Project description:In the filamentous fungus Podospora anserina, cell death by incompatibility can be monitored using the conditional self-incompatible (SI) het-R het-V strain. SI strains are homokaryotic strains bearing incompatible het genes in all nuclei. The co-expression of these het genes triggers cell death in all the cells and hence in the entire mycelium. The het-R het-V SI strain, bearing the two incompatible het-R and het-V genes, proved particularly convenient as cell death triggering is thermosensitive (J. Labarère et al., 1973, C R Acad Sci Hebd Seances Acad Sci D, 276) and is induced by a simple shift in growth temperature (from 32°C to 26°C).The development of this cell death reaction has been recorded for 4 h after transfer to the restrictive temperature. A time course (T0h to T4h) of the transcriptomic response to the temperature shift has been explored in parallel within SI (het-R het-V) and wild type (compatible het-r hetV) s strains.
Project description:In the filamentous fungus Podospora anserina, cell death by incompatibility can be monitored using the conditional self-incompatible (SI) het-R het-V strain. SI strains are homokaryotic strains bearing incompatible het genes in all nuclei. The co-expression of these het genes triggers cell death in all the cells and hence in the entire mycelium. The het-R het-V SI strain, bearing the two incompatible het-R and het-V genes, proved particularly convenient as cell death triggering is thermosensitive (J. LabarM-CM-(re et al., 1973, C R Acad Sci Hebd Seances Acad Sci D, 276) and is induced by a simple shift in growth temperature (from 32M-BM-0C to 26M-BM-0C).The development of this cell death reaction has been recorded for 4 h after transfer to the restrictive temperature. A time course (T0h to T4h) of the transcriptomic response to the temperature shift has been explored in parallel within SI (het-R het-V) and wild type (compatible het-r hetV) s strains. With Platform GPL10116 : reference design with 2 strains: a WT strain and het-R het-V self-incompatible strain (J. LabarM-CM-(re et al., 1973, C R Acad Sci Hebd Seances Acad Sci D, 276). Each strain with four biological replicates; the common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5.
Project description:The underlying molecular mechanisms of programmed cell death (PCD) associated with fungal allorecognition, a form of innate immunity, remain largely unknown. In this study, transcriptome analysis was used to infer mechanisms activated during barrage formation in vic3-incompatible strains of C. parasitica, the chestnut blight fungus. Pronounced differential expression occurred in barraging strains of genes involved in mating pheromone (mf2-1, mf2-2), secondary metabolite production, detoxification (including oxidative stress), apoptosis-related, RNA interference and HET-domain genes. Evidence for secondary metabolite production and reactive oxygen species (ROS) accumulation is supported through UPLC-HRMS analysis and cytological staining, respectively. Differential expression of mating-related genes and HET-domain genes was further examined by RT-qPCR of incompatible interactions involving each of the 6 vegetative incompatibility (vic) loci in C. parasitica and revealed distinct recognition process networks. We infer that vegetative incompatibility in C. parasitica activates defence reactions that involve secondary metabolism, resulting in increased toxicity of the extra- and intracellular environment. Accumulation of ROS (and other potential toxins) may result in detoxification failure and activation of apoptosis, sporulation, and the expression of associated pheromone genes. The incompatible reaction leaves abundant traces of a process-specific metabolome as conidiation is initiated.
Project description:Compared to what is known in model species, reproductive biology in citrus is still poorly understood. Although in recent years several efforts have been made to study pollen-pistil interaction and self-incompatibility, little information is available about the molecular mechanisms regulating these processes. We performed microarray analysis for the identification of candidate genes involved in pollen-pistil interaction and self-incompatibility in clementine (Citrus clementina Hort. ex Tan.). The analysis was performed comparing the transcriptome of laser-microdissected stylar canal cells isolated from two clementine genotypes differing for self-incompatibility response (‘Comune’, self-incompatible; and ‘Monreal’, a self compatible mutation of ‘Comune’).
