Effector secretion is crucial for root endophytes to establish and protect their ecological niche. We used time-resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root-associated bacteria. We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization. Some effectors responded to both plants and microbes, suggesting dual roles in intermicrobial competition and plant-microbe interactions. A subset of putative antimicrobial effectors, including a GH18-CBM5 chitinase, was induced exclusively by microbes. Functional analyses of this chitinase revealed its antimicrobial and plant-protective properties. We conclude that dynamic effector gene expression underpins the ability of Sebacinales to thrive in diverse ecological niches with a single fungal chitinase contributing substantially to niche defense.

Programmed cell death occurring during plant development (dPCD) is a fundamental process integral for plant growth and reproduction. Here, we investigate the connection between developmentally controlled PCD and fungal accommodation in Arabidopsis thaliana roots, focusing on the root cap-specific transcription factor ANAC033/SOMBRERO (SMB) and the senescence-associated nuclease BFN1. Mutations of both dPCD regulators increase colonization by the beneficial fungus Serendipita indica , primarily in the differentiation zone. smb-3 mutants additionally exhibit hypercolonization around the meristematic zone and a delay of S. indica -induced root-growth promotion. This demonstrates that root cap dPCD and rapid post-mortem clearance of cellular corpses represent a physical defense mechanism restricting microbial invasion of the root. Additionally, reporter lines and transcriptional analysis revealed that BFN1 expression is downregulated during S. indica colonization in mature root epidermal cells, suggesting a transcriptional control mechanism that facilitates the accommodation of beneficial microbes in the roots.

The domestication of crops, coupled with agroecosystem development, is associated with major environmental changes and provides an ideal model of phenotypic plasticity. Here, we examined 32 genotypes of three tetraploid wheat (Triticum turgidum L.) subspecies, wild emmer, emmer, and durum wheat, which are representative of the key stages in the domestication of tetraploid wheat. We developed a pipeline that integrates RNA-Seq data and population genomics to assess gene expression plasticity and identify selection signatures under diverse nitrogen availability conditions. Our analysis revealed differing gene expression responses to nitrogen availability across primary (wild emmer to emmer) and secondary (emmer to durum wheat) domestication. Notably, nitrogen triggered the expression of twice as many genes in durum wheat compared to that in emmer and wild emmer. Unique selection signatures were identified at each stage: primary domestication mainly influenced genes related to biotic interactions, whereas secondary domestication affected genes related to amino acid metabolism, in particular lysine. Selection signatures were found in differentially expressed genes (DEGs), notably those associated with nitrogen metabolism, such as the gene encoding glutamate dehydrogenase (GDH). Overall, our study highlights the pivotal role of nitrogen availability in the domestication and adaptive responses of a major food crop, with varying effects across different traits and growth conditions. Nitrogen had a pivotal role in tetraploid wheat domestication and adaptive responses, leading to diverse impacts on gene expression and selection signatures during primary and secondary domestication.

SummaryThe intracellular colonization of plant roots by the beneficial fungal endophyteSerendipita indicafollows a biphasic strategy. After an early biotrophic phase, the interaction transitions to a host cell death phase restricted to the epidermal and cortex layers of the root. Host cell death contributes to the successful accommodation of the fungus during the beneficial interaction inArabidopsis thaliana. How host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5’-nucleotidaseSiE5NT and the nucleaseSiNucA, act synergistically in the plant apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. The uptake of extracellular dAdo, but not the structurally related adenosine (Ado), activates a previously undescribed cell death mechanism inA. thaliana. Mutation of the equilibrative nucleoside transporter ENT3 inA. thalianaresults in resistance to cell death triggered by extracellular dAdo and reduced fungal-mediated cell death during root colonization. A library screen ofA. thalianaT-DNA insertion lines identified a toll/interleukin-1 receptor nucleotide-binding leucine-rich repeat (TIR-NLR) protein as an additional intracellular component in dAdo-triggered cell death. Mutation of this previously uncharacterised TIR-NLR, which we have named ISI (induced byS.indica), affects host cell death, fungal colonization and growth promotion, suggesting a key role in the regulation of root cell death and plant-microbe interaction. Our data show that the combined activity of two fungal apoplastic enzymes leads to the production of a metabolite that, upon uptake, triggers TIR-NLR-modulated plant cell death, providing a link to immunometabolism in plants.Short summaryEfficient intraradical colonization by the beneficial fungal endophyteSerendipita indicarequires restricted host cell death. How this symbiotic host cell death is initiated and controlled is largely unknown. Here we show that two fungal enzymes, the ecto-5’-nucleotidaseSiE5NT and the nucleaseSiNucA, act synergistically in the apoplast at the onset of cell death to produce deoxyadenosine (dAdo), a potent cell death inducer in animal systems. Uptake of extracellular dAdo activates a previously undescribed cell death mechanism in plants. Mutation of theA. thalianaequilibrative nucleoside transporterENT3leads to resistance to cell death triggered by uptake of extracellular dAdo and to reduced fungal-mediated cell death during colonization. A library screen ofA. thalianaT-DNA insertion lines identified a TIR-NLR protein as an additional intracellular component in dAdo-triggered cell death, providing a link to immunometabolism in plants.In a nutshellRegulated host cell death is part of the plant defense strategy against pathogens, but it is also involved in the accommodation of certain beneficial microbes in the roots. We have identified extracellular metabolites and intracellular metabolic signals that contribute to colonization by beneficial root fungal endophytes and uncovered a conserved cell death mechanism likely co-opted for establishing plant-endophyte symbiosis.

