or ID (mostly moderate), and four also had a language impairment mainly affecting expression. Behavioral disturbances were frequent, and endocrine disorders were more frequent in females. In conclusion, our findings provide evidence of the role of KDM5C in ID in females highlighting the increasing implication of XLID genes in females, even in sporadic affected individuals. Disease expression of XLID in females should be taken into consideration for genetic counseling.
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http://dx.doi.org/10.1111/cge.13755DOI Listing
July 2020

De novo TBR1 variants cause a neurocognitive phenotype with ID and autistic traits: report of 25 new individuals and review of the literature.

Eur J Hum Genet 2020 06 31;28(6):770-782. Epub 2020 Jan 31.

Department of Pediatrics, The Barbara Bush Children's Hospital, Maine Medical Center, Portland, OR, USA.

TBR1, a T-box transcription factor expressed in the cerebral cortex, regulates the expression of several candidate genes for autism spectrum disorders (ASD). Although TBR1 has been reported as a high-confidence risk gene for ASD and intellectual disability (ID) in functional and clinical reports since 2011, TBR1 has only recently been recorded as a human disease gene in the OMIM database. Currently, the neurodevelopmental disorders and structural brain anomalies associated with TBR1 variants are not well characterized. Through international data sharing, we collected data from 25 unreported individuals and compared them with data from the literature. We evaluated structural brain anomalies in seven individuals by analysis of MRI images, and compared these with anomalies observed in TBR1 mutant mice. The phenotype included ID in all individuals, associated to autistic traits in 76% of them. No recognizable facial phenotype could be identified. MRI analysis revealed a reduction of the anterior commissure and suggested new features including dysplastic hippocampus and subtle neocortical dysgenesis. This report supports the role of TBR1 in ID associated with autistic traits and suggests new structural brain malformations in humans. We hope this work will help geneticists to interpret TBR1 variants and diagnose ASD probands.
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http://dx.doi.org/10.1038/s41431-020-0571-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253452PMC
June 2020

Genome sequencing in cytogenetics: Comparison of short-read and linked-read approaches for germline structural variant detection and characterization.

Mol Genet Genomic Med 2020 03 27;8(3):e1114. Epub 2020 Jan 27.

Centre National de Recherche en Génomique Humaine (CNRGH), CEA, Evry, France.

Background: Structural variants (SVs) include copy number variants (CNVs) and apparently balanced chromosomal rearrangements (ABCRs). Genome sequencing (GS) enables SV detection at base-pair resolution, but the use of short-read sequencing is limited by repetitive sequences, and long-read approaches are not yet validated for diagnosis. Recently, 10X Genomics proposed Chromium, a technology providing linked-reads to reconstruct long DNA fragments and which could represent a good alternative. No study has compared short-read to linked-read technologies to detect SVs in a constitutional diagnostic setting yet. The aim of this work was to determine whether the 10X Genomics technology enables better detection and comprehension of SVs than short-read WGS.

Methods: We included 13 patients carrying various SVs. Whole genome analyses were performed using paired-end HiSeq X sequencing with (linked-read strategy) or without (short-read strategy) Chromium library preparation. Two different bioinformatic pipelines were used: Variants are called using BreakDancer for short-read strategy and LongRanger for long-read strategy. Variant interpretations were first blinded.

Results: The short-read strategy allowed diagnosis of known SV in 10/13 patients. After unblinding, the linked-read strategy identified 10/13 SVs, including one (patient 7) missed by the short-read strategy.

Conclusion: In conclusion, regarding the results of this study, 10X Genomics solution did not improve the detection and characterization of SV.
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http://dx.doi.org/10.1002/mgg3.1114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057128PMC
March 2020

Deciphering exome sequencing data: Bringing mitochondrial DNA variants to light.

Hum Mutat 2019 12 26;40(12):2430-2443. Epub 2019 Aug 26.

INSERM-University of Burgundy-Franche Comté, UMR1231 GAD, Dijon, France.

The expanding use of exome sequencing (ES) in diagnosis generates a huge amount of data, including untargeted mitochondrial DNA (mtDNA) sequences. We developed a strategy to deeply study ES data, focusing on the mtDNA genome on a large unspecific cohort to increase diagnostic yield. A targeted bioinformatics pipeline assembled mitochondrial genome from ES data to detect pathogenic mtDNA variants in parallel with the "in-house" nuclear exome pipeline. mtDNA data coming from off-target sequences (indirect sequencing) were extracted from the BAM files in 928 individuals with developmental and/or neurological anomalies. The mtDNA variants were filtered out based on database information, cohort frequencies, haplogroups and protein consequences. Two homoplasmic pathogenic variants (m.9035T>C and m.11778G>A) were identified in 2 out of 928 unrelated individuals (0.2%): the m.9035T>C (MT-ATP6) variant in a female with ataxia and the m.11778G>A (MT-ND4) variant in a male with a complex mosaic disorder and a severe ophthalmological phenotype, uncovering undiagnosed Leber's hereditary optic neuropathy (LHON). Seven secondary findings were also found, predisposing to deafness or LHON, in 7 out of 928 individuals (0.75%). This study demonstrates the usefulness of including a targeted strategy in ES pipeline to detect mtDNA variants, improving results in diagnosis and research, without resampling patients and performing targeted mtDNA strategies.
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http://dx.doi.org/10.1002/humu.23885DOI Listing
December 2019

