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Plants have developed strategies to deal with the great variety of challenges they are exposed to. Among them, common targets are the regulation of transcription and translation to finely modulate protein levels during both biotic and abiotic stresses. Increasing evidence suggests that ribosomes are highly adaptable modular supramolecular structures which remodel to adapt to stresses. Each Arabidopsis thaliana ribosome consists of approximately 81 distinct ribosomal proteins (RPs), each of which is encoded by two to seven genes. To investigate the identity of ribosomal proteins of the small subunit (RPS) and of the large subunit (RPL) as well as ribosomes-associated proteins, we analysed by LC/MS/MS immunopurified ribosomes from A. thaliana leaves treated with isonicotinic acid (INA), an inducer of plant innate immunity. We quantified a total of 2084 proteins. 165 ribosome-associated proteins showed increased abundance while 52 were less abundant. Of the 52 identified RPS (from a possibility of 104 encoding genes), 15 were deregulated. Similarly, from the 148 possible RPL, 80 were detected and 9 were deregulated. Our results revealed potential candidates involved in innate immunity that could be interesting targets for functional genomic studies.

Under field conditions, plants are often simultaneously exposed to several abiotic and biotic stresses resulting in significant reductions in growth and yield; thus, developing a multi-stress tolerant variety is imperative. Previously, we reported the neofunctionalization of a novel PNP family protein, Putranjiva roxburghii purine nucleoside phosphorylase (PRpnp) to trypsin inhibitor to cater to the needs of plant defense. However, to date, no study has revealed the potential role and mechanism of either member of this protein group in plant defense. Here, we overexpressed PRpnp in Citrus aurantifolia which showed nuclear-cytoplasmic localization, where it functions in maintaining the intracellular purine reservoir. Overexpression of PRpnp significantly enhanced tolerance to salt, oxidative stress, alkaline pH, drought, and two pests, Papilio demoleus and Scirtothrips citri in transgenic plants. Global gene expression studies revealed that PRpnp overexpression up-regulated differentially expressed genes (DEGs) related to ABA- and JA- biosynthesis and signaling, plant defense, growth, and development. LC-MS/MS analysis validated higher endogenous ABA and JA accumulation in transgenic plants. Taken together, our results suggest that PRpnp functions by enhancing the endogenous ABA and JA, which interact synergistically and it also inhibits trypsin proteases in the insect gut. Also, like other purine salvage genes, PRpnp also regulates CK metabolism and increases the levels of CK-free bases in transgenic Mexican lime. We also suggest that PRpnp can be used as a potential candidate to develop new varieties with improved plant vigor and enhanced multiple stress resistance.

Plants have developed strategies to deal with the great variety of challenges they are exposed to. Among them, common targets are the regulation of transcription and translation to finely modulate protein levels during both biotic and abiotic stresses. Increasing evidence suggests that ribosomes are highly adaptable modular supramolecular structures which remodel to adapt to stresses. Each Arabidopsis thaliana ribosome consists of approximately 80 distinct ribosomal proteins (RPs), each of which being encoded by two to seven genes. To investigate the identity of ribosomal proteins of the small subunit (RPS) and of the large subunit (RPL) as well as ribosomes-associated proteins, we analysed by LC/MS/MS immunopurified ribosomes from A. thaliana leaves treated with isonicotinic acid (INA), an inducer of plant innate immunity. We quantified a total of 2084 proteins. 165 ribosome-associated proteins showed increased abundance while 52 were less abundant. Of the 55 identified RPS (from a possibility of 104 encoding genes), 16 were deregulated. Similarly, from the 148 possible RPL, 81 were detected and 9 were deregulated. Our results revealed potential candidates involved in innate immunity that could be interesting targets for functional genomic studies.

