Construction of central and axial chirality via Pd(II)/Bim-catalyzed asymmetric dearomative Michael reaction of polycyclic tropones.

A highly enantioselective Pd/Bim-catalyzed dearomative Michael reaction applying polycyclic tropones as non-benzenoid aromatic Michael acceptors and arylboronic acids as aryl pronucleophiles has been developed. The bridged biaryls bearing central and axial chirality, including pentacyclic cyclohepta[b]indoles and 6,7-dihydrodibenzo[a,c][7]annulen-5-ones, are generally generated in good to high yields and excellent enantioselectivities and can be readily transformed into useful derivatives.

Precipitation input increases biodiversity of planktonic communities in the Qinghai-Tibet Plateau.

Planktonic communities in aquatic ecosystems are crucial water quality indicators, with their growth dependent on runoff chemical and hydraulic characteristics (e.g., nutrient availability and turbidity). Previous studies have indicated that runoff components (i.e., proportions of precipitation, groundwater, snowmelt, etc.) play a vital role in regulating runoff characteristics, potentially affecting planktonic communities. However, the response of these communities to runoff components, particularly in mountainous regions, remains underexplored. In this study, we conducted four sampling campaigns from 2017 to 2020 in a watershed on the Qinghai-Tibet Plateau. Combined with laboratory incubation experiments, we examined the impact of various runoff components on the diversity and abundance of phytoplankton and zooplankton. We found that a higher proportion of precipitation in runoff contributed to an increase in the diversity of plankton communities. Laboratory experiments with unified water samples incubated with different runoff components demonstrated that the significant influence of precipitation on planktonic diversity primarily stems from the influx of abundant exogenous particulate material into rivers. Using a path analysis, we further confirmed that the impact of precipitation on diversity is primarily through chemical pathways, notably by increasing nutrient concentrations. Our study enhances our understanding of the interactions between the hydrological cycle and aquatic ecosystems, offering valuable insights for effectively maintaining and managing these natural environments.

The polycaprolactone and silk fibroin nanofibers with Janus-structured sheaths for antibacterial and antioxidant by loading Taxifolin.

Electrospinning is a widely recognized method for producing Janus or core-shell nanofibers. In this study, nanofibrous membranes were fabricated through co-axial electrospinning utilizing polycaprolactone (PCL) and silk fibroin (SF) as the Janus shell, and taxifolin (TAX) and SF as the core. The resulting nanofibers had diameters of 816 ± 161 nm and core diameters of 73 ± 5 nm. The morphology and properties of the PCL-SF@SF/TAX nanofibers were subsequently analyzed. The results demonstrated that the nanofibrous membranes achieved physical and chemical characteristics potential for tissue engineering and drug delivery. Specifically, the membranes exhibited a Young's modulus of 9.64 ± 0.29 MPa, a water contact angle of 79.1 ± 1.3°, and a weight loss of 17.3 ± 1.0 % over a period of 28 days. The incorporation of TAX endowed the membranes with antibacterial properties, effectively combating Escherichia coli and Staphylococcus aureus. Furthermore, the membranes demonstrated antioxidant capabilities, with a DPPH radical scavenging efficiency of 38.5 ± 5.6 % and a Trolox-equivalent antioxidant capacity of 0.24 ± 0.01 mM. The release of the antioxidant was sustained over 28 days, following first-order release kinetics. The nanofibrous membranes, referred to as PSST, exhibit promising potential for use as biomaterials, characterized by their antibacterial activity, antioxidant and cytocompatibility.

AlphaFold-guided redesign of a plant pectin methylesterase inhibitor for broad-spectrum disease resistance.

