Thermal Stability of Au Rhombic Dodecahedral Nanocrystals Can Be Greatly Enhanced by Coating Their Surface with an Ultrathin Shell of Pt.

Rhombic dodecahedral nanocrystals have been considered particularly difficult to synthesize because they are enclosed by {110}, a low-index facet with the greatest surface energy. Recently, we demonstrated the use of seed-mediated growth for the facile and robust synthesis of Au rhombic dodecahedral nanocrystals (AuRD). While the unique shape and surface structure of AuRD are desirable for potential applications in plasmonics and catalysis, respectively, their high surface energy makes them highly susceptible to thermal degradation. Here we demonstrate that it is feasible to greatly improve the thermal stability with some sacrifice to the plasmonic properties of the original AuRD by coating their surface with an ultrathin shell made of Pt. Our in situ electron microscopy analysis indicates that the ultrathin Pt coating can increase the thermal stability from 60 up to 450 °C, a trend that is also supported by the results from a computational study.

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Gene expression changes throughout the life cycle allow a bacterial plant pathogen to persist in diverse environmental habitats.

Bacterial pathogens exhibit a remarkable ability to persist and thrive in diverse ecological niches. Understanding the mechanisms enabling their transition between habitats is crucial to control dissemination and potential disease outbreaks. Here, we use Ralstonia solanacearum, the causing agent of the bacterial wilt disease, as a model to investigate pathogen adaptation to water and soil, two environments that act as bacterial reservoirs, and compare this information with gene expression in planta. Gene expression in water resembled that observed during late xylem colonization, with an intriguing induction of the type 3 secretion system (T3SS). Alkaline pH and nutrient scarcity-conditions also encountered during late infection stages-were identified as the triggers for this T3SS induction. In the soil environment, R. solanacearum upregulated stress-responses and genes for the use of alternate carbon sources, such as phenylacetate catabolism and the glyoxylate cycle, and downregulated virulence-associated genes. We proved through gain- and loss-of-function experiments that genes associated with the oxidative stress response, such as the regulator OxyR and the catalase KatG, are key for bacterial survival in soil, as their deletion cause a decrease in culturability associated with a premature induction of the viable but non culturable state (VBNC). This work identifies essential factors necessary for R. solanacearum to complete its life cycle and is the first comprehensive gene expression analysis in all environments occupied by a bacterial plant pathogen, providing valuable insights into its biology and adaptation to unexplored habitats.

Fast and Non-equilibrium Uptake of Hydrogen by Pd Icosahedral Nanocrystals.

We report for the first time that Pd nanocrystals can absorb H via a "single-phase pathway" when particles with a proper combination of shape and size are used. Specifically, when Pd icosahedral nanocrystals of 7- and 12-nm in size are exposed to H atoms, the H-saturated twin boundaries can divide each particle into 20 smaller single-crystal units in which the formation of phase boundaries is no longer favored. As such, absorption of H atoms is dominated by the single-phase pathway and one can readily obtain PdHx with anyx in the range of 0-0.7. When switched to Pd octahedral nanocrystals, the single-phase pathway is only observed for particles of 7 nm in size. We also establish that the H-absorption kinetics will be accelerated if there is a tensile strain in the nanocrystals due to the increase in lattice spacing. Besides the unique H-absorption behaviors, the PdHx (x=0-0.7) icosahedral nanocrystals show remarkable thermal and catalytic stability toward the formic acid oxidation due tothe decrease in chemical potential for H atoms in a Pd lattice under tensile strain.

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

Bioavailability of Cariban® Capsules: A Modified-Release Fixed-Dose Combination of Doxylamine and Pyridoxine to Relieve Nausea and Vomiting During Pregnancy.

