AtSTP8, an endoplasmic reticulum-localised monosaccharide transporter from Arabidopsis, is recruited to the extrahaustorial membrane during powdery mildew infection.

Biotrophic pathogens are believed to strategically manipulate sugar transport in host cells to enhance their access to carbohydrates. However, mechanisms of sugar translocation from host cells to biotrophic fungi such as powdery mildew across the plant-haustorium interface remain poorly understood. To investigate this question, systematic subcellular localisation analysis was performed for all the 14 members of the monosaccharide sugar transporter protein (STP) family in Arabidopsis thaliana. The best candidate AtSTP8 was further characterised for its transport properties in Saccharomyces cerevisiae and potential role in powdery mildew infection by gene ablation and overexpression in Arabidopsis. Our results showed that AtSTP8 was mainly localised to the endoplasmic reticulum (ER) and appeared to be recruited to the host-derived extrahaustorial membrane (EHM) induced by powdery mildew. Functional complementation assays in S. cerevisiae suggested that AtSTP8 can transport a broad spectrum of hexose substrates. Moreover, transgenic Arabidopsis plants overexpressing AtSTP8 showed increased hexose concentration in leaf tissues and enhanced susceptibility to powdery mildew. Our data suggested that the ER-localised sugar transporter AtSTP8 may be recruited to the EHM where it may be involved in sugar acquisition by haustoria of powdery mildew from host cells in Arabidopsis.

Multiple intramolecular trafficking signals in RESISTANCE TO POWDERY MILDEW 8.2 are engaged in activation of cell death and defense.

The extrahaustorial membrane (EHM) is a host-derived interfacial membrane encasing the haustorium of powdery mildew fungi. Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically targeted to the EHM via two EHM-targeting signals. Here, we demonstrate that proper coordination between the trafficking forces engaged via the EHM-targeting signals and the nuclear localization signals (NLSs), as well as the nuclear export signals (NESs), in RPW8.2 is critical for the activation of cell death and defense. We show that in the absence of pathogens, RPW8.2 is partitioned between the cytoplasm and the nucleus, and turned over via both the 26S proteasome- and the vacuole-dependent pathways. Enhanced cytoplasmic localization of RPW8.2 by tagging it with a NES led to lethal cell death. By contrast, enhanced nuclear localization of RPW8.2 by adding an NLS to it resulted in resistance to powdery mildew. Whereas expression of the NES-containing C-terminal domain of RPW8.2 in the cytoplasm is sufficient to trigger cell death, no such cell death-inducing activity is found with RPW8.2 variants that contain the two EHM-targeting signals along with the NES-containing C-terminal domain. In addition, we present evidence for the involvement of a leaf senescence pathway in RPW8.2-mediated cell death and defense. Taken together, our data suggest that RPW8.2 is subject to adjustment by distinct and perhaps coordinated mechanisms for its localization and function via interaction with the multiple intramolecular trafficking signals, which should provide further insights into RPW8.2-activated, EHM-focused resistance against powdery mildew.

Dual and Opposing Roles of Xanthine Dehydrogenase in Defense-Associated Reactive Oxygen Species Metabolism in Arabidopsis.

While plants produce reactive oxygen species (ROS) for stress signaling and pathogen defense, they need to remove excessive ROS induced during stress responses in order to minimize oxidative damage. How can plants fine-tune this balance and meet such conflicting needs? Here, we show that XANTHINE DEHYDROGENASE1 (XDH1) in Arabidopsis thaliana appears to play spatially opposite roles to serve this purpose. Through a large-scale genetic screen, we identified three missense mutations in XDH1 that impair XDH1's enzymatic functions and consequently affect the powdery mildew resistance mediated by RESISTANCE TO POWDERY MILDEW8 (RPW8) in epidermal cells and formation of xanthine-enriched autofluorescent objects in mesophyll cells. Further analyses revealed that in leaf epidermal cells, XDH1 likely functions as an oxidase, along with the NADPH oxidases RbohD and RbohF, to generate superoxide, which is dismutated into H2O2 The resulting enrichment of H2O2 in the fungal haustorial complex within infected epidermal cells helps to constrain the haustorium, thereby contributing to RPW8-dependent and RPW8-independent powdery mildew resistance. By contrast, in leaf mesophyll cells, XDH1 carries out xanthine dehydrogenase activity to produce uric acid in local and systemic tissues to scavenge H2O2 from stressed chloroplasts, thereby protecting plants from stress-induced oxidative damage. Thus, XDH1 plays spatially specified dual and opposing roles in modulation of ROS metabolism during defense responses in Arabidopsis.

Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew.

BACKGROUND: Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. RESULTS: We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620-6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116-175 genes encoding CSEPs with limited gene amplification. CONCLUSIONS: Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.

Homologues of the RPW8 Resistance Protein Are Localized to the Extrahaustorial Membrane that Is Likely Synthesized De Novo.

Upon penetration of the host cell wall, the powdery mildew fungus develops a feeding structure named the haustorium in the invaded host cell. Concomitant with haustorial biogenesis, the extrahaustorial membrane (EHM) is formed to separate the haustorium from the host cell cytoplasm. The Arabidopsis resistance protein RPW8.2 is specifically targeted to the EHM where it activates haustorium-targeted resistance against powdery mildew. RPW8.2 belongs to a small family with six members in Arabidopsis (Arabidopsis thaliana). Whether Homologs of RPW8 (HR) 1 to HR4 are also localized to the EHM and contribute to resistance has not been determined. Here, we report that overexpression of HR1, HR2, or HR3 led to enhanced resistance to powdery mildew, while genetic depletion of HR2 or HR3 resulted in enhanced susceptibility, indicating that these RPW8 homologs contribute to basal resistance. Interestingly, we found that N-terminally YFP-tagged HR1 to HR3 are also EHM-localized. This suggests that EHM-targeting is an ancestral feature of the RPW8 family. Indeed, two RPW8 homologs from Brassica oleracea tested also exhibit EHM-localization. Domain swapping analysis between HR3 and RPW8.2 suggests that sequence diversification in the N-terminal 146 amino acids of RPW8.2 probably functionally distinguishes it from other family members. Moreover, we found that N-terminally YFP-tagged HR3 is also localized to the plasma membrane and the fungal penetration site (the papilla) in addition to the EHM. Using this unique feature of YFP-HR3, we obtained preliminary evidence to suggest that the EHM is unlikely derived from invagination of the plasma membrane, rather it may be mainly synthesized de novo.

Arabidopsis phospholipase Dα1 and Dδ oppositely modulate EDS1- and SA-independent basal resistance against adapted powdery mildew.

Plants use a tightly regulated immune system to fight off various pathogens. Phospholipase D (PLD) and its product, phosphatidic acid, have been shown to influence plant immunity; however, the underlying mechanisms remain unclear. Here, we show that the Arabidopsis mutants pldα1 and pldδ, respectively, exhibited enhanced resistance and enhanced susceptibility to both well-adapted and poorly adapted powdery mildew pathogens, and a virulent oomycete pathogen, indicating that PLDα1 negatively while PLDδ positively modulates post-penetration resistance. The pldα1δ double mutant showed a similar infection phenotype to pldα1, genetically placing PLDα1 downstream of PLDδ. Detailed genetic analyses of pldδ with mutations in genes for salicylic acid (SA) synthesis (SID2) and/or signaling (EDS1 and PAD4), measurement of SA and jasmonic acid (JA) levels, and expression of their respective reporter genes indicate that PLDδ contributes to basal resistance independent of EDS1/PAD4, SA, and JAsignaling. Interestingly, while PLDα1-enhanced green fluorescent protein (eGFP) was mainly found in the tonoplast before and after haustorium invasion, PLDδ-eGFP's focal accumulation to the plasma membrane around the fungal penetration site appeared to be suppressed by adapted powdery mildew. Together, our results demonstrate that PLDα1 and PLDδ oppositely modulate basal, post-penetration resistance against powdery mildew through a non-canonical mechanism that is independent of EDS1/PAD4, SA, and JA.

Orbitalwise Coordination Number for Predicting Adsorption Properties of Metal Nanocatalysts.

We present the orbitalwise coordination number CN^{α} (α=s or d) as a reactivity descriptor for metal nanocatalysts. With the noble metal Au (5d^{10}6s^{1}) as a specific case, the CN^{s} computed using the two-center s-electron hopping integrals to neighboring atoms provides an accurate and robust description of the trends in CO and O adsorption energies on extended surfaces terminated with different facets and nanoparticles of varying size and shape, outperforming existing bond-counting methods. Importantly, the CN^{s} has a solid physiochemical basis via a direct connection to the moment characteristics of the projected density of states onto the s orbital of a Au adsorption site. Furthermore, the CN^{s} shows promise as a viable descriptor for predicting adsorption properties of Au alloy nanoparticles with size-dependent lattice strains and coinage metal ligands.