Secondary actionable findings identified by exome sequencing: expected impact on the organisation of care from the study of 700 consecutive tests.

Eur J Hum Genet 2019 08 24;27(8):1197-1214. Epub 2019 Apr 24.

Centre National de Recherche en Génomique Humaine, Evry, France.

With exome/genome sequencing (ES/GS) integrated into the practice of medicine, there is some potential for reporting incidental/secondary findings (IFs/SFs). The issue of IFs/SFs has been studied extensively over the last 4 years. In order to evaluate their implications in care organisation, we retrospectively evaluated, in a cohort of 700 consecutive probands, the frequency and burden of introducing the search for variants in a maximum list of 244 medically actionable genes (genes that predispose carriers to a preventable or treatable disease in childhood/adulthood and genes for genetic counselling issues). We also focused on the 59 PharmGKB class IA/IB pharmacogenetic variants. We also compared the results in different gene lists. We identified variants (likely) affecting protein function in genes for care in 26 cases (3.7%) and heterozygous variants in genes for genetic counselling in 29 cases (3.8%). Mean time for the 700 patients was about 6.3 min/patient for medically actionable genes and 1.3 min/patient for genes for genetic counselling, and a mean time of 37 min/patients for the reinterpreted variants. These results would lead to all 700 pre-test counselling sessions being longer, to 55 post-test genetic consultations and to 27 secondary specialised medical evaluations. ES also detected 42/59 pharmacogenetic variants or combinations of variants in the majority of cases. An extremely low metabolizer status in genes relevant for neurodevelopmental disorders (CYP2C9 and CYP2C19) was found in 57/700 cases. This study provides information regarding the need to anticipate the implementation of genomic medicine, notably the work overload at various steps of the process.
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http://dx.doi.org/10.1038/s41431-019-0384-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777608PMC
August 2019

Clinical whole-exome sequencing for the diagnosis of rare disorders with congenital anomalies and/or intellectual disability: substantial interest of prospective annual reanalysis.

Genet Med 2018 06 2;20(6):645-654. Epub 2017 Nov 2.

Centre de Génétique et Centre de Référence "Anomalies du Développement et Syndromes Malformatifs," Hôpital d'Enfants, Centre Hospitalier Universitaire de Dijon, Dijon, France.

PurposeCongenital anomalies and intellectual disability (CA/ID) are a major diagnostic challenge in medical genetics-50% of patients still have no molecular diagnosis after a long and stressful diagnostic "odyssey." Solo clinical whole-exome sequencing (WES) was applied in our genetics center to improve diagnosis in patients with CA/ID.MethodsThis retrospective study examined 416 consecutive tests performed over 3 years to demonstrate the effectiveness of periodically reanalyzing WES data. The raw data from each nonpositive test was reanalyzed at 12 months with the most recent pipeline and in the light of new data in the literature. The results of the reanalysis for patients enrolled in the third year are not yet available.ResultsOf the 416 patients included, data for 156 without a diagnosis were reanalyzed. We obtained 24 (15.4%) additional diagnoses: 12 through the usual diagnostic process (7 new publications, 4 initially misclassified, and 1 copy-number variant), and 12 through translational research by international data sharing. The final yield of positive results was 27.9% through a strict diagnostic approach, and 2.9% through an additional research strategy.ConclusionThis article highlights the effectiveness of periodically combining diagnostic reinterpretation of clinical WES data with translational research involving data sharing for candidate genes.
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http://dx.doi.org/10.1038/gim.2017.162DOI Listing
June 2018

Soil networks become more connected and take up more carbon as nature restoration progresses.

Nat Commun 2017 02 8;8:14349. Epub 2017 Feb 8.

NIOO-KNAW, Microbial Ecology, Droevendaalsesteeg 10, Wageningen 6708 PB, The Netherlands.

