Tree tobacco (Nicotiana glauca) cuticular wax composition is essential for leaf retention during drought, facilitating a speedy recovery following rewatering.

Despite decades of extensive study, the role of cuticular lipids in sustaining plant fitness is far from being understood. We utilized genome-edited tree tobacco (Nicotiana glauca) to investigate the significance of different classes of epicuticular wax in abiotic stress such as cuticular water loss, drought, and light response. We generated mutants displaying a range of wax compositions. Four wax mutants and one cutin mutant were extensively investigated for alterations in their response to abiotic factors. Although the mutations led to elevated cuticular water loss, the wax mutants did not display elevated transpiration or reduced growth under nonstressed conditions. However, under drought, plants lacking alkanes were unable to reduce their transpiration, leading to leaf death, impaired recovery, and stem cracking. By contrast, plants deficient in fatty alcohols exhibited elevated drought tolerance, which was part of a larger trend of plant phenotypes not clustering by a glossy/glaucous appearance in the parameters examined in this study. We conclude that although alkanes have little effect on whole N. glauca transpiration and biomass gain under normal, nonstressed conditions, they are essential during drought responses, since they enable plants to seal their cuticle upon stomatal closure, thereby reducing leaf death and facilitating a speedy recovery.

Dissecting Human Blood Immune Cells Response to Intracellular Infection Using Single-Cell RNA Sequencing.

Complex interactions between diverse host immune cells can determine the outcome of pathogen infections. Advances in single-cell RNA sequencing (scRNA-seq) allow detection of the transcriptional patterns of different immune cells at steady state and after infection. To reveal the complex interactions of the human immune system in response to diverse intracellular pathogens, we developed a protocol for scRNA-seq of ex vivo infected human peripheral blood mononuclear cells (PBMCs). We demonstrate here infection with Salmonella enterica serovar Typhimurium, but this protocol can be used for any other pathogen of interest, and expand our knowledge of human host-pathogen biology.

A non-classical monocyte-derived macrophage subset provides a splenic replication niche for intracellular Salmonella.

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single-cell RNA sequencing (scRNA-seq) have identified multiple subsets within the mononuclear population, but implications to their function during infection are limited. Here, we surveyed the mononuclear niche of intracellular Salmonella Typhimurium (S.Tm) during early systemic infection in mice. We described eclipse-like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue-resident red-pulp macrophages. A second phase involved extensive bacterial replication within a macrophage population characterized by CD9 expression. We demonstrated that CD9+ macrophages induced pathways for detoxificating oxidized lipids, that may be utilized by intracellular S.Tm. We established that CD9+ macrophages originated from non-classical monocytes (NCM), and NCM-depleted mice were more resistant to S.Tm infection. Our study defines macrophage subset-specific host-pathogen interactions that determine early infection dynamics and infection outcome of the entire organism.

Host succinate is an activation signal for Salmonella virulence during intracellular infection.

Key to the success of intracellular pathogens is the ability to sense and respond to a changing host cell environment. Macrophages exposed to microbial products undergo metabolic changes that drive inflammatory responses. However, the role of macrophage metabolic reprogramming in bacterial adaptation to the intracellular environment has not been explored. Here, using metabolic profiling and dual RNA sequencing, we show that succinate accumulation in macrophages is sensed by intracellular Salmonella Typhimurium (S. Tm) to promote antimicrobial resistance and type III secretion. S Tm lacking the succinate uptake transporter DcuB displays impaired survival in macrophages and in mice. Thus, S Tm co-opts the metabolic reprogramming of infected macrophages as a signal that induces its own virulence and survival, providing an additional perspective on metabolic host-pathogen cross-talk.

Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells.

Complex interactions between different host immune cell types can determine the outcome of pathogen infections. Advances in single cell RNA-sequencing (scRNA-seq) allow probing of these immune interactions, such as cell-type compositions, which are then interpreted by deconvolution algorithms using bulk RNA-seq measurements. However, not all aspects of immune surveillance are represented by current algorithms. Here, using scRNA-seq of human peripheral blood cells infected with Salmonella, we develop a deconvolution algorithm for inferring cell-type specific infection responses from bulk measurements. We apply our dynamic deconvolution algorithm to a cohort of healthy individuals challenged ex vivo with Salmonella, and to three cohorts of tuberculosis patients during different stages of disease. We reveal cell-type specific immune responses associated not only with ex vivo infection phenotype but also with clinical disease stage. We propose that our approach provides a predictive power to identify risk for disease, and human infection outcomes.

Immune cell type 'fingerprints' at the basis of outcome diversity of human infection.