Plant cell walls are a critical site where plants and pathogens continuously struggle for physiological dominance. Here we show that dynamic remodeling of pectin methylesterification of plant cell walls is a component of the physiological and co-evolutionary struggles between hosts and pathogens. A Phytophthora sojae secreted pectin methylesterase (PsPME1) decreases the degree of pectin methylesterification, thus synergizing with an endo-polygalacturonase (PsPG1) to weaken plant cell walls. To counter PsPME1-mediated susceptibility, a plant-derived pectin methylesterase inhibitor protein, GmPMI1, protects pectin to maintain a high methylesterification status. GmPMI1 protects plant cell walls from enzymatic degradation by inhibiting both soybean and P. sojae pectin methylesterases during infection. However, constitutive expression of GmPMI1 disrupted the tradeoff between host growth and defense responses. So, we used AlphaFold structure tools to design a modified form of GmPMI1 (GmPMI1R) which specifically targets and inhibits pectin methylesterases secreted from pathogens but not from the plants. Transient expression of GmPMI1R enhanced plant resistance to oomycetes and fungal pathogens. In summary, our work highlights biochemical modification of the cell wall as an important focal point in the physiological and co-evolutionary conflict between the hosts and microbes and serves as an important proof-of-concept for how rapid advancements in AI-driven structure-based tools can accelerate the prediction of new strategies for plant protection.

Prediction of the global occurrence of maize diseases and estimation of yield loss under climate change.

BACKGROUND: Climate change significantly impacts global maize production via yield reduction, posing a threat to global food security. Disease-related crop damage reduces quality and yield and results in economic losses. However, the occurrence of diseases caused by climate change, and thus crop yield loss, has not been given much attention. RESULTS: This study aims to investigate the potential impact of six major diseases on maize yield loss over the next 20 to 80 years under climate change. To this end, the Maximum Entropy model was implemented, based on Coupled Model Intercomparison Project 6 data. The results indicated that temperature and precipitation are identified as primary limiting factors for disease onset. Southern corn rust was projected to be the most severe disease in the future; with a few of the combined occurrence of all the selected diseases covered in this study were predicted to progressively worsen over time. Yield losses caused by diseases varied per continent, with North America facing the highest loss, followed by Asia, South America, Europe, Africa, and Oceania. CONCLUSION: This study provides a basis for regional projections and global control of maize diseases under future climate conditions. © 2024 Society of Chemical Industry.

Amino acid mutation of succinate dehydrogenase complex induced resistance to benzovindiflupyr in Magnaporthe oryzae.

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhBH245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance.

Synergistic enhancement of oxygen vacancy enrichment and morphology regulation in CeO2-NiCo2O4 heterostructure catalysts for high-performance cathodes in direct borohydride-hydrogen peroxide fuel cells.

Hydrogen peroxide (H2O2) emerges as a viable oxidant for fuel cells, necessitating the development of an efficient and cost-effective electrocatalyst for the hydrogen peroxide reduction reaction (HPRR). In this study, we synthesized a self-supporting, highly active HPRR electrocatalyst comprising two morphologically distinct components: CeO2-NiCo2O4 nanowires and CeO2-NiCo2O4 metal organic framework derivatives, via a two-step hydrothermal process followed by air calcination. X-ray diffraction and transmission electron microscopy analysis confirmed the presence of CeO2 and NiCo2O4, revealing the amalgamated interface between them. CeO2 exhibits multifunctionality in regulating the surface electronic configuration of NiCo2O4, fostering synergistic connections, and introducing oxygen deficiencies to enhance the catalytic efficacy in HPRR. Electrochemical measurements demonstrate a reduction current density of 789.9 mA·cm-2 at -0.8 V vs. Ag/AgCl. The assembly of direct borohydride-hydrogen peroxide fuel cell (DBHPFC) exhibits a peak power density of 45.2 mW·cm-2, demonstrating durable stability over a continuous operation period of 120 h. This investigation providing evidence that the fabrication of heterostructured catalysts based on CeO2 for HPRR is a viable approach for the development of high-efficiency electrocatalysts in fuel cell technology.

Multifunctional bilayer nanofibrous membrane enhances periodontal regeneration via mesenchymal stem cell recruitment and macrophage polarization.