Soil organisms have an important role in aboveground community dynamics and ecosystem functioning in terrestrial ecosystems. However, most studies have considered soil biota as a black box or focussed on specific groups, whereas little is known about entire soil networks. Here we show that during the course of nature restoration on abandoned arable land a compositional shift in soil biota, preceded by tightening of the belowground networks, corresponds with enhanced efficiency of carbon uptake. In mid- and long-term abandoned field soil, carbon uptake by fungi increases without an increase in fungal biomass or shift in bacterial-to-fungal ratio. The implication of our findings is that during nature restoration the efficiency of nutrient cycling and carbon uptake can increase by a shift in fungal composition and/or fungal activity. Therefore, we propose that relationships between soil food web structure and carbon cycling in soils need to be reconsidered.
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http://dx.doi.org/10.1038/ncomms14349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309817PMC
February 2017

Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum.

Nat Commun 2016 09 7;7:12662. Epub 2016 Sep 7.

Centre National de la Recherche Scientifique, UMR 7257, F-13288 Marseille, France.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.
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http://dx.doi.org/10.1038/ncomms12662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023957PMC
September 2016

The Bacterial and Fungal Diversity of an Aged PAH- and Heavy Metal-Contaminated Soil is Affected by Plant Cover and Edaphic Parameters.

Microb Ecol 2016 Apr 6;71(3):711-24. Epub 2015 Oct 6.

CNRS, LIEC UMR7360, Faculté des Sciences et Technologies, BP70239, 54506, Vandoeuvre-lès-Nancy, France.

Industrial wasteland soils with aged PAH and heavy metal contaminations are environments where pollutant toxicity has been maintained for decades. Although the communities may be well adapted to the presence of stressors, knowledge about microbial diversity in such soils is scarce. Soil microbial community dynamics can be driven by the presence of plants, but the impact of plant development on selection or diversification of microorganisms in these soils has not been established yet. To test these hypotheses, aged-contaminated soil samples from a field trial were collected. Plots planted with alfalfa were compared to bare soil plots, and bacterial and fungal diversity and abundance were assessed after 2 and 6 years. Using pyrosequencing of 16S rRNA gene and ITS amplicons, we showed that the bacterial community was dominated by Proteobacteria, Actinobacteria, and Bacteroidetes and was characterized by low Acidobacteria abundance, while the fungal community was mainly represented by members of the Ascomycota. The short-term toxic impact of pollutants usually reduces the microbial diversity, yet in our samples bacterial and fungal species richness and diversity was high suggesting that the community structure and diversity adapted to the contaminated soil over decades. The presence of plants induced higher bacterial and fungal diversity than in bare soil. It also increased the relative abundance of bacterial members of the Actinomycetales, Rhizobiales, and Xanthomonadales orders and of most fungal orders. Multivariate analysis showed correlations between microbial community structure and heavy metal and PAH concentrations over time, but also with edaphic parameters (C/N, pH, phosphorus, and nitrogen concentrations).
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http://dx.doi.org/10.1007/s00248-015-0682-8DOI Listing
April 2016

The Mutualist Laccaria bicolor Expresses a Core Gene Regulon During the Colonization of Diverse Host Plants and a Variable Regulon to Counteract Host-Specific Defenses.

Mol Plant Microbe Interact 2015 Mar;28(3):261-73

The coordinated transcriptomic responses of both mutualistic ectomycorrhizal (ECM) fungi and their hosts during the establishment of symbiosis are not well-understood. This study characterizes the transcriptomic alterations of the ECM fungus Laccaria bicolor during different colonization stages on two hosts (Populus trichocarpa and Pseudotsuga menziesii) and compares this to the transcriptomic variations of P. trichocarpa across the same time-points. A large number of L. bicolor genes (≥ 8,000) were significantly regulated at the transcriptional level in at least one stage of colonization. From our data, we identify 1,249 genes that we hypothesize is the 'core' gene regulon necessary for the mutualistic interaction between L. bicolor and its host plants. We further identify a group of 1,210 genes that are regulated in a host-specific manner. This variable regulon encodes a number of genes coding for proteases and xenobiotic efflux transporters that we hypothesize act to counter chemical-based defenses simultaneously activated at the transcriptomic level in P. trichocarpa. The transcriptional response of the host plant P. trichocarpa consisted of differential waves of gene regulation related to signaling perception and transduction, defense response, and the induction of nutrient transfer in P. trichocarpa tissues. This study, therefore, gives fresh insight into the shifting transcriptomic landscape in both the colonizing fungus and its host and the different strategies employed by both partners in orchestrating a mutualistic interaction.
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http://dx.doi.org/10.1094/MPMI-05-14-0129-FIDOI Listing
March 2015

Transcriptome analysis of poplar rust telia reveals overwintering adaptation and tightly coordinated karyogamy and meiosis processes.

Front Plant Sci 2013 21;4:456. Epub 2013 Nov 21.

INRA, UMR 1136, Interactions Arbres-Microorganismes Champenoux, France ; UMR 1136, Université de Lorraine, Interactions Arbres-Microorganismes Vandoeuvre-lès-Nancy, France.