BACKGROUND: Nausea and vomiting is a very prevalent condition during pregnancy. Combination of doxylamine and pyridoxine is placed as first-line pharmacological option for its treatment in most clinical guidelines. Among different release forms available, Cariban® is a fixed-dose combination of doxylamine/pyridoxine 10/10 mg, formulated as modified-release capsules. OBJECTIVES: In the present study, we aimed to characterize the bioavailability performance of Cariban® in vitro and in vivo. METHODS: An in vitro dissolution test was performed to evaluate the release profile of Cariban®, together with immediate- and delayed-release formulations available on the market. A single-center, single-dose, open-label bioavailability study following Cariban® administration in 12 healthy adult female patients was carried out to explore the drug behavior in vivo (protocol NBR-002-13; EUDRA-CT 2013-005422-35). These data were additionally used to perform a computational pharmacokinetic simulation of the posology approved for this drug. RESULTS: Cariban® capsules demonstrate a prolonged-release performance, with an early, gradual, and progressive release of both actives until reaching a complete dissolution after 4-5 h in solution. The pharmacokinetic features of these capsules show that doxylamine and pyridoxine metabolites are early absorbed, being all detectable in plasma within 1 h following oral administration. Computational pharmacokinetic simulation predicts that different posology provides distinct profiles of metabolites in plasma, with 1-1-2 (morning-midafternoon-night) being the one that concentrates higher plasma levels but lower dose dumping for 24 h. CONCLUSION: Cariban® behaves as a prolonged-release formulation, which correlates with rapid absorption and arising of the actives in the plasma, but also long-lasting and sustained bioavailability, especially when administered following the complete posology. These results would underlie its demonstrated efficacy to relieve nausea and vomiting of pregnancy (NVP) under clinical settings.

The Tomato Feruloyl Transferase FHT Promoter Is an Accurate Identifier of Early Development and Stress-Induced Suberization.

As a wall polymer, suberin has a multifaceted role in plant development and stress responses. It is deposited between the plasma membrane and the primary cell wall in specialized tissues such as root exodermis, endodermis, phellem, and seed coats. It is formed de novo in response to stresses such as wounding, salt injury, drought, and pathogen attack and is a complex polyester mainly consisting of fatty acids, glycerol, and minor amounts of ferulic acid that are associated to a lignin-like polymer predominantly composed of ferulates. Metabolomic and transcriptomic studies have revealed that cell wall lignification precedes suberin deposition. The ferulic acid esterified to ω-hydroxy fatty acids, synthetized by the feruloyl transferase FHT (or ASFT), presumably plays a role in coupling both polymers, although the precise mechanism is not understood. Here, we use the promoter of tomato suberin feruloyl transferase (FHT/ASFT) fused to GUS (ß-glucuronidase) to demonstrate that ferulate deposition agrees with the site of promoter FHT activation by using a combination of histochemical staining and UV microscopy. Hence, FHT promoter activation and alkali UV microscopy can be used to identify the precise localization of early suberizing cells rich in ferulic acid and can additionally be used as an efficient marker of early suberization events during plant development and stress responses. This line can be used in the future as a tool to identify emerging suberization sites via ferulate deposition in tomato plants, which may contribute to germplasm screening in varietal improvement programs.

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

Recognition 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.

Phase-Controlled Synthesis of Ru Nanocrystals via Template-Directed Growth: Surface Energy versus Bulk Energy.

Despite the successful control of crystal phase using template-directed growth, much remains unknown about the underlying mechanisms. Here, we demonstrate that the crystal phase taken by the deposited metal depends on the lateral size of face-centered cubic (fcc)-Pd nanoplate templates with 12 nm plates giving fcc-Ru while 18-26 nm plates result in hexagonal closed-packed (hcp)-Ru. Although Ru overlayers with a metastable fcc- (high in bulk energy) or stable hcp-phase (low in bulk energy) can be epitaxially deposited on the basal planes, the lattice mismatch will lead to jagged hcp- (high in surface energy) and smooth fcc-facets (low in surface energy), respectively, on the side faces. As the proportion of basal and side faces on the nanoplates varies with lateral size, the crystal phase will change depending on the relative contributions from the surface and bulk energies. The Pd@fcc-Ru outperforms the Pd@hcp-Ru nanoplates toward ethylene glycol and glycerol oxidation reactions.

Induced ligno-suberin vascular coating and tyramine-derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato.

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance.

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses.

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N1-107 ) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant-pathogen co-evolution.

The Bacterial Wilt Reservoir Host Solanum dulcamara Shows Resistance to Ralstonia solanacearum Infection.