A comprehensive mutational analysis of the Arabidopsis resistance protein RPW8.2 reveals key amino acids for defense activation and protein targeting.

The Arabidopsis thaliana resistance to powdery mildew8.2 (RPW8.2) protein is specifically targeted to the extrahaustorial membrane (EHM) encasing the haustorium, or fungal feeding structure, where RPW8.2 activates broad-spectrum resistance against powdery mildew pathogens. How RPW8.2 activates defenses at a precise subcellular locale is not known. Here, we report a comprehensive mutational analysis in which more than 100 RPW8.2 mutants were functionally evaluated for their defense and trafficking properties. We show that three amino acid residues (i.e., threonine-64, valine-68, and aspartic acid-116) are critical for RPW8.2-mediated cell death and resistance to powdery mildew (Golovinomyces cichoracearum UCSC1). Also, we reveal that two arginine (R)- or lysine (K)-enriched short motifs (i.e., R/K-R/K-x-R/K) make up the likely core EHM-targeting signals, which, together with the N-terminal transmembrane domain, define a minimal sequence of 60 amino acids that is necessary and sufficient for EHM localization. In addition, some RPW8.2 mutants localize to the nucleus and/or to a potentially novel membrane that wraps around plastids or plastid-derived stromules. Results from this study not only reveal critical amino acid elements in RPW8.2 that enable haustorium-targeted trafficking and defense, but also provide evidence for the existence of a specific, EHM-oriented membrane trafficking pathway in leaf epidermal cells invaded by powdery mildew.

Ectopic expression of RESISTANCE TO POWDERY MILDEW8.1 confers resistance to fungal and oomycete pathogens in Arabidopsis.

Broad-spectrum disease resistance is a highly valuable trait in plant breeding and attracts special attention in research. The Arabidopsis gene locus RESISTANCE TO POWDERY MILDEW 8 (RPW8) contains two adjacent homologous genes, RPW8.1 and RPW8.2, and confers broad-spectrum resistance to powdery mildew. Remarkably, the RPW8.2 protein is specifically localized to the extrahaustorial membrane (EHM) encasing the feeding structure of powdery mildew whereby RPW8.2 activates haustorium-targeted defenses. Here, we show that ectopic expression of the yellow fluorescent protein (YFP)-tagged RPW8.1 from the native promoter leads to unique cell death lesions and enhances resistance to virulent fungal and oomycete pathogens that cause powdery mildew and downy mildew diseases, respectively. In powdery mildew-infected plants, RPW8.1-YFP accumulates at higher levels in the mesophyll cells underneath the infected epidermal cells where RPW8.2-YFP is mainly expressed. This cell type-preferential protein accumulation pattern largely correlates with that of H(2)O(2) accumulation, suggesting that RPW8.1 may spatially collaborate with RPW8.2 in activation of resistance to powdery mildew. Interestingly, when ectopically expressed from the RPW8.2 promoter, RPW8.1-YFP is also targeted to the EHM of powdery mildew and the transgenic plants display resistance to both powdery mildew and downy mildew. Using YFP as a reporter, we further reveal that the RPW8.1 promoter is constitutively active but induced to higher levels in cells at the infection site, whereas the RPW8.2 promoter is activated specifically in cells at the infection site. Taken together, our results suggest that RPW8.1 (and its promoter) is functionally distinct from RPW8.2 and may have a higher potential in engineering broad-spectrum resistance in plants.

Functional identification of multiple nucleocytoplasmic trafficking signals in the broad-spectrum resistance protein RPW8.2.

Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important intramolecular regulatory elements for protein nucleocytoplasmic trafficking. This regulation confers spatial specificity to signal initiation and transduction in eukaryotic cells and thus is fundamental to the viability of all eukaryotic organisms. Here, we developed a simple and rapid method in which activity of putative NLSs or NESs was reported by subcellular localization of two tandem fluorescent proteins in fusion with the respective NLSs or NESs after agroinfiltration-mediated transient expression in leaves of Nicotiana benthamiana (Nb). We further demonstrated that the predicted NES from amino acid residue (aa) 9 to 22 and the NLS from aa91 to 101 in the broad-spectrum disease resistance protein RPW8.2 possess nuclear export and import activity, respectively. Additionally, by testing overlapping fragments covering the full length of RPW8.2, we identified another NLS from aa65 to 74 with strong nuclear import activity and two tandem non-canonical NESs in the C-terminus with strong nuclear export activity. Taken together, our results demonstrated the utility of a simple method to evaluate potential NLSs and NESs in plant cells and suggested that RPW8.2 may be subject to opposing nucleocytoplasmic trafficking forces for its subcellular localization and functional execution.