Sinonasal tumours are heterogeneous malignancies, presenting different histological features and clinical behaviour. Many studies emphasize the role of specific miRNA in the development and progression of cancer, and their expression profiles could be used as prognostic biomarkers to predict the survival. Recently, using the next-generation sequencing (NGS)-based miRNome analysis the miR-34/miR-449 cluster was identified as miRNA superfamily involved in the pathogenesis of sinonasal cancers (SNCs). In the present study, we established an Argonaute-2 (AGO2): mRNA immunoprecipitation followed by high-throughput sequencing to analyse the regulatory role of miR-34/miR-449 in SNCs. Using this approach, we identified direct target genes (targetome), which were involved in regulation of RNA-DNA metabolic, transcript and epigenetic processes. In particular, the STK3, C9orf78 and STRN3 genes were the direct targets of both miR-34c and miR-449a, and their regulation are predictive of tumour progression. This study provides the first evidence that miR-34/miR-449 and their targets are deregulated in SNCs and could be proposed as valuable prognostic biomarkers.

Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.

Associations between plants and beneficial root-endophytic fungi enhance plant performance by improving nutrient uptake, abiotic stress tolerance and disease resistance. To successfully colonize different host plants and defend their host niche against competing microbes, but also to cooperate with beneficial bacterial members of the microbiota, root endophytes such as Sebacinales secrete a multitude of tightly regulated effector-proteins and carbohydrate-active enzymes. However, the functions, specificity, and regulation of these proteins remain poorly understood. In this study, we employ time-resolved transcriptomics to analyse the gene expression profiles of two Sebacinales members interacting with organisms from different kingdoms of life. We identified crucial genes for plant colonization and intermicrobial competition, including a fungal GH18-CBM5 chitinase specifically upregulated in response to the phytopathogenic fungus Bipolaris sorokiniana. This chitinase protects the plant hosts against the pathogen, reducing fungal biomass and disease symptoms in barley and Arabidopsis thaliana. Our findings shed light on interaction partner specific gene expression in Sebacinales endophytes, with potential applications in enhancing plant health and resilience.

The domestication of crops is associated with the genome-wide loss of nucleotide diversity caused by selection and genetic drift. Here we investigated 32 genotypes representing key stages in the domestication of tetraploid wheat, i.e. wild emmer, emmer and durum wheat. RNA-Seq analysis was combined with estimates of evolvability, heritability and Q ST to characterize the plasticity of gene expression and its adaptive role in primary and secondary domestication under different nitrogen growth conditions. We confirmed the loss of nucleotide diversity and found that primary and secondary domestication affected gene expression diversity in different ways, influenced by nitrogen availability. We found that nitrogen starvation induced the expression of a larger number of genes in durum wheat compared to emmer and wild emmer. Q ST distributions and Q ST –F ST comparisons revealed distinct selection signatures at each domestication stage. While primary domestication affected the expression of genes involved in biotic interactions, secondary domestication was associated with changes in expression of genes involved in metabolism of amino acids, particularly lysine. Our combined results revealed genes specifically involved in nitrogen metabolism, such as glutamate dehydrogenase , with important role in early development, underwent selection during secondary domestication. Therefore, our findings show that nitrogen availability had a pivotal role during the domestication and adaptive responses of one of our major food crops, with varying effects across different traits.