Most rust fungi have a complex life cycle involving up to five different spore-producing stages. The telial stage that produces melanized overwintering teliospores is one of these and plays a fundamental role for generating genetic diversity as karyogamy and meiosis occur at that stage. Despite the importance of telia for the rust life cycle, almost nothing is known about the fungal genetic programs that are activated in this overwintering structure. In the present study, the transcriptome of telia produced by the poplar rust fungus Melampsora larici-populina has been investigated using whole genome exon oligoarrays and RT-qPCR. Comparative expression profiling at the telial and uredinial stages identifies genes specifically expressed or up-regulated in telia including osmotins/thaumatin-like proteins (TLPs) and aquaporins that may reflect specific adaptation to overwintering as well numerous lytic enzymes acting on plant cell wall, reflecting extensive cell wall remodeling at that stage. The temporal dynamics of karyogamy was followed using combined RT-qPCR and DAPI-staining approaches. This reveals that fusion of nuclei and induction of karyogamy-related genes occur simultaneously between the 25 and 39 days post inoculation time frame. Transcript profiling of conserved meiosis genes indicates a preferential induction right after karyogamy and corroborates that meiosis begins prior to overwintering and is interrupted in Meiosis I (prophase I, diplonema stage) until teliospore germination in early spring.
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http://dx.doi.org/10.3389/fpls.2013.00456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835972PMC
December 2013

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

Proc Natl Acad Sci U S A 2013 Dec 25;110(50):20117-22. Epub 2013 Nov 25.

Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1136, Interactions Arbres/Microorganismes, Centre de Nancy, Université de Lorraine, 54280 Champenoux, France.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.
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http://dx.doi.org/10.1073/pnas.1313452110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864322PMC
December 2013

Laser microdissection and microarray analysis of Tuber melanosporum ectomycorrhizas reveal functional heterogeneity between mantle and Hartig net compartments.

Environ Microbiol 2013 Jun 5;15(6):1853-69. Epub 2013 Feb 5.

UMR 1136 INRA/Université de Lorraine, Interactions Arbres/Micro-organismes, INRA, Institut National de la Recherche Agronomique, Centre INRA de Nancy, 54280 Champenoux, France.

The ectomycorrhizal (ECM) symbiosis, a mutualistic plant-fungus association, plays a fundamental role in forest ecosystems by enhancing plant growth and by providing host protection from root diseases. The cellular complexity of the symbiotic organ, characterized by the differentiation of structurally specialized tissues (i.e. the fungal mantle and the Hartig net), is the major limitation to study fungal gene expression in such specific compartments. We investigated the transcriptional landscape of the ECM fungus Tuber melanosporum during the major stages of its life cycle and we particularly focused on the complex symbiotic stage by combining the use of laser capture microdissection and microarray gene expression analysis. We isolated the fungal/soil (i.e. the mantle) and the fungal/plant (i.e. the Hartig net) interfaces from transverse sections of T. melanosporum/Corylus avellana ectomycorrhizas and identified the distinct genetic programmes associated with each compartment. Particularly, nitrogen and water acquisition from soil, synthesis of secondary metabolites and detoxification mechanisms appear to be important processes in the fungal mantle. In contrast, transport activity is enhanced in the Hartig net and we identified carbohydrate and nitrogen-derived transporters that might play a key role in the reciprocal resources' transfer between the host and the symbiont.
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http://dx.doi.org/10.1111/1462-2920.12080DOI Listing
June 2013

The AINTEGUMENTA LIKE1 homeotic transcription factor PtAIL1 controls the formation of adventitious root primordia in poplar.

Plant Physiol 2012 Dec 17;160(4):1996-2006. Epub 2012 Oct 17.

Institut National de la Recherche Agronomique and Lorraine University, Unité Mixte de Recherche Institut National de la Recherche Agronomique/Lorraine University 1136 Interactions Arbres/Micro-organismes, Institut National de la Recherche Agronomique-Nancy, 54280 Champenoux, France.

Adventitious rooting is an essential but sometimes rate-limiting step in the clonal multiplication of elite tree germplasm, because the ability to form roots declines rapidly with age in mature adult plant tissues. In spite of the importance of adventitious rooting, the mechanism behind this developmental process remains poorly understood. We have described the transcriptional profiles that are associated with the developmental stages of adventitious root formation in the model tree poplar (Populus trichocarpa). Transcriptome analyses indicate a highly specific temporal induction of the AINTEGUMENTA LIKE1 (PtAIL1) transcription factor of the AP2 family during adventitious root formation. Transgenic poplar samples that overexpressed PtAIL1 were able to grow an increased number of adventitious roots, whereas RNA interference mediated the down-expression of PtAIL1 expression, which led to a delay in adventitious root formation. Microarray analysis showed that the expression of 15 genes, including the transcription factors AGAMOUS-Like6 and MYB36, was overexpressed in the stem tissues that generated root primordia in PtAIL1-overexpressing plants, whereas their expression was reduced in the RNA interference lines. These results demonstrate that PtAIL1 is a positive regulator of poplar rooting that acts early in the development of adventitious roots.
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http://dx.doi.org/10.1104/pp.112.204453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510126PMC
December 2012

RNA-Seq of early-infected poplar leaves by the rust pathogen Melampsora larici-populina uncovers PtSultr3;5, a fungal-induced host sulfate transporter.