Despite the availability of antibiotics and immunization, infectious diseases remain a major cause of malignancy and death worldwide. Yet, it is well documented that for most infectious agents, clinical disease develops in only a small minority of infected individuals. There is, in fact, great heterogeneity in infection outcome, from complete clearance of the pathogen to severe illness. Understanding this variation remains elusive, despite its great potential to equip us with new tools for the treatment of infectious diseases. Here, we propose a novel perspective for studying this diversity in human infection outcome, one that utilizes single-cell analysis technologies. Recent advances in single-cell RNA-seq technologies allow the detection of rare subpopulations that play important roles in host-pathogen interactions. We propose that applying single-cell RNA-seq to the study of infection can provide a 'fingerprint' of the immune cell types that are associated with the ability of the host to clear a pathogen and, thereby, broaden our current understanding of variation in susceptibility to infection within the population.

Composition of cuticular waxes coating flag leaf blades and peduncles of Triticum aestivum cv. Bethlehem.

The work herein presents comprehensive analyses of the cuticular wax mixtures covering the flag leaf blade and peduncle of bread wheat (Triticum aestivum) cv. Bethlehem. Overall, Gas Chromatography-Mass Spectrometry and Flame Ionization Detection revealed a wax coverage of flag leaf blades (16 µg/cm(2)) a third that of peduncles (49 µg/cm(2)). Flag leaf blade wax was dominated by 1-alkanols, while peduncle wax contained primarily ß-diketone and hydroxy-ß-diketones, thus suggesting differential regulation of the acyl reduction and ß-diketone biosynthetic pathways in the two analyzed organs. The characteristic chain length distributions of the various wax compound classes are discussed in light of their individual biosynthetic pathways and biosynthetic relationships between classes. Along with previously reported wheat wax compound classes (fatty acids, 1-alkanols, 1-alkanol esters, aldehydes, alkanes, ß-diketone, hydroxy-ß-diketones, alkylresorcinols and methyl alkylresorcinols), esters of 2-alkanols and three types of aromatic esters (benzyl, phenethyl and p-hydroxyphenethyl) are also reported. In particular, 2-heptanol esters were identified. Detailed analyses of the isomer distributions within 1-alkanol and 2-alkanol ester homologs revealed distinct patterns of esterified acids and alcohols, suggesting several wax ester synthases with very different substrate preferences in both wheat organs. Terpenoids, including two terpenoid esters, were present only in peduncle wax.

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines ß-Diketone Biosynthesis and Glaucousness.

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular ß-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of ß-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating ß-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a ß-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in ß-diketone biosynthesis, demonstrating a gene cluster also in the ß-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

Scratching the surface: genetic regulation of cuticle assembly in fleshy fruit.

The hydrophobic cuticular membrane of land plants performs a number of important roles during fruit development, including protection from a range of abiotic and biotic stresses. The components of the fleshy fruit cuticle are synthesized and secreted from the epidermal cells. While the biosynthetic and transport pathways of the cuticle have been thoroughly investigated for a number of decades, the regulatory mechanisms allowing fine tuning of cuticle deposition are only now beginning to be elucidated. Transcription factors belonging to the APETALA2, homeodomain-leucine zipper IV, and MYB families have been shown to be important regulators of both cuticle biosynthesis and epidermal cell differentiation, highlighting the connection between these processes. The involvement of MADS-box transcription factors demonstrates the link between fruit ripening and cuticle deposition. Epigenetic and post-transcriptional regulatory mechanisms also play a role in the control of cuticle biosynthesis, in addition to phytohormones, such as abscisic acid, that have been shown to stimulate cuticle deposition. These various levels of genetic regulation allow the plant constantly to maintain and adjust the cuticle in response to environmental and developmental cues.

Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.

During 5'-cap-dependent translation, methylated 5'-cap and 3'-poly(A) tail work synergistically in a poly(A) binding protein (PABP)-dependent manner to facilitate translation via promoting the formation of a closed mRNA loop. On the other hand, during internal translation initiation, the requirement for and the roles of 3'-poly(A) tail and PABP vary depending on specific characteristics of each internal ribosomal entry site (IRES). In this study, we analyzed the effect of 3'-poly(A) tail and phylogenetically divergent PABPs on a polypurine tract-containing IRES element derived from the coat protein gene of crucifer-infecting tobamovirus (CrTMV IRES(CP)). We find that mutations in the internal polypurine tract decrease IRES activity in a heterologous (mammalian) system in vivo. Moreover, these mutations decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays in vitro. Partial PABP depletion and reconstitution assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p provide further evidence that CrTMV IRES(CP) requires PABP for maximal activity. Furthermore, stronger enhancement in the presence of 3'-poly(A) and the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the CrTMV IRES(CP) and the 3'-poly(A).