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. We know little about why only some cultivars in crop plants are tissue culture friendly. In this study, tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency. Multiomics studies involving transcriptomics, proteomics, metabolomics, and biochemical analysis were performed using GP and D91 callus to unravel the regulatory mechanisms. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MSMS revealed 3062 protein groups and 16989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis also revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91.Highlights:Multi omics analysis revealed chloroplast play crucial role in providing in vitro regeneration capability in contrasting genotypes

Abiotic and biotic stresses induce the formation of reactive oxygen species (ROS), which subsequently causes the excessive accumulation of aldehydes in cells. Stress-derived aldehydes are commonly designated as reactive electrophile species (RES) as a result of the presence of an electrophilic α, β-unsaturated carbonyl group. Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent enzymes that metabolize a wide range of endogenous and exogenous aliphatic and aromatic aldehyde molecules by oxidizing them to their corresponding carboxylic acids. The ALDH enzymes are found in nearly all organisms, and plants contain fourteen ALDH protein families. In this review, we performed a critical analysis of the research reports over the last decade on plant ALDHs. Newly discovered roles for these enzymes in metabolism, signaling and development have been highlighted and discussed. We concluded with suggestions for future investigations to exploit the potential of these enzymes in biotechnology and to improve our current knowledge about these enzymes in gene signaling and plant development.

RNA sequencing allows the measuring of gene expression at a resolution unmet by expression arrays or RT-qPCR. It is however necessary to normalize sequencing data by library size, transcript size and composition, among other factors, before comparing expression levels. The use of internal control genes or spike-ins is advocated in the literature for scaling read counts, but the methods for choosing reference genes are mostly targeted at RT-qPCR studies and require a set of pre-selected candidate controls or pre-selected target genes.Here, we report an R-based pipeline to select internal control genes based solely on read counts and gene sizes. This novel method first normalizes the read counts to Transcripts per Million (TPM) and then excludes weakly expressed genes using the DAFS script to calculate the cut-off. It then selects as references the genes with lowest TPM covariance. We used this method to pick custom reference genes for the differential expression analysis of three transcriptome sets from transgenic Arabidopsis plants expressing heterologous fungal effector proteins tagged with GFP (using GFP alone as the control). The custom reference genes showed lower covariance and fold change as well as a broader range of expression levels than commonly used reference genes. When analyzed with NormFinder, both typical and custom reference genes were considered suitable internal controls, but the custom selected genes were more stably expressed. geNorm produced a similar result in which most custom selected genes ranked higher (i.e. were more stably expressed) than commonly used reference genes.The proposed method is innovative, rapid and simple. Since it does not depend on genome annotation, it can be used with any organism, and does not require pre-selected reference candidates or target genes that are not always available.

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347’s role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

Highlights • 1.82% of asymptomatic individuals may be SARS-CoV-2 carriers. • SARS-CoV-2 may spread asymptomatically in a population under social distancing restrictions., Several countries have undertaken social distancing measures to stop SARS-CoV-2’ spread. Asymptomatic carriers’ prevalence is unknown and would provide essential information on hidden viral circulation. In our cross-sectional study, 1.82% of 330 asymptomatic confined individuals living in the community carried SARS-CoV-2 despite no contact with declared cases, raising concerns about unnoticed transmission., GRAPHICAL ABSTRACT Asymptomatic frequency estimate during lockdown. * according to public data during the time of the study. Image, graphical abstract

Camera pose estimation in known scenes is a 3D geometry task recently tackled by multiple learning algorithms. Many regress precise geometric quantities, like poses or 3D points, from an input image. This either fails to generalize to new viewpoints or ties the model parameters to a specific scene. In this paper, we go Back to the Feature: we argue that deep networks should focus on learning robust and invariant visual features, while the geometric estimation should be left to principled algorithms. We introduce PixLoc, a scene-agnostic neural network that estimates an accurate 6-DoF pose from an image and a 3D model. Our approach is based on the direct alignment of multiscale deep features, casting camera localization as metric learning. PixLoc learns strong data priors by end-to-end training from pixels to pose and exhibits exceptional generalization to new scenes by separating model parameters and scene geometry. The system can localize in large environments given coarse pose priors but also improve the accuracy of sparse feature matching by jointly refining keypoints and poses with little overhead. The code will be publicly available at https://github.com/cvg/pixloc., Comment: Accepted to CVPR 2021

Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with “highly unsaturated and phenolic compounds” and “peptides” enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

