An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana

Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way

Protease activities triggered by Ralstonia solanacearum infection in susceptible and tolerant tomato lines

Altres ajuts: CERCA Programme/Generalitat de CatalunyaActivity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum

An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana

Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way

Efficient randomization of biological networks while preserving functional characterization of individual nodes

[Background]: Networks are popular and powerful tools to describe and model biological processes. Many computational methods have been developed to infer biological networks from literature, high-throughput experiments, and combinations of both. Additionally, a wide range of tools has been developed to map experimental data onto reference biological networks, in order to extract meaningful modules. Many of these methods assess results’ significance against null distributions of randomized networks. However, these standard unconstrained randomizations do not preserve the functional characterization of the nodes in the reference networks (i.e. their degrees and connection signs), hence including potential biases in the assessment.[Results]: Building on our previous work about rewiring bipartite networks, we propose a method for rewiring any type of unweighted networks. In particular we formally demonstrate that the problem of rewiring a signed and directed network preserving its functional connectivity (F-rewiring) reduces to the problem of rewiring two induced bipartite networks. Additionally, we reformulate the lower bound to the iterations’ number of the switching-algorithm to make it suitable for the F-rewiring of networks of any size. Finally, we present BiRewire3, an open-source Bioconductor package enabling the F-rewiring of any type of unweighted network. We illustrate its application to a case study about the identification of modules from gene expression data mapped on protein interaction networks, and a second one focused on building logic models from more complex signed-directed reference signaling networks and phosphoproteomic data.[Conclusions]: BiRewire3 it is freely available at https://www.bioconductor.org/packages/BiRewire/, and it should have a broad application as it allows an efficient and analytically derived statistical assessment of results from any network biology tool.FI has been partially supported by the European Bionformatics Institute and Wellcome Trust Sanger Institute post-doctoral (ESPOD) program.Peer reviewe

Efficient randomization of biological networks while preserving functional characterization of individual nodes

Background: Networks are popular and powerful tools to describe and model biological processes. Many computational methods have been developed to infer biological networks from literature, high-throughput experiments, and combinations of both. Additionally, a wide range of tools has been developed to map experimental data onto reference biological networks, in order to extract meaningful modules. Many of these methods assess results' significance against null distributions of randomized networks. However, these standard unconstrained randomizations do not preserve the functional characterization of the nodes in the reference networks (i.e. their degrees and connection signs), hence including potential biases in the assessment.- Results: Building on our previous work about rewiring bipartite networks, we propose a method for rewiring any type of unweighted networks. In particular we formally demonstrate that the problem of rewiring a signed and directed network preserving its functional connectivity (F-rewiring) reduces to the problem of rewiring two induced bipartite networks. Additionally, we reformulate the lower bound to the iterations' number of the switching-algorithm to make it suitable for the F-rewiring of networks of any size. Finally, we present BiRewire3, an open-source Bioconductor package enabling the F-rewiring of any type of unweighted network. We illustrate its application to a case study about the identification of modules from gene expression data mapped on protein interaction networks, and a second one focused on building logic models from more complex signed-directed reference signaling networks and phosphoproteomic data.- Conclusions: BiRewire3 it is freely available at https://www.bioconductor.org/packages/BiRewire/, and it should have a broad application as it allows an efficient and analytically derived statistical assessment of results from any network biology tool

Protease Activities Triggered by Ralstonia solanacearum Infection in Susceptible and Tolerant Tomato Lines.

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum

Protease activities triggered by Ralstonia solanacearum infection in susceptible and tolerant tomato lines

Altres ajuts: CERCA Programme/Generalitat de CatalunyaActivity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum

Protease Activities Triggered by Ralstonia solanacearum Infection in Susceptible and Tolerant Tomato Lines.

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum

Dataset of Robust transcriptional indicators of plant immune cell death revealed by spatio-temporal transcriptome analyses

Leaf tissue was separately collected from the IN and OUT area of infiltration at 5 different time points: 0, 1, 2, 4 and 6 hours by making use of a sterile scalpel Leaf tissue was stored in 2 mL Eppendorf tubes and snapped-frozen in liquid nitrogen until the time of RNA extraction. Each sample collected consisted of tissue from six leaves derived from three different plants. For generation of three biological replicates from each condition (area, treatment and time), three independent experiments were performed. total sum of 60 samples -2 treatments (mock/infected), 5 time points (0, 1, 2, 4 and 6 hpi), 2 areas (IN/OUT) and 3 biological replicates- were used for RNA-sequencing. -- Organism: Arabidopsis thaliana. -- Experiment type: Expression profiling by high throughput sequencingResources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198022We aim to monitor global transcriptomic changes during the HR in Arabidopsis Col-0 leaf upon localized infiltration of Pst DC3000 (avrRpm1) (5*107cfu/mL) and mock (10 Mm MgCl2) in a spatio-temporal manner. The infiltrated cells were harvested in parallel with the immediately adjacent uninfected cells. Samples were collected at 0,1,2,4,6 post-inoculation in three biological replicates.Peer reviewe

Robust transcriptional indicators of immune cell death revealed by spatiotemporal transcriptome analyses

Altres ajuts: CERCA Programme/Generalitat de CatalunyaRecognition of a pathogen by the plant immune system often triggers a form of regulated cell death traditionally known as the hypersensitive response (HR). This type of cell death occurs precisely at the site of pathogen recognition, and it is restricted to a few cells. Extensive research has shed light on how plant immune receptors are mechanistically activated. However, two central key questions remain largely unresolved: how does cell death zonation take place, and what are the mechanisms that underpin this phenomenon? Consequently, bona fide transcriptional indicators of HR are lacking, which prevents deeper insight into its mechanisms before cell death becomes macroscopic and precludes early or live observation. In this study, to identify the transcriptional indicators of HR we used the paradigmatic Arabidopsis thaliana-Pseudomonas syringae pathosystem and performed a spatiotemporally resolved gene expression analysis that compared infected cells that will undergo HR upon pathogen recognition with bystander cells that will stay alive and activate immunity. Our data revealed unique and time-dependent differences in the repertoire of differentially expressed genes, expression profiles, and biological processes derived from tissue undergoing HR and that of its surroundings. Furthermore, we generated a pipeline based on concatenated pairwise comparisons between time, zone, and treatment that enabled us to define 13 robust transcriptional HR markers. Among these genes, the promoter of an uncharacterized AAA-ATPase was used to obtain a fluorescent reporter transgenic line that displays a strong spatiotemporally resolved signal specifically in cells that will later undergo pathogen-triggered cell death. This valuable set of genes can be used to define cells that are destined to die upon infection with HR-triggering bacteria, opening new avenues for specific and/or high-throughput techniques to study HR processes at a single-cell level