Project description:The cultivated almond exhibits self-incompatibility of the gametophytic type regulated by the S-locus, and expressed in pistil (S-RNase) and in pollen (SFB protein). The aim of this study is to clarify the transcription pattern of these 2 S-genes and to identify additional components of the gametophytic self-incompatibility system in almond. With this aim, A2-198 (self compatible) and ITAP-1 (self incompatible) almond selections were used: RNA-seq of pistils of these two accessions both un-pollinated and pollinated with A2-198 pollen were carried out.
Project description:Graft compatibility is the ability of two plants to form cohesive vascular connections. Tomato and pepper grafts are incompatible but the underlying causes of this phenomenon remain unknown. We utilzied a broad array of techniques to profile graft compatibility including viability, biophysical stability, and growth. Cell death in the junction was quantified using trypan blue and TUNNEL assays. Transcriptomic analysis of cell death in the junction was preformed using RNA-sequncing. Finally a meta-transcriptomic analysis was conducted with published datasets to further explore the genetic signature of graft incompatibility.We found that all varieties of pepper tested across two species were incompatible with tomato. Tomato and pepper graft incompatibility is characterized by stem instability, reduced growth, and persistent cell death in the graft junction. We showed that tomato and pepper heterografts have prolonger transcriptional activity, with defense processes highly enrched. We identfied a large subset of NLRs and genes involved in programmed cell death which were upregulated in incompatible tissue. We also identified a set of genes with orthologs in both tomato and pepper which are upregulated in incompatible grafts including biosythesis of steroidal glycoalkaloids. Finally we utilized various biological stressors to explore the genetic signature of grafting. We found a significant overlap in the genetic profile of grafting and plant parsitism. We also identified over 1000 genes uniquely upregulated in incompatible grafting including genes in involved in DNA-damage repair. Based on the broad upregulation of NLRs and genes involved with programmed cell death, prolonged cell death in the junction, and DNA damage, we have determined that tomato and pepper graft incompatibility is likely caused by a form of genetic incompatibility which triggers an autoimmune-like response.
Project description:Allorecognition, the capacity to discriminate self from conspecific non-self, is a ubiquitous organismal feature typically governed by genes evolving under balancing selection. Here, we show that in the fungus Podospora anserina, allorecognition loci controlling vegetative incompatibility (het genes), define two reproductively isolated groups through pleiotropic effects on sexual compatibility. These two groups emerge from the antagonistic interactions of the unlinked loci het-r (encoding a NOD-like receptor) and het-v (encoding a methyltransferase and an MLKL/HeLo domain protein). Using a combination of genetic and ecological data, supported by simulations, we provide a concrete and molecularly defined example whereby the origin and coexistence of reproductively isolated groups in sympatry is driven by pleiotropic genes under balancing selection.
Project description:A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology. 1) A library of 10 oligonucleotides per CDS has been design by the manufacturer. 2) A computational step has been performed to determine 3' position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS. 3) Microarray v.2 (GPL10115), manufactured with this design, have been used for a selection procedure based on the use of transcriptomic experimental data generated with diverse culture conditions. To maximize the number of expressed CDS, experimental data were generated with a large range of growth conditions covering crucial physiological changes from key steps of vegetative growth and sexual development.For each condition, four biological replicates were done to increase the statistical power of the analysis. An oligonucleotide scoring has been settled to select the best oligonucleotide per CDS. The final array design, Microarray v.3 (GPL10116), contained 10,539 probes for nuclear CDS. The array was enriched with 17 mitochondrial CDS probes that did not undergo the experimental screening. Each probe was replicated four times on each 44K array to improve the statistical significance of results. Before using in industrious transcriptomic experiment, ultimate qualification steps have been carried out. First, DNA extracted from the/ S/ and /s /strains was hybridized with the final design Microarray v.III. The apha_ORF and /het/ genes were identified as polymorphic between /s/ and /S/ strains (|FC|>2; a/ =/0.001) confirming previous genetic and molecular analyses (Turcq B etal.,1990*,* /Current genetics, /Sellem et al., 1990, Mol. Gen. Genet.). In second qualification step, a self-to-self hybridization experiment, replicated three times, has been /per/formed with a commonreference: 89.3% of the probes displayed a signal-to-standard-deviation ratio >3. The final microarray design is referenced as AMADID 018343. Microarray slides are available to the research community from Agilent.