Effects of surface activation on the structural and catalytic properties of ruthenium nanoparticles supported on mesoporous silica.

Using colloid-based methods to prepare supported catalytic metallic nanoparticles (NPs) often faces the challenge of removing the stabilizer used during synthesis and activating the catalyst without modifying the particles or the support. We explored three surface activation protocols (thermal oxidation at 150 °C, thermal reduction at 350 °C, and argon-protected calcination at 650 °C) to activate ruthenium NPs supported on mesoporous silica (MSU-F), and assessed their effects on the structural and catalytic properties of the catalysts, and their activity by the aqueous phase hydrogenation of pyruvic acid. The NPs were synthesized by polyol reduction using poly-N-vinyl-2-pyrrolidone (PVP) as a stabilizer, and supported on MSU-F by sonication-assisted deposition. The NPs maintained their original morphology on the support during activation. Ar-protected calcination was the most efficient of the three for completely removing PVP from particle surfaces, and provided the highest degree of particle crystallinity and a metal dispersion comparable to commercial Ru/SiO2. Its catalytic performance was significantly higher than the other two protocols, although all three thermally activated catalysts achieved higher activity than the commercial catalyst at the same Ru loading. Post-reaction analysis also showed that the supported catalyst activated at 650 °C retained its morphology during the reaction, which is an important requirement for recyclability.

Efficient enrichment of uranium (VI) in aqueous solution using magnesium-aluminum layered double hydroxide composite phosphate-modified hydrothermal biochar: Mechanism and adsorption.

This study presents the successful synthesis of Magnesium-aluminum layered double hydroxide composite phosphate-modified hydrothermal biochar for efficient removal of U(VI) from aqueous solutions. A novel synthesis approach involving phosphate thermal polymerization-hydrothermal method was employed, deviating from conventional pyrolysis methods, to produce hydrothermal biochar. The combination of solvent thermal polymerization technique with hydrothermal process facilitated efficient loading of layered double hydroxide (LDH) components onto the biochar surface, ensuring simplicity, low energy consumption and enhanced modifiability. Bamboo waste was utilized as the precursor for biochar, highlighting its superior green and sustainable characteristics. Additionally, this study elucidated the interactions between phosphate-modified hydrothermal biochar and LDH components with U(VI). Physicochemical analysis demonstrated that the composite biochar possessed a high surface area and abundant oxygen-containing functional groups. XPS and FTIR analyses confirmed the efficient adsorption of U(VI), attributed to chelation interactions between phosphate groups, magnesium hydroxyl groups, hydroxyl groups and U(VI), as well as the co-precipitation of U(VI) with multi-hydroxyl aluminum cations captured by LDH. The composite biochar reached adsorption equilibrium with U(VI) within 80 min and exhibited excellent fitting to the pseudo-second-order kinetic model and Langmuir model. Under conditions of pH = 4 and 298 K, it displayed significantly high maximum adsorption capacity of approximately 388.81 mg g⁻1, surpassing untreated biochar by 17-fold. The adsorption process was found to be endothermic and spontaneous and even after five consecutive adsorption-desorption cycles, the removal efficiency of U(VI) remained stable at 75.46%. These findings underscore the promising application prospects of Magnesium-aluminum layered double hydroxide composite phosphate-modified hydrothermal biochar in efficiently separating U(VI) from uranium-containing wastewater, emphasizing its environmental and economic value.

The geological origins and soil properties of loess-like silty clay: a case study in the jinan area.

This study, using Jinan as a case study, systematically investigates the characteristics and geological genesis of loess-like silty clay in the middle and lower reaches of the Yellow River. The primary distribution of loess-like silty clay is revealed through field surveys, laboratory experiments, and previous literature reviews. The chemical and physical properties of the loess-like silty clay were examined, in addition to investigations into its mineral composition, microstructural characteristics, and engineering mechanical properties, in order to enhance comprehension of its attributes and formation mechanisms. The research suggests that the distinctive soil environment in the area has been influenced by numerous instances of the Yellow River overflow and channel shifts over its history, as well as the impacts of climate change, geological factors, and human activities. The primary sources of material for the loess-like silty clay consist of loess, Hipparion Red Clay, and paleosol layers. The discussion also addresses the impact of regional climate on the formation of mineral components. The aforementioned findings hold significant implications for advancing the understanding of historical climatic and paleogeographic shifts, as well as for addressing engineering challenges associated with the distribution of loess-like silty clay.