Ralstonia solanacearum causes bacterial wilt, a devastating plant disease, responsible for serious losses on many crop plants. R. solanacearum phylotype II-B1 strains have caused important outbreaks in temperate regions, where the pathogen has been identified inside asymptomatic bittersweet (Solanum dulcamara) plants near rivers and in potato fields. S. dulcamara is a perennial species described as a reservoir host where R. solanacearum can overwinter, but their interaction remains uncharacterised. In this study, we have systematically analysed R. solanacearum infection in S. dulcamara, dissecting the behaviour of this plant compared with susceptible hosts such as tomato cv. Marmande, for which the interaction is well described. Compared with susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the stem. Taken together, these results demonstrate that S. dulcamara behaves as partially resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study proves that tolerance (i.e., the capacity to reduce the negative effects of infection) is not required for a wild plant to act as a reservoir host. We propose that inherent resistance (impediment to colonisation) and a perennial habit enable bittersweet plants to behave as reservoirs for R. solanacearum.

Molecular Detection of Ralstonia solanacearum to Facilitate Breeding for Resistance to Bacterial Wilt in Potato.

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs.

Sustainable natural bioresources in crop protection: antimicrobial hydroxycoumarins induce membrane depolarization-associated changes in the transcriptome of Ralstonia solanacearum.

BACKGROUND: Ralstonia solanacearum is one of the most devastating pathogens affecting crop production worldwide. The hydroxycoumarins (umbelliferone, esculetin and daphnetin) represent sustainable natural bioresources on controlling plant bacterial wilt. However, the antibacterial mechanism of hydroxycoumarins against plant pathogens still remains poorly understood. RESULTS: Here we characterized the effect of three hydroxycoumarins on the transcriptome of R. solanacearum. All three hydroxycoumarins were able to kill R. solanacearum, but their antibacterial activity impacted differently the bacterial transcriptome, indicating that their modes of action might be different. Treatment of R. solanacearum cultures with hydroxycoumarins resulted in a large number of differentially expressed genes (DEGs), involved in basic cellular functions and metabolic process, such as down-regulation of genes involved in fatty acid synthesis, lipopolysaccharides biosynthesis, RNA modification, ribosomal submits, oxidative phosphorylation and electrontransport, as well as up-regulation of genes involved in transcriptional regulators, drug efflux, and oxidative stress responses. Future studies based on in vitro experiments are proposed to investigate lipopolysaccharides biosynthesis pathway leading to R. solanacearum cell death caused by hydroxycoumarins. Deletion of lpxB substantially inhibited the growth of R. solanacearum, and reduced virulence of pathogen on tobacco plants. CONCULSION: Our transcriptomic analyses show that specific hydroxycoumarins suppressed gene expression involved in fatty acid synthesis, RNA modification, ribosomal submits, oxidative phosphorylation and electrontransport. These findings provide evidence that hydroxycoumarins inhibit R. solanacearum growth through multi-target effect. Hydroxycoumarins could serve as sustainable natural bioresources against plant bacterial wilt through membrane destruction targeting the lipopolysaccharides biosynthesis pathway.

Identification of RipAZ1 as an avirulence determinant of Ralstonia solanacearum in Solanum americanum.

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease.

A genome-wide association study reveals cytokinin as a major component in the root defense responses against Ralstonia solanacearum.

Bacterial wilt caused by the soil-borne pathogen Ralstonia solancearum is economically devastating, with no effective methods to fight the disease. This pathogen invades plants through their roots and colonizes their xylem, clogging the vasculature and causing rapid wilting. Key to preventing colonization are the early defense responses triggered in the host's root upon infection, which remain mostly unknown. Here, we have taken advantage of a high-throughput in vitro infection system to screen natural variability associated with the root growth inhibition phenotype caused by R. solanacearum in Arabidopsis during the first hours of infection. To analyze the genetic determinants of this trait, we have performed a genome-wide association study, identifying allelic variation at several loci related to cytokinin metabolism, including genes responsible for biosynthesis and degradation of cytokinin. Further, our data clearly demonstrate that cytokinin signaling is induced early during the infection process and cytokinin contributes to immunity against R. solanacearum. This study highlights a new role for cytokinin in root immunity, paving the way for future research that will help in understanding the mechanisms underpinning root defenses.