PLoS One 2012 30;7(8):e44408. Epub 2012 Aug 30.

Unité Mixte de Recherche 1136 'Interactions Arbres/Microorganismes', INRA (Institut National de la Recherche Agronomique)/Université de Lorraine, Centre INRA de Nancy, Champenoux, France.

Biotroph pathogens establish intimate interactions with their hosts that are conditioned by the successful secretion of effectors in infected tissues and subsequent manipulation of host physiology. The identification of early-expressed pathogen effectors and early-modulated host functions is currently a major goal to understand the molecular basis of biotrophy. Here, we report the 454-pyrosequencing transcriptome analysis of early stages of poplar leaf colonization by the rust fungus Melampsora larici-populina. Among the 841,301 reads considered for analysis, 616,879 and 649 were successfully mapped to Populus trichocarpa and M. larici-populina genome sequences, respectively. From a methodological aspect, these results indicate that this single approach is not appropriate to saturate poplar transcriptome and to follow transcript accumulation of the pathogen. We identified 19 pathogen transcripts encoding early-expressed small-secreted proteins representing candidate effectors of interest for forthcoming studies. Poplar RNA-Seq data were validated by oligoarrays and quantitatively analysed, which revealed a highly stable transcriptome with a single transcript encoding a sulfate transporter (herein named PtSultr3;5, POPTR_0006s16150) showing a dramatic increase upon colonization by either virulent or avirulent M. larici-populina strains. Perspectives connecting host sulfate transport and biotrophic lifestyle are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431362PMC
February 2013

Comprehensive proteome analysis in Cenococcum geophilum Fr. as a tool to discover drought-related proteins.

J Proteomics 2012 Jun 8;75(12):3707-19. Epub 2012 May 8.

Section of Forest Genetics, Technische Universität München, Hans-Carl-von-Carlowitz-Platz 2, D-85354 Freising, Germany.

Cenococcum geophilum is a widely distributed ectomycorrhizal fungus potentially playing a significant role in resistance and resilience mechanisms of its tree hosts exposed to drought stress. In this study, we performed a large scale protein analysis in pure cultures of C. geophilum in order to gain first global insights into the proteome assembly of this fungus. Using 1-D gel electrophoresis coupled with ESI-MS/MS, we indentified 638 unique proteins. Most of these proteins were related to the metabolic and cellular processes, and the transport machinery of cells. In a second step, we examined the influence of water deprivation on the proteome of C. geophilum pure cultures at three time points of gradually imposed drought. The results indicated that 12 proteins were differentially abundant in mycelia subjected to drought compared to controls. The induced responses in C. geophilum point towards regulation of osmotic stress, maintainance of cell integrity, and counteracting increased levels of reactive oxygen species formed during water deprivation.
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http://dx.doi.org/10.1016/j.jprot.2012.04.039DOI Listing
June 2012

Genome-wide analysis of cell wall-related genes in Tuber melanosporum.

Curr Genet 2012 Jun 6;58(3):165-77. Epub 2012 Apr 6.

Istituto per la Protezione delle Piante del CNR, UOS Torino, Viale Mattioli, 25, 10125, Turin, Italy.

A genome-wide inventory of proteins involved in cell wall synthesis and remodeling has been obtained by taking advantage of the recently released genome sequence of the ectomycorrhizal Tuber melanosporum black truffle. Genes that encode cell wall biosynthetic enzymes, enzymes involved in cell wall polysaccharide synthesis or modification, GPI-anchored proteins and other cell wall proteins were identified in the black truffle genome. As a second step, array data were validated and the symbiotic stage was chosen as the main focus. Quantitative RT-PCR experiments were performed on 29 selected genes to verify their expression during ectomycorrhizal formation. The results confirmed the array data, and this suggests that cell wall-related genes are required for morphogenetic transition from mycelium growth to the ectomycorrhizal branched hyphae. Labeling experiments were also performed on T. melanosporum mycelium and ectomycorrhizae to localize cell wall components.
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http://dx.doi.org/10.1007/s00294-012-0374-6DOI Listing
June 2012

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system.