Endophytic root colonisation of the host plant Arabidopsis thaliana by the beneficial root endophyte Serendipita indica is characterised by an initial biotrophic phase followed by a restricted host cell death-associated phase. During colonisation two secreted S. indica effectors synergistically produce the cell death inducer deoxyadenosine (dAdo). Although the mechanism of regulated cell death is important for fungal accommodation, the host molecular pathways that restrict S. indica -induced cell death and allow the maintenance of a mutualistic relationship are largely unknown. Here we show that autophagy, a major degradation pathway plays a key role in the beneficial interaction between S. indica and Arabidopsis. Autophagy restricts fungal colonisation and exerts a protective function against dAdo-induced cell death. Impairment of dAdo uptake in an autophagy mutant background confers resistance to dAdo-induced cell death and precludes the symbiosis-mediated cell death. Taken together, we propose that autophagy-driven pro-survival responses are required to facilitate a long-lasting mutualistic interaction between S. indica and Arabidopsis.

The aetiology of leiomyoma is debated; however, dysregulated progenitor cells or miRNAs appear to be involved. Previous profiling analysis of miRNA in healthy myometrium- (M-MSCs) and leiomyoma- (L-MSCs) derived mesenchymal stem cells (MSCs) identified 15 miRNAs differentially expressed between M-MSCs and L-MSCs. Here, we try to elucidate whether these differentially regulated 15 miRNAs arise as a conversion of M-MSCs along the differentiation process or whether they may originate from divergent cell commitment. To trace the origin of the dysregulation, a comparison was made of the expression of miRNAs previously identified as differentially regulated in M-MSCs and L-MSCs with that detected in MSCs from amniotic fluid (considered as a substitute for embryonic cells). The results do not allow for a foregone conclusion: the miRNAs converging to the adherens junction pathway showed a gradual change along the differentiation process, and the miRNAs which coincided with the other three pathways (ECM-receptor interaction, TGFβ and cell cycle) showed a complex, not linear, regulation and, therefore, a trend along the hypothetical differentiation process was not deduced. However, the role of miRNAs appears to be predominant in the onset of leiomyoma and may follow two different mechanisms (early commitment; exacerbation); furthermore, miRNAs can support the observed (epigenetic) predisposition.

In legumes, pod shattering occurs when mature pods dehisce along the sutures, and detachment of the valves promotes seed dispersal. In Phaseolus vulgaris (L)., the major locus qPD5.1-Pv for pod indehiscence was identified recently. We developed a BC4/F4 introgression line population and narrowed the major locus down to a 22.5 kb region. Here, gene expression and a parallel histological analysis of dehiscent and indehiscent pods identified an AtMYB26 orthologue as the best candidate for loss of pod shattering, on a genomic region ~11 kb downstream of the highest associated peak. Based on mapping and expression data, we propose early and fine up-regulation of PvMYB26 in dehiscent pods. Detailed histological analysis establishes that pod indehiscence is associated with the lack of a functional abscission layer in the ventral sheath, and that the key anatomical modifications associated with pod shattering in common bean occur early during pod development. We finally propose that loss of pod shattering in legumes resulted from histological convergent evolution and that it is the result of selection at orthologous loci.

Ageing is among the main risk factors for human disease onset and the identification of the hallmarks of senescence remains a challenge for the development of appropriate therapeutic target in the elderly. Here, we compare senescence-related changes in two cell populations of mesenchymal stromal cells by analysing their miRNA profiling: Human Dental Pulp Stromal Cells (hDPSCs) and human Periosteum-Derived Progenitor Cells (hPDPCs). After these cells were harvested, total RNA extraction and whole genome miRNA profiling was performed, and DIANA-miRPath analysis was applied to find the target/pathways. Only 69 microRNAs showed a significant differential expression between dental pulp and periosteum progenitor cells. Among these, 24 were up regulated, and 45 were downregulated in hDPSCs compared to hPDPCs. Our attention was centered on miRNAs (22 upregulated and 34 downregulated) involved in common pathways for cell senescence (i.e. p53, mTOR pathways), autophagy (i.e. mTOR and MAPK pathways) and cell cycle (i.e. MAPK pathway). The p53, mTOR and MAPK signaling pathways comprised 43, 37 and 112 genes targeted by all selected miRNAs, respectively. Our finding is consistent with the idea that the embryological origin influences cell behavior and the ageing process. Our study strengthens the hypothesis that ageing is driven by numerous mediators interacting through an intricate molecular network, which affects adult stem cells self-renewal capability.