The continuous stimulation of periodontitis leads to a decrease in the number of stem cells within the lesion area and significantly impairing their regenerative capacity. Therefore, it is crucial to promote stem cell homing and regulate the local immune microenvironment to suppress inflammation for the regeneration of periodontitis-related tissue defects. Here, we fabricated a novel multifunctional bilayer nanofibrous membrane using electrospinning technology. The dense poly(caprolactone) (PCL) nanofibers served as the barrier layer to resist epithelial invasion, while the polyvinyl alcohol/chitooligosaccharides (PVA/COS) composite nanofiber membrane loaded with calcium beta-hydroxy-beta-methylbutyrate (HMB-Ca) acted as the functional layer. Material characterization tests revealed that the bilayer nanofibrous membrane presented desirable mechanical strength, stability, and excellent cytocompatibility. In vitro, PCL@PVA/COS/HMB-Ca (P@PCH) can not only directly promote rBMSCs migration and differentiation, but also induce macrophage toward pro-healing (M2) phenotype-polarization with increasing the secretion of anti-inflammatory and pro-healing cytokines, thus providing a favorable osteoimmune environment for stem cells recruitment and osteogenic differentiation. In vivo, the P@PCH membrane effectively recruited host MSCs to the defect area, alleviated inflammatory infiltration, and accelerated bone defects repair. Collectively, our data indicated that the P@PCH nanocomposite membrane might be a promising biomaterial candidate for guided tissue regeneration in periodontal applications.

Deciphering the key factors affecting pesticide residue risk in vegetable ecosystem.

Soil contamination, particularly from pesticide residues, presents a significant challenge to the sustainable development of agricultural ecosystems. Identifying the key factors influencing soil pesticide residue risk and implementing effective measures to mitigate their risks at the source are essential. Here, we collected soil samples and conducted a comprehensive survey among local farmers in the Three Gorges Reserve Area, a major agricultural production region in Southwest China. Subsequently, employing a dual analytical approach combining structural equation modeling (SEM) and random forest modeling (RFM), we examined the effects of various factors on pesticide residue accumulation in vegetable ecosystems. Our SEM analysis revealed that soil characteristics (path coefficient 0.85) and cultivation factor (path coefficient 0.84) had the most significant effect on pesticide residue risk, while the farmer factors indirectly influenced pesticide residues by impacting both cultivation factors and soil characteristics. Further exploration using RFM identified the three most influential factors contributing to pesticide residue risk as cation exchange capacity (CEC) (account for 18.84%), cultivation area (account for 14.12%), and clay content (account for 13.01%). Based on these findings, we carried out experimental trials utilizing Integrated Pest Management (IPM) technology, resulting in a significant reduction in soil pesticide residues and notable improvements in crop yields. Therefore, it is recommended that governmental efforts should prioritize enhanced training for vegetable farmers, promotion of eco-friendly plant protection methods, and regulation of agricultural environments to ensure sustainable development.

Can restoring water and sediment fluxes across a mega-dam cascade alleviate a sinking river delta?

Hydropower, although an attractive renewable energy source, can alter the flux of water, sediments, and biota, producing detrimental impacts in downstream regions. The Mekong River illustrates the impacts of large dams and the limitations of conventional dam regulating strategies. Even under the most optimistic sluicing scenario, sediment load at the Mekong Delta could only recover to 62.3 ± 8.2 million tonnes (1 million tonnes = 109 kilograms), short of the (100 to 160)-million tonne historical level. Furthermore, unless retrofit to reroute sediments, the dams are doomed to continue trapping sediment for at least 170 years and thus starve downstream reaches of sediment, contributing to the impending disappearance of the Mekong Delta. Therefore, we explicitly challenge the widespread use of large dead storages-the portion of the reservoirs that cannot be emptied-in dam designs. Smaller dead storages can ease sediment starvation in downstream regions, thereby buffering against sinking deltas or relative sea level rises.

Molecular mechanism of reduced biological fitness of fludioxonil-resistant strains of Botrytis cinerea based on transcriptome analysis.