Rust fungi are plant pathogens that cause epidemics that threaten the production of important plant species, such as wheat, soy, coffee and poplar.Melampsora larici-populina(Mlp) causes the poplar rust and encodes at least 1 184 candidate effectors (CEs), however their functions are poorly known. In this study, we usedArabidopsisplants constitutively expressing CEs ofMlpto discover processes targeted by these fungal proteins. For this purpose, we sequenced the transcriptome and used mass spectrometry to analyse the metabolome ofArabidopsisplants expressing individually one of the 14 selected CEs and of a control line. We found 2 299 deregulated genes across the experiment. Among the down-regulated genes, the KEGG pathways “MAPK signaling pathway” and “Plant-pathogen interaction” were respectively over-represented in six and five of the 14 transgenic lines. Moreover, genes related to hormone response and defense were down-regulated across all transgenic lines are. We further observed that there were 680 metabolites deregulated in at least one CE-expressing transgenic line, with highly unsaturated and phenolic compounds enriched in up-regulated metabolites and peptides enriched among down-regulated metabolites. Interestingly, we found that transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Taken together, our results indicate that the sequence of effectors and their belonging to families may not be a good predictor of their impact on the plant.ImportanceRust fungi are plant pathogens that threaten the production of important crops, including wheat, soy, coffee and poplar. Effectors are used by pathogens to control the host, however in the case ofMelampsora larici-populina, the causal agent of the poplar rust, and other rust fungi these proteins are poorly known. We usedArabidopsisplants expressing candidate effectors (CEs) ofMlpto better understand the interaction between this pathogen and its hosts. We found that expression of unrelated CEs led to similar patterns of gene and metabolite deregulation, while transgenic lines expressing CEs belonging to the same family showed different groups of different genes and metabolites deregulated. Thus, our results suggest that functional annotation of effectors based on sequence similarity may be misleading.

In commercial settings, cannabis is generally propagated through cuttings, a process referred in the industry as cloning. Some producers perform either topping or fimming to trigger the production of axillary shoots, which will enhance the number of flowers per plants and thus increase the yield of the cannabis plants. Topping or fimming is generally performed after the cuttings have been transferred to rooting media for two weeks. We have tested a new method to increase the shoot number per plant. The modification of the standard topping method consist of performing the topping on mother plants, prior to taking the cuttings for cloning, and the cuttings are taken one week after the topping is performed. The resulting plantlets develop axillary shoots much faster and the time of production from cuttings to harvesting is decreased by 7-10 days. The method proposed herein requires minimal adjustment to the existing workflow and the plants produce as much as when standard topping is performed. Moreover, this method cuts backs on the production time and nearly two weeks are saved compared to the standard topping procedure since the plantlets do not need to recover after topping. Application of this new procedure results in faster production time and ultimately enhanced productivity.

Melampsora larici-populina (Mlp), the causal agent of Populus leaf rust, secretes an array of effectors into the host through the haustorium to gain nutrients and suppress immunity. The precise mechanisms by which these effectors promote virulence remain unclear. To address this question, we developed a transgenic Arabidopsis line expressing a candidate effector, Mlp124357. Constitutive expression of the effector increased plant susceptibility to pathogens. A GxxxG motif present in Mlp124357 is required for its subcellular localization at the vacuolar membrane of the plant cell, as replacement of the glycine residues with alanines led to the delocalization of Mlp124357 to the nucleus and cytoplasm. We used immunoprecipitation and mass spectrometry (MS) to identify Mlp124357 interaction partners. Only one of the putative interaction partners knock-out line caused delocalization of the effector, indicating that Arabidopsis protein disulfide isomerase-11 (AtPDI-11) is required for the effector localization. This interaction was further confirmed by a complementation test, a yeast-two hybrid assay and a molecular modeling experiment. Moreover, localization results and infection assays suggest that AtPDI-11 act as a helper for Mlp124357. In summary, our findings established that one of Mlp effectors resides at the vacuole surface and modulates plant susceptibility.