Fungal Biol 2012 Feb 27;116(2):261-75. Epub 2011 Nov 27.

Dipartimento di Protezione e Valorizzazione Agroalimentare, University of Bologna, Via Fanin 46, 40127 Bologna, Italy.

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.
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http://dx.doi.org/10.1016/j.funbio.2011.11.009DOI Listing
February 2012

A comprehensive analysis of genes encoding small secreted proteins identifies candidate effectors in Melampsora larici-populina (poplar leaf rust).

Mol Plant Microbe Interact 2012 Mar;25(3):279-93

Unité Mixte de Recherche 1136 Institut National de la Recherche Agronomique-Nancy Université, Interactions Arbres/Microorganismes, INRA Nancy, 54280 Champenoux, France.

The obligate biotrophic rust fungus Melampsora larici-populina is the most devastating and widespread pathogen of poplars. Studies over recent years have identified various small secreted proteins (SSP) from plant biotrophic filamentous pathogens and have highlighted their role as effectors in host-pathogen interactions. The recent analysis of the M. larici-populina genome sequence has revealed the presence of 1,184 SSP-encoding genes in this rust fungus. In the present study, the expression and evolutionary dynamics of these SSP were investigated to pinpoint the arsenal of putative effectors that could be involved in the interaction between the rust fungus and poplar. Similarity with effectors previously described in Melampsora spp., richness in cysteines, and organization in large families were extensively detailed and discussed. Positive selection analyses conducted over clusters of paralogous genes revealed fast-evolving candidate effectors. Transcript profiling of selected M. laricipopulina SSP showed a timely coordinated expression during leaf infection, and the accumulation of four candidate effectors in distinct rust infection structures was demonstrated by immunolocalization. This integrated and multifaceted approach helps to prioritize candidate effector genes for functional studies.
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http://dx.doi.org/10.1094/MPMI-09-11-0238DOI Listing
March 2012

454-pyrosequencing of Coffea arabica leaves infected by the rust fungus Hemileia vastatrix reveals in planta-expressed pathogen-secreted proteins and plant functions in a late compatible plant-rust interaction.

Mol Plant Pathol 2012 Jan 1;13(1):17-37. Epub 2011 Jun 1.

IRD, Institut de Recherche pour le Développement, UMR 186 IRD-Cirad-UM2 Résistance des Plantes aux Bioagresseurs, BP 64501, 34394 Montpellier Cedex 5, France.

Coffee (Coffea arabica L.), one of the key export and cash crops in tropical and subtropical countries, suffers severe losses from the rust fungus Hemileia vastatrix. The transcriptome of H. vastatrix was analysed during a compatible interaction with coffee to obtain an exhaustive repertoire of the genes expressed during infection and to identify potential effector genes. Large-scale sequencing (454-GS-FLEX Titanium) of mixed coffee and rust cDNAs obtained from 21-day rust-infected leaves generated 352 146 sequences which assembled into 22 774 contigs. In the absence of any reference genomic sequences for Coffea or Hemileia, specific trinucleotide frequencies within expressed sequence tags (ESTs) and blast homology against a set of dicots and basidiomycete genomes were used to distinguish pathogen from plant sequences. About 30% (6763) of the contigs were assigned to H. vastatrix and 61% (13 951) to C. arabica. The majority (60%) of the rust sequences did not show homology to any genomic database, indicating that they were potential novel fungal genes. In silico analyses of the 6763 H. vastatrix contigs predicted 382 secreted proteins and identified homologues of the flax rust haustorially expressed secreted proteins (HESPs) and bean rust transferred protein 1 (RTP1). These rust candidate effectors showed conserved amino-acid domains and conserved patterns of cysteine positions suggestive of conserved functions during infection of host plants. Quantitative reverse transcription-polymerase chain reaction profiling of selected rust genes revealed dynamic expression patterns during the time course of infection of coffee leaves. This study provides the first valuable genomic resource for the agriculturally important plant pathogen H. vastatrix and the first comprehensive C. arabica EST dataset.
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http://dx.doi.org/10.1111/j.1364-3703.2011.00723.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638645PMC
January 2012

Melampsora larici-populina transcript profiling during germination and timecourse infection of poplar leaves reveals dynamic expression patterns associated with virulence and biotrophy.

Mol Plant Microbe Interact 2011 Jul;24(7):808-18

Institut National de la Recherche Agronomique, Champenoux, France.