BACKGROUND: Fludioxonil is a fungicide used to control gray mold. However, the frequency of resistance in the field is low, and highly resistant strains are rarely isolated. The biological fitness of the resistant strain is lower than that of the wild strain. Therefore, the molecular mechanism underlying the decrease in the fitness of the fludioxonil-resistant strain of Botrytis cinerea was explored to provide a theoretical basis for resistance monitoring and management. RESULTS: Transcriptome analysis was performed on five different-point mutant resistant strains of fludioxonil, focusing on mining and screening candidate genes that lead to reduced fitness of the resistant strains and the functional verification of these genes. The differentially expressed genes (DEGs) of the five point-mutation resistant strains intersected with 1869 DEGs. Enrichment analysis showed that three downregulated genes (Bcin05g07030, Bcgad1, and Bcin03g05840) were enriched in multiple metabolic pathways and were downregulated in both domesticated strains. Bcin05g07030 and Bcin03g05840 were involved in mycelial growth and development, pathogenicity, and conidial yield, and negatively regulated oxidative stress and cell wall synthesis. Bcgad1 was involved in mycelial growth and development, conidial yield, oxidative stress, and cell wall synthesis. Furthermore, Bcin05g07030 was involved in osmotic stress and spore germination, whereas Bcin03g05840 and Bcgad1 negatively regulated osmotic stress and cell wall integrity. CONCLUSION: These results enable us to further understand the molecular mechanism underlying the decrease in the biological fitness of B. cinerea fludioxonil-resistant strains. © 2024 Society of Chemical Industry.

Induction of Salmonella Enteritidis into a Viable but Nonculturable State by Cinnamaldehyde and Its Resuscitation.

Trans-cinnamaldehyde (TC), a typical plant-derived compound, has been widely used in the control of foodborne pathogen contamination. Nevertheless, the risk associated with the occurrence of viable but nonculturable (VBNC) bacteria induced by TC remains unclear. The results of this study showed that Salmonella Enteritidis (S. Enteritidis) entered the VBNC state after being induced by TC at a minimum inhibitory concentration of 312.5 µg/mL and survived for at least 22 days under TC treatment. Enhanced resistance was found against heat treatment (75°C, 30 s), antibiotics (i.e., ampicillin, ceftriaxone sodium, chloramphenicol), and hydrogen peroxide (3%) in VBNC S. Enteritidis. A synergistic effect against VBNC S. Enteritidis occurred when TC was combined with acid treatment, including lactic acid and acetic acid (pH = 3.5). VBNC and resuscitated S. Enteritidis by sodium pyruvate treatment (100 mM) were found to retain the infectious ability to Caco-2 cells. Relative expression levels of the stress-related genes relA, spoT, ppx, lon, katG, sodA, dnaK, and grpE were upregulated in VBNC S. Enteritidis. Accumulation of reactive oxygen species (ROS) and protein aggregates was observed in VBNC cells. Besides, the resuscitation of VBNC cells was accompanied with clearance of ROS and protein aggregates. In summary, this study presents a comprehensive characterization of stress tolerance and resuscitation of VBNC S. Enteritidis induced by cinnamaldehyde, and the results provide useful information for the development of effective control strategy against VBNC pathogenic bacteria in food production.

Postnatal Development of Synaptic Plasticity at Hippocampal CA1 Synapses: Correlation of Learning Performance with Pathway-Specific Plasticity.

To determine the critical timing for learning and the associated synaptic plasticity, we analyzed developmental changes in learning together with training-induced plasticity. Rats were subjected to an inhibitory avoidance (IA) task prior to weaning. While IA training did not alter latency at postnatal day (PN) 16, there was a significant increase in latency from PN 17, indicating a critical day for IA learning between PN 16 and 17. One hour after training, acute hippocampal slices were prepared for whole-cell patch clamp analysis following the retrieval test. In the presence of tetrodotoxin (0.5 µM), miniature excitatory postsynaptic currents (mEPSCs) and inhibitory postsynaptic currents (mIPSCs) were sequentially recorded from the same CA1 neuron. Although no changes in the amplitude of mEPSCs or mIPSCs were observed at PN 16 and 21, significant increases in both excitatory and inhibitory currents were observed at PN 23, suggesting a specific critical day for training-induced plasticity between PN 21 and 23. Training also increased the diversity of postsynaptic currents at PN 23 but not at PN 16 and 21, demonstrating a critical day for training-induced increase in the information entropy of CA1 neurons. Finally, we analyzed the plasticity at entorhinal cortex layer III (ECIII)-CA1 or CA3-CA1 synapses for each individual rat. At either ECIII-CA1 or CA3-CA1 synapses, a significant correlation between mean α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/N-methyl-D-aspartic acid (AMPA/NMDA) ratio and learning outcomes emerged at PN 23 at both synapses, demonstrating a critical timing for the direct link between AMPA receptor-mediated synaptic plasticity and learning efficacy. Here, we identified multiple critical periods with respect to training-induced synaptic plasticity and delineated developmental trajectories of learning mechanisms at hippocampal CA1 synapses.