Overexpressing a heat shock factor gene ( TaHsfA6b T) from wheat provides thermotolerance in barley by constitutive expression of heat and other abiotic stress-response genes. Temperature is one of the most crucial abiotic factors defining the yield potential of temperate cereal crops, such as barley. The regulators of heat shock response (HSR), heat stress transcription factors (Hsfs), modulate the transcription level of heat-responsive genes to protect the plants from heat stress. In this study, an Hsf from wheat (TaHsfA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHsfA6b showed improvement in thermotolerance. The constitutive overexpression of a TaHsfA6b gene upregulated the expression of major heat shock proteins and other abiotic stress-responsive genes. RNA-seq and qRT-PCR analysis confirmed the upregulation of Hsps, chaperonins, DNAJ, LEA protein genes, and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of reactive oxygen species (ROS) occurred in wild-type (WT) plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms, showing lesser ROS accumulation under high temperature. No negative phenotypic changes were observed in overexpression lines. These results suggest that TaHsfA6b is a regulator of HSR and its overexpression altered the expression patterns of some main stress-related genes and enhanced the thermotolerance of this cereal crop.

Armillaria sinapina, a fungal pathogen of primary timber species of North American forests, causes white root rot disease that ultimately kills the trees. A more detailed understanding of the molecular mechanisms underlying this illness will support future developments on disease resistance and management, as well as in the decomposition of cellulosic material for further use. In this study, RNA-Seq technology was used to compare the transcriptome profiles of A. sinapina fungal culture grown in yeast malt broth medium supplemented or not with betulin, a natural compound of the terpenoid group found in abundance in white birch bark. This was done to identify enzyme transcripts involved in the metabolism (redox reaction) of betulin into betulinic acid, a potent anticancer drug. De novo assembly and characterization of A. sinapina transcriptome was performed using Illumina technology. A total of 170,592,464 reads were generated, then 273,561 transcripts were characterized. Approximately, 53% of transcripts could be identified using public databases with several metabolic pathways represented. A total of 11 transcripts involved in terpenoid biosynthesis were identified. In addition, 25 gene transcripts that could play a significant role in lignin degradation were uncovered, as well as several redox enzymes of the cytochromes P450 family. To our knowledge, this research is the first transcriptomic study carried out on A. sinapina.

Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study, we compared direct rRT-PCR method (without RNA extraction) using SeeGene AllplexTM 2019-nCoV rRT-PCR with the RealStar® SARS-CoV-2 rRT-PCR kit (Altona Diagnostics). Furthermore, we assessed the impact of swab storage media composition on PCR efficiency. We show that SeeGene and Altona’s assays provide similar efficiency. Importantly, we provide evidence that RNA extraction can be successfully bypassed when samples are stored in UTM medium or in molecular water but not when samples are stored in saline solution and in Hanks medium.

Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study we evaluated the reliability and the efficiency of a direct RT-QPCR method (without RNA extraction) using SeeGene Allplex™ 2019-nCoV RT-QPCR and the influence of swab storage media composition on further viral detection.We show that SeeGene’s assay provides similar efficiency as the RealStar® SARS-CoV-2 RT-PCR kit (Altona Diagnostics), and that RNA extraction is not necessary nor advantageous if samples are stored in UTM or molecular water but is recommended if samples are stored in saline solution and in Hanks medium.

Boreal forests provide important ecosystem services, most notably being the mitigation of increasing atmospheric CO2 emissions. Microbial biodiversity, particularly the local diversity of fungi, has been shown to promote multiple functions of the boreal forests of Northeastern China. However, this microbial biodiversity-multifunctionality relationship has yet to be explored in Northeastern Canada, where historical environment have shaped a different regional pool of microbial diversity. This study focuses on the relationship between the soil microbiome and ecosystem multifunctionality, as well as the influence of pH and redox potential (Eh) on the regulation of such relationship. Structural equation modelling (SEM) was used to explore the different causal relationships existing in the studied ecosystems. In a managed part of the Canadian boreal forest, 156 forest polygons were sampled to (1) estimate the &alpha, and &beta, diversity of fungal and bacterial communities and (2) measure 12 ecosystem functions mainly related to soil nutrient storage and cycling. Both bacteria and fungi influenced ecosystem multifunctionality, but on their own respective functions. Bacterial &beta, diversity was the most important factor increasing primary productivity and soil microbial biomass, while reducing soil emitted atmospheric CO2. Environmental characteristics, particularly low levels of organic matter in soil, were shown to have the strongest positive impact on boreal ecosystem multifunctionality. Overall, our results were consistent with those obtained in Northeastern China, however, some differences need to be further explored especially considering the history of forest management in Northeastern Canada.