Background: Leiomyoma are the most common indication for hysterectomy in the world and have a strong economic impact on health care systems; many different mechanisms have been considered for their aetiology, such as inflammation, dysregulated progenitor cells or different regulation of miRNAs. After performing a whole genome miRNA profiling in progenitor cells (PCs) derived from healthy myometrium (MPCs) and from leyomioma (LPCs), only 15 miRNAs were identified as differentially expressed between MPCs and LPCs. Progenitor cells from Amniotic Fluid (AFPCs) are considered the most undifferentiated cells after the embryonic ones. Here we try to clarify if the miRNAs differently regulated between leiomyoma and myometrium cells arise as a conversion of MPCs along the differentiation process or if they may originate from a divergent cell commitment. To track the origin of the dysregulation, miRNA expression was analyzed in AFPCs (considered as surrogate for embryonic cells), MPCs and LPCs.MPCs, LPCs and AFPCs were isolated and subjected to whole genoma miRNA profiling; the expression of the 15 miRNAs previously identified as differentially regulated in MPCs and LPCs was compared to that detected in AFPCs. Results : Clustering analysis sub-grouped the 15 miRNAs into 4 major clusters that converge to the KEGG pathways: Adherens junction, ECM-receptor interaction, TGFβ signaling and cell cycle. miRNAs are differentially regulated in MPCs and LPCs compared to AFPCs and 10/15 of them show statistically significant variations between MPCs and LPCs. Conclusion(s) : Our results seem to point that a linear physiological differentiation axis exists from AFPCS to MPCs that, under particular insults, pathologically continues toward LPCs.Our results seem to point that a linear physiological differentiation axis exists from AFPCS to MPCs that, under particular insults, pathologically continues toward LPCs.

Eukaryotic genomes are pervasively transcribed and only a small portion of the transcribed sequences belongs to protein coding genes. High-throughput sequencing technology contributed to consolidate this perspective, allowing the identification of numerous noncoding RNAs with key roles in biological processes. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt with limited phylogenetic conservation, expressed at low levels and characterized by tissue/organ specific expression profiles. Although a large set of lncRNAs has been identified, the functional roles of lncRNAs are only beginning to be recognized and the molecular mechanism of lncRNA-mediated gene regulation remains largely unexplored, particularly in plants where their annotation and characterization are still incomplete. Using public and proprietary poly-(A)+ RNA-seq data as well as a collection of full length ESTs from several organs, developmental stages and stress conditions in three Brachypodium distachyon inbred lines, we describe the identification and the main features of thousands lncRNAs. Here we provide a genome-wide characterization of lncRNAs, highlighting their intraspecies conservation and describing their expression patterns among several organs/tissues and stress conditions. This work represents a fundamental resource to deepen our knowledge on long noncoding RNAs in C3 cereals, allowing the Brachypodium community to exploit these results in future research programs.

To elucidate the physiology underlying the development of superficial scald in pears,susceptible"Blanquilla"fruit was treated with different compounds that either promoted(ethylene)or repressed(1-methylcyclopropene and lovastatin)the incidence of this disorder after 4 months of cold storage.Our data show that scald was negligible for the fruit treated with 1-methylcyclopropene or lovastatin,but highly manifested in untreated(78％incidence)or ethylene-treated fruit(97％incidence).The comparison between the fruit metabolomic profile and transcriptome evidenced a distinct reprogramming associated with each treatment.In all treated samples,cold storage led to an activation of a cold-acclimation-resistance mechanism,including the biosynthesis of very-long-chain fatty acids,which was especially evident in 1-methylcyclopropane-treated fruit.Among the treatments applied,only 1-methylcyclopropene inhibited ethylene production,hence supporting the involvement of this hormone in the development of scald.However,a common repression effect on the PPO gene combined with higher sorbitol content was found for both lovastatin and 1-methylcyclopropene-treated samples,suggesting also a non-ethylene-mediated process preventing the development of this disorder.The results presented in this work represent a step forward to better understand the physiological mechanisms governing the etiology of superficial scald in pears.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recent advances in high throughput sequencing technology have revealed a pervasive and complex transcriptional activity of all eukaryotic genomes and have allowed the identification and characterization of several classes of noncoding RNAs (ncRNAs) with key roles in various biological processes. Among ncRNAs, long ncRNAs (lncRNAs) are transcripts typically longer than 200 nucleotides whose members tend to be expressed at low levels, show a lack of phylogenetic conservation and exhibit tissue-specific, cell-specific, or stress-responsive expression profiles. Although a large set of lncRNAs has been identified both in animal and plant systems, the regulatory roles of lncRNAs are only beginning to be recognized and the molecular basis of lncRNA mediated gene regulation remains largely unexplored, particularly in plants. Here, we describe an efficient methodology to identify long noncoding RNAs using next-generation sequencing data.