Binding Mode and Molecular Mechanism of the Two-Component Histidine Kinase Bos1 of Botrytis cinerea to Fludioxonil and Iprodione.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1. The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 µg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply.

Functional Evolution of Pseudofabraea citricarpa as an Adaptation to Temperature Change.

Citrus target spot, caused by Pseudofabraea citricarpa, was formerly considered a cold-tolerant fungal disease. However, it has now spread from high-latitude regions to warmer low-latitude regions. Here, we conducted physiological observations on two different strains of the fungus collected from distinct regions, and evaluated their pathogenicity. Interestingly, the CQWZ collected from a low-latitude orchard, exhibited higher temperature tolerance and pathogenicity when compared to the SXCG collected from a high-latitude orchard. To further understand the evolution of temperature tolerance and virulence in these pathogens during the spread process, as well as the mechanisms underlying these differences, we performed genomic comparative analysis. The genome size of CQWZ was determined to be 44,004,669 bp, while the genome size of SXCG was determined to be 45,377,339 bp. Through genomic collinearity analysis, we identified two breakpoints and rearrangements during the evolutionary process of these two strains. Moreover, gene annotation results revealed that the CQWZ possessed 376 annotated genes in the "Xenobiotics biodegradation and metabolism" pathway, which is 79 genes more than the SXCG. The main factor contributing to this difference was the presence of salicylate hydroxylase. We also observed variations in the oxidative stress pathways and core pathogenic genes. The CQWZ exhibited the presence of a heat shock protein (HSP SSB), a catalase (CAT2), and 13 core pathogenic genes, including a LysM effector, in comparison to the SXCG. Furthermore, there were significant disparities in the gene clusters responsible for the production of seven metabolites, such as Fumonisin and Brefeldin. Finally, we identified the regulatory relationship, with the HOG pathway at its core, that potentially contributes to the differences in thermotolerance and virulence. As the global climate continues to warm, crop pathogens are increasingly expanding to new territories. Our findings will enhance understanding of the evolution mechanisms of pathogens under climate change.

CRISPR-targeted mutagenesis of mitogen-activated protein kinase phosphatase 1 improves both immunity and yield in wheat.

Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over- or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plant Arabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock-down of TaMKP1 by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused by Puccinia striiformis f. sp. tritici (Pst) and powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), indicating that TaMKP1 negatively regulates disease resistance in wheat. Unexpectedly, while Tamkp1 mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild-type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK-mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield.

Genomic epidemiology of hypervirulent Listeria monocytogenes CC619: Population structure, phylodynamics and virulence.

Listeria monocytogenes is a ubiquitous foodborne pathogen causing human and animal listeriosis with high mortality. Neurological and maternal-neonatal listeriosis outbreaks in Europe and the United States were frequently associated with clonal complexes CC1, CC2 and CC6 harboring Listeria Pathogenicity Island-1 (LIPI-1), as well as CC4 carrying both LIPI-1 and LIPI-4. However, human listeriosis in China was predominantly linked to CC87 and CC619 from serotype 1/2b. To understand the genetic evolution and distribution patterns of CC619, we characterized the epidemic history, population structure, and transmission feature of CC619 strains through analysis of 49,421 L. monocytogenes genomes globally. We found that CC619 was uniquely distributed in China, and closely related with perinatal infection. As CC619 strains were being mainly isolated from livestock and poultry products, we hypothesized that pigs and live chicken were the reservoirs of CC619. Importantly, all CC619 strains not only harbored the intact LIPI-1 and LIPI-4, but these also carried LIPI-3 that could facilitate host colonization and invasion. The deficiency of LIPI-3 or LIPI-4 markedly decreased L. monocytogenes colonization capacity in a model of intragastric infection in the mouse. Altogether, our findings suggest that the hypervirulent CC619 harboring three pathogenicity islands LIPI-1, LIPI-3 and LIPI-4 is a putatively persistent population in various foods, environment, and human population, warranting the further research for deciphering its pathogenicity and strengthening epidemiological surveillance.