In plant–pathogen interactions, expression and localization of effectors in the aqueous apoplastic region play a crucial role in the establishment or suppression of pathogen development. Silicon (Si) has been shown to protect plants in several host–pathogen interactions, but its mode of action remains a source of debate. Its deposition in the apoplastic area of plant cells suggests that it might interfere with receptor–effector recognition. In this study, soybean plants treated or not with Si were inoculated with Phytophthora sojae and differences in the ensuing infection process were assessed through different microscopy techniques, transcript analysis of effector and defense genes, and effector (Avr6) localization through immunolocalization and fluorescence labeling. In plants grown without Si, the results showed the rapid (4 d post-inoculation) host recognition by P. sojae through the development of haustorium-like bodies, followed by expression and release of effectors into the apoplastic region. In contrast, Si treatment resulted in limited pathogen development, and significantly lower expression and presence of Avr6 in the apoplastic region. Based on immunolocalization and quantification of Avr6 through fluorescence labeling, our results suggest that the presence of Si in the apoplast interferes with host recognition and/or limits receptor–effector interactions, which leads to an incompatible interaction.

Plant vacuoles are multifunctional organelles with dynamic and transient membranous structures, such as trans-vacuolar strands and bulbs. Bulbs are highly mobile structures that travel along trans-vacuolar strands. A candidate effector protein from Melampsora larici-populina (Mlp124357) fused with the enhanced green fluorescent protein (eGFP) was used to investigate the properties of central vacuoles and their bulbs. We discovered the coexistence of two bulb populations in Arabidopsis cells. In addition to previously-described bulbs, which present even marker protein distribution on the bulb surface, we discerned bulbs displaying irregular fusion protein distribution. Using fluorescence recovery after photobleaching (FRAP), we also demonstrated that bulbs do not exchange proteins with the tonoplast once they are formed. These results show that more than one type of bulb may co-exist in the same cell and provide evidence of micro-domains on the bulb surface. They also reveal that proteins do not flow freely from the tonoplast to the bulb membrane, giving new insight into the biology of tonoplast-derived substructures.

Lobster mushroom is a wild edible mushroom with potential commercial value. It is the product resulting of the infection, most commonly of Russula brevipes, by Hypomyces lactifluorum. This study undertook quantitative polymerase chain reaction analysis of tissues sampled at different infection stages to investigate R. brevipes – H. lactifluorum interaction. We followed the colonization of R. brevipes sporocarps by H. lactifluorum that leads to the edible lobster mushrooms. In parallel, metabolomics analysis was performed to detect differences in metabolite profile among non-infected R. brevipes sporocarp and lobster mushroom. The results show that H. lactifluorum DNA is not restricted to the margin but is distributed relatively evenly across the sporocarp of the lobster mushroom. Russula brevipes DNA was also present throughout the sporocarp but was less abundant at the margins and increased inwards. Russula brevipes DNA also declined as the infection progressed. Metabolomics analysis revealed that the flesh of lobster mushroom, which remains identical in appearance to the flesh of the host, undergoes transformation that alters its metabolite profile, most notably of lipids and terpene compounds. These results define a parasitic relationship between the two species that entails a decline of R. brevipes DNA and a modification of its metabolite profile.

Bacterial contamination of drinking water is becoming a major issue for increasing populations around the world. Current water treatment technologies based on chlorination are effective but generate toxic disinfection by-products. In this study, antibacterial electrospun chitosan-PEO/TEMPO-oxidized cellulose composite was tested for the first time for the deactivation of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli bacteria and their removal by filtration from aqueous solution. Results of the disk diffusion method, confocal microscopy and scanning electron microscopy show that both bactericidal and bacteriostatic behavior against both bacteria depend on the content and mobility of protonated amino groups. It was also found that these properties are enhanced when the biocomposite is doped with copper ions. Filtration tests in a dead-end stainless-steel cell show that both bacteria are completely removed from aqueous solutions (> 95% during the first filtration and 100% after subsequent filtrations). Finally, the composites tested can be used up to three times without significant loss of permeability. However, it was CS-PEO/TOC Cu2+ bio-based composite which displayed the slightest reduction in permeability (2.20%) after three filtrations.