Melampsora larici-populina is responsible for poplar leaf rust disease and causes severe epidemics in poplar plantations in Europe. The poplar rust genome has been recently sequenced and, in order to find the genetic determinants associated with its biotrophic lifestyle, we generated a whole-genome custom oligoarray and analyzed transcript profiles of M. larici-populina during the infection timecourse in poplar leaves. Different stages were investigated during the asexual development of the rust fungus, including resting and germinating urediniospores and seven in planta stages in the telial host. In total, 76% of the transcripts were detected during leaf infection as well as in urediniospores, whereas 20% were only detected in planta, including several transporters and many small secreted proteins (SSP). We focused our analysis on gene categories known to be related to plant colonization and biotrophic growth in rust pathogens, such as SSP, carbohydrate active enzymes (CAZymes), transporters, lipases, and proteases. Distinct sets of SSP transcripts were expressed all along the infection process, suggesting highly dynamic expression of candidate rust effectors. In contrast, transcripts encoding transporters and proteases were mostly expressed after 48 h postinoculation, when numerous haustoria are already formed in the leaf mesophyll until uredinia formation, supporting their role in nutrient acquisition during biotrophic growth. Finally, CAZymes and lipase transcripts were predominantly expressed at late stages of infection, highlighting their importance during sporulation.
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http://dx.doi.org/10.1094/MPMI-01-11-0006DOI Listing
July 2011

Obligate biotrophy features unraveled by the genomic analysis of rust fungi.

Proc Natl Acad Sci U S A 2011 May 2;108(22):9166-71. Epub 2011 May 2.

Unité Mixte de Recherche 1136, Institut National de la Recherche Agronomique/Nancy Université, Interactions Arbres/Micro-organismes, Centre de Nancy, 54280 Champenoux, France.

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.
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http://dx.doi.org/10.1073/pnas.1019315108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3107277PMC
May 2011

Genome-wide inventory of metal homeostasis-related gene products including a functional phytochelatin synthase in the hypogeous mycorrhizal fungus Tuber melanosporum.

Fungal Genet Biol 2011 Jun 19;48(6):573-84. Epub 2010 Nov 19.

Department of Biochemistry and Molecular and Functional Biology, University of Parma, 43100 Parma, Italy.

Ectomycorrhizal fungi are thought to enhance mineral nutrition of their host plants and to confer increased tolerance toward toxic metals. However, a global view of metal homeostasis-related genes and pathways in these organisms is still lacking. Building upon the genome sequence of Tuber melanosporum and on transcriptome analyses, we set out to systematically identify metal homeostasis-related genes in this plant-symbiotic ascomycete. Candidate gene products (101) were subdivided into three major functional classes: (i) metal transport (58); (ii) oxidative stress defence (32); (iii) metal detoxification (11). The latter class includes a small-size metallothionein (TmelMT) that was functionally validated in yeast, and phytochelatin synthase (TmelPCS), the first enzyme of this kind to be described in filamentous ascomycetes. Recombinant TmelPCS was shown to support GSH-dependent, metal-activated phytochelatin synthesis in vitro and to afford increased Cd/Cu tolerance to metal hypersensitive yeast strains. Metal transporters, especially those related to Cu and Zn trafficking, displayed the highest expression levels in mycorrhizae, suggesting extensive translocation of both metals to root cells as well as to fungal metalloenzymes (e.g., laccase) that are strongly upregulated in symbiotic hyphae.
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http://dx.doi.org/10.1016/j.fgb.2010.11.003DOI Listing
June 2011

Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes.

BMC Genomics 2010 Nov 12;11:630. Epub 2010 Nov 12.

INRA, Nancy Université, UMR1137 Ecologie et Ecophysiologie Forestières, IFR 110 EFABA, F-54280 Champenoux, France.

Background: Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance. Here we assess the genotype-dependent component of the drought-induced transcriptome response in two poplar genotypes differing in drought tolerance. Drought-induced responses were analysed in leaves and root apices and were compared with available transcriptome data from other Populus species.

Results: Using a multi-species designed microarray, a genomic DNA-based selection of probesets provided an unambiguous between-genotype comparison. Analyses of functional group enrichment enabled the extraction of processes physiologically relevant to drought response. The drought-driven changes in gene expression occurring in root apices were consistent across treatments and genotypes. For mature leaves, the transcriptome response varied weakly but in accordance with the duration of water deficit. A differential clustering algorithm revealed similar and divergent gene co-expression patterns among the two genotypes. Since moderate stress levels induced similar physiological responses in both genotypes, the genotype-dependent transcriptional responses could be considered as intrinsic divergences in genome functioning. Our meta-analysis detected several candidate genes and processes that are differentially regulated in root and leaf, potentially under developmental control, and preferentially involved in early and long-term responses to drought.

Conclusions: In poplar, the well-known drought-induced activation of sensing and signalling cascades was specific to the early response in leaves but was found to be general in root apices. Comparing our results to what is known in arabidopsis, we found that transcriptional remodelling included signalling and a response to energy deficit in roots in parallel with transcriptional indices of hampered assimilation in leaves, particularly in the drought-sensitive poplar genotype.
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http://dx.doi.org/10.1186/1471-2164-11-630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091765PMC
November 2010

Genome-wide search and functional identification of transcription factors in the mycorrhizal fungus Tuber melanosporum.