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula.

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

Heat Resistance, Virulence, and Gene Expression of Desiccation-Adapted Salmonella Enteritidis During Long-Term Storage in Low-Water Activity Foods.

Desiccation stress could induce crossprotection and even affect virulence of Salmonella enterica. However, the influence of food matrices with low-water activity on desiccation adaptation of Salmonella still remains unclear. This study investigated the survival and adaptation of Salmonella Enteritidis in skim milk powder, ginger powder, and chocolate powder under desiccation storage conditions for a total of 12 weeks. High survival rates of Salmonella Enteritidis in all food matrices maintained over the long-term desiccation storage. Desiccation-adapted Salmonella Enteritidis enhanced heat resistance (p < 0.05) with the increase of storage time. Food composition plays an important role in the induction of crossresistance of desiccation-adapted Salmonella. After desiccation storage, Salmonella Enteritidis in ginger powder was most tolerant to heat treatment. Salmonella Enteritidis in skim milk powder was most resistant to the gastrointestinal simulation environment, and had strongest adhesion to Caco-2 cells. The effects of food composition on gene expression (rpoS, proV, otsA, otsB, grpE, dnaK, rpoH, and sigDE) in desiccation-adapted Salmonella Enteritidis were not significant (p > 0.05). At initial desiccation storage, osmotic protection-related genes (fadA, proV, otsA, and otsB), stress response regulator (rpoS), and heat-resistance-related genes (grpE, dnaK, and rpoH) were all significantly upregulated (p < 0.05). However, after 4-week storage, the expression level of desiccation-related genes, proV, otsA, otsB, grpE, dnaK, and rpoH, significantly decreased (p < 0.05). This study enables a better understanding of Salmonella's responses to long-term desiccation stress in different kinds of low-water activity foods.

Plant hypersensitive induced reaction protein facilitates cell death induced by secreted xylanase associated with the pathogenicity of Sclerotinia sclerotiorum.

Necrotrophic fungal plant pathogens employ cell death-inducing proteins (CDIPs) to facilitate infection. However, the specific CDIPs and their mechanisms in pathogenic processes of Sclerotinia sclerotiorum, a necrotrophic pathogen that causes disease in many economically important crop species, have not yet been clearly defined. This study found that S. sclerotiorum secretes SsXyl2, a glycosyl hydrolase family 11 xylanase, at the late stage of hyphal infection. SsXyl2 targets the apoplast of host plants to induce cell death independent of xylanase activity. Targeted disruption of SsXyl2 leads to serious impairment of virulence, which can be recovered by a catalytically impaired SsXyl2 variant, thus supporting the critical role of cell death-inducing activity of SsXyl2 in establishing successful colonization of S. sclerotiorum. Remarkably, infection by S. sclerotiorum induces the accumulation of Nicotiana benthamiana hypersensitive-induced reaction protein 2 (NbHIR2). NbHIR2 interacts with SsXyl2 at the plasma membrane and promotes its localization to the cell membrane and cell death-inducing activity. Furthermore, gene-edited mutants of NbHIR2 displayed increased resistance to the wild-type strain of S. sclerotiorum, but not to the SsXyl2-deletion strain. Hence, SsXyl2 acts as a CDIP that manipulates host cell physiology by interacting with hypersensitive induced reaction protein to facilitate colonization by S. sclerotiorum. These findings provide valuable insights into the pathogenic mechanisms of CDIPs in necrotrophic pathogens and lead to a more promising approach for breeding resistant crops against S. sclerotiorum.