New Phytol 2011 Feb 8;189(3):736-750. Epub 2010 Nov 8.

Department of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy.

• Developmental transitions associated with the life cycle of plant-symbiotic fungi, such as the ascomycete Tuber melanosporum, are likely to require an extensive reprogramming of gene expression brought about by transcription factors (TFs). To date, little is known about the transcriptome alterations that accompany developmental shifts associated with symbiosis or fruiting body formation. • Taking advantage of the black truffle genome sequence, we used a bioinformatic approach, coupled with functional analysis in yeast and transcriptome profiling, to identify and catalogue T. melanosporum TFs, the so-called 'regulome'. • The T. melanosporum regulome contains 102 homologs of previously characterized TFs, 57 homologs of hypothetical TFs, and 42 putative TFs apparently unique to Tuber. The yeast screen allowed the functional discovery of four TFs and the validation of about one-fifth of the in silico predicted TFs. Truffle proteins apparently unrelated to transcription were also identified as potential transcriptional regulators, together with a number of plant TFs. • Twenty-nine TFs, some of which associated with particular developmental stages, were found to be up-regulated in ECMs or fruiting bodies. About one-quarter of these up-regulated TFs are expressed at surprisingly high levels, thus pointing to a striking functional specialization of the different stages of the Tuber life cycle.
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http://dx.doi.org/10.1111/j.1469-8137.2010.03525.xDOI Listing
February 2011

Isolation and characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum.

New Phytol 2011 Feb 20;189(3):710-722. Epub 2010 Oct 20.

National Research Council, Plant Genetics Institute - Perugia Division, Via della Madonna Alta 130, I-06128 Perugia, Italy.

• The genome of Tuber melanosporum has recently been sequenced. Here, we used this information to identify genes involved in the reproductive processes of this edible fungus. The sequenced strain (Mel28) possesses only one of the two master genes required for mating, that is, the gene that codes for the high mobility group (HMG) transcription factor (MAT1-2-1), whereas it lacks the gene that codes for the protein containing the α-box- domain (MAT1-1-1), suggesting that this fungus is heterothallic. • A PCR-based approach was initially employed to screen truffles for the presence of the MAT1-2-1 gene and amplify the conserved regions flanking the mating type (MAT) locus. The MAT1-1-1 gene was finally identified using primers designed from the conserved regions of strains that lack the MAT1-2-1 gene. • Mating type-specific primer pairs were developed to screen asci and gleba from truffles of different origins and to genotype single ascospores within the asci. These analyses provided definitive evidence that T. melanosporum is a heterothallic species with a MAT locus that is organized similarly to those of ancient fungal lineages. • A greater understanding of the reproductive mechanisms that exist in Tuber spp. allows for optimization of truffle plantation management strategies.
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http://dx.doi.org/10.1111/j.1469-8137.2010.03492.xDOI Listing
February 2011

Laser capture microdissection of uredinia formed by Melampsora larici-populina revealed a transcriptional switch between biotrophy and sporulation.

Mol Plant Microbe Interact 2010 Oct;23(10):1275-86

Unité Mixte de Recherche 1136 INRA/Nancy Université Interactions Arbres/Micro-organismes, Champenoux, France.

The foliar rust caused by the basidiomycete Melampsora larici-populina is the main disease affecting poplar plantations in Europe. The biotrophic status of rust fungi is a major limitation to study gene expression of cell or tissue types during host infection. At the uredinial stage, infected poplar leaves contain distinct rust tissues such as haustoria, infection hyphae, and uredinia with sporogenous hyphae and newly formed asexual urediniospores. Laser capture microdissection (LCM) was used to isolate three areas corresponding to uredinia and subjacent zones in the host mesophyll for expression analysis with M. larici-populina whole-genome exon oligoarrays. Optimization of tissue preparation prior to LCM allowed isolation of RNA of good integrity for genome-wide expression profiling. Our results indicate that the poplar rust uredinial stage is marked by distinct genetic programs related to biotrophy in the host palisade mesophyll and to sporulation in the uredinium. A strong induction of transcripts encoding small secreted proteins, likely containing rust effectors, is observed in the mesophyll, suggesting a late maintenance of suppression of host defense in the tissue containing haustoria and infection hyphae. On the other hand, cell cycle and cell defense rescue transcripts are strongly accumulated in the sporulation area. This combined LCM-transcriptomic approach brings new insights on the molecular mechanisms underlying urediniospore formation in rust fungi.
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http://dx.doi.org/10.1094/MPMI-05-10-0111DOI Listing
October 2010
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