Clarification of the infection pattern of Xanthomonas citri subsp. citri on citrus fruit by artificial inoculation.

BACKGROUND: Citrus canker is a significant bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that severely impedes the healthy development of the citrus industry. Especially when citrus fruit is infected by Xcc, it will reduce or even lost its commercial value. However, due to the prolonged fruiting cycle and intricate structure, much less research progress had been made in canker disease on fruit than on leaf. In fact, limited understanding has been achieved on canker development and the response to Xcc infection in fruit. RESULTS: Herein, the progression of canker disease on sweet orange fruit was tracked in the field. Results indicated that typical lesions initially appear on the sepal, style residue, nectary disk, epicarp, and peduncle of young fruits after petal fall. The susceptibility of fruits to Xcc infection diminished as the fruit developed, with no new lesions forming at the ripening stage. The establishment of an efficient method for inoculating Xcc on fruit as well as the artificial inoculation throughout the fruit's developmental cycle clarified this infection pattern. Additionally, microscopic observations during the infection process revealed that Xcc invasion caused structural changes on the surface and cross-section of the fruit. CONCLUSIONS: An efficient system for inoculation on citrus fruit with Xcc was established, by which it can serve for the evaluation of citrus germplasm for canker disease resistance and systematic research on the interactions between Xcc and citrus fruits.

Characterization of the binding ability of the odorant binding protein BminOBP9 of Bactrocera minax to citrus volatiles.

BACKGROUND: Bactrocera minax, one of the most important citrus pests, oviposits exclusively on citrus fruit. In the insect olfactory system, odorant-binding proteins (OBPs) facilitate the initial recognition role of host odor molecules. The aim of this study was to characterize the functional OBPs of B. minax and identify specific volatile organic compounds in the Citrus genus as OBP targets. RESULTS: BminOBP9 (BminGOBP99a), a closely related homolog of BdorGOBP99a, which reduces the egg-laying behavior of Bactrocera dorsalis through silencing technology, was cloned, expressed, and purified. The binding ability of BminOBP9 to 11 citrus volatiles was then examined using fluorescence competition binding assays (FCBA). The results demonstrated that BminOBP9 could bind to all tested citrus volatiles, as could BdorGOBP99a, ZcucGOBP99a, and ZtauGOBP99a. Interestingly, the binding ability of BminOBP9 was the strongest among the four, suggesting that BminOBP9 may have a function in the specific recognition of citrus volatiles. Furthermore, we aligned the above four proteins and found nine distinctive amino acid sites in BminOBP9. To identify the unique binding sites of BminOBP9, we produced the nine mutants using site-directed mutagenesis. Further FCBA showed that the binding ability of the nine mutants to citrus volatiles significantly reduced, and six of them (substitutes S24P, L36F, E53K, N68D, D112A, and S118R) had the weakest binding ability. CONCLUSION: The results demonstrated that BminOBP9 was the specific protein involved in the perception of citrus host volatiles by B. minax. Moreover, BminOBP9 could prove efficient in screening the candidate odors for pest management. © 2020 Society of Chemical Industry.

Safety and Efficacy of a Novel Intubating Laryngeal Mask during the recovery period following Supratentorial Tumour Surgery.

OBJECTIVE: To assess safety and efficacy of a novel intubation laryngeal mask airway (ILMA) during the recovery period following supratentorial tumour surgery. METHODS: Patients who underwent supratentorial tumour surgery at our centre from January 2012 to December 2016 were eligible for this prospective randomised, parallel group study. We developed a novel ILMA using closely fitting laryngeal masks (No. 4/5) with 7.0/7.5 mm endotracheal tubes (ETT) plus screw fixators and anti-pollution sleeves. RESULTS: In total, 100 patients were intubated with the novel ILMA and 100 the ETT. There were no differences between groups in haemodynamic variables, oxygen saturation, exhaled CO2, or bispectral index all recorded during the 72-hour recovery period. However, there were significantly fewer incidences of coughing, less fluid drainage and lower haemoglobin levels in surgical fluid in the ILMA group compared with the ETT group. CONCLUSION: Our novel ILMA device was associated with reduced coughing, fluid drainage and blood in surgical drain during the recovery period following supratentorial tumour surgery.

Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance.

The RPW8 locus from Arabidopsis thaliana Ms-0 includes two functional paralogous genes (RPW8.1 and RPW8.2) and confers broad-spectrum resistance via the salicylic acid-dependent signaling pathway to the biotrophic fungal pathogens Golovinomyces spp. that cause powdery mildew diseases on multiple plant species. To identify proteins involved in regulation of the RPW8 protein function, a yeast two-hybrid screen was performed using RPW8.2 as bait. The 14-3-3 isoform lambda (designated GF14lambda) was identified as a potential RPW8.2 interactor. The RPW8.2-GF14lambda interaction was specific and engaged the C-terminal domain of RPW8.2, which was confirmed by pulldown assays. The physiological impact of the interaction was revealed by knocking down GF14lambda by T-DNA insertion, which compromised basal and RPW8-mediated resistance to powdery mildew. In addition, over-expression of GF14lambda resulted in hypersensitive response-like cell death and enhanced resistance to powdery mildew via the salicylic acid-dependent signaling pathway. The results from this study suggest that GF14lambda may positively regulate the RPW8.2 resistance function and play a role in enhancing basal resistance in Arabidopsis.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp. citri to achieve resistance to citrus canker disease.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a serious bacterial disease that affects citrus production worldwide. Citron C-05 (Citrus medica) is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc. However, it has not been determined when, where, and how Xcc is restricted in the tissues of Citron C-05 during the infection process. In the present study, we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc (eGFP-Xcc) strain in Citron C-05 along with five susceptible biotypes (i.e., lemon, pummelo, sour orange, sweet orange, and ponkan mandarin) upon inoculation via the spraying or leaf infiltration of a bacterial suspension. The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes, Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection. Not surprisingly, resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death, as well as greater upregulation of several defense-related genes, including a pathogenesis-related gene (PR1) and a glutathione S-transferase gene (GST1), compared with sweet orange as a susceptible control. Taken together, our results not only provide further valuable details of the spatiotemporal dynamics of the host entry, propagation, and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

Correlation between controlled lung collapse and early lung injury in dogs.

A new type of pulmonary sequestration ventilator was used to compare the relationship between controlled lung collapse and early lung injury in thoracic surgery for dogs. Eighteen experimental dogs were randomly divided into three groups (G1-G3 groups). After general anesthesia, the shunt balance in lung was controlled and the pulmonary sequestration tube was placed in the femoral artery and vein, and the Swan-Ganz tube was placed into the right internal jugular vein as well. Two-lung ventilation (TLV) was first performed for 20 min, followed by one-lung ventilation (OLV). The degree of collapse was 100% (G1), 90% (G2), and 50% (G3). Blood samples were extracted from femoral artery and jugular vein prior to collapse (T0), and at 30 (T1), 60 (T2), and 120 (T3) min after collapse for blood gas analysis to determine the shunt ratio (Qs/Qt). Blood samples were also subjected to enzyme linked immunosorbent assay (ELISA) to determine serum tumor necrosis factor-α (TNF-α), intercellular immune adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) levels. Arterial blood pressure, heart rate, pulmonary artery pressure and other physiological indicators were monitored during the experiment. Lung tissues were collected at T3 to calculate the wet/dry weight ratio (W/D). Histopathological changes were observed and compared by microscopic observation and blind scoring of pathological section after hematoxylin and eosin (H&E) staining. There were no significant differences in the physiological indexes between the two groups during TLV (P>0.05). Mean pulmonary arterial pressure (MPAP) in G2 and G3 groups was significantly more stable than that in G1 group after OLV (P<0.05); shunt ratio Qs/Qt, W/D, and serum TNF-α, ICAM-1 and IL-6 levels in the lung were decreased; and the degrees of pulmonary edema, hemorrhage, inflammatory cell infiltration and lung injury were also decreased. There was no statistically significant difference in each index at each time-point between G2 and G3 groups (P>0.05). Compared with complete lung collapse (collapse degree: 100%), controlled lung collapse (collapse degree: 90% and 50%) can better reduce the intraoperative lung injury, but there was no significant difference between the collapse degrees of 90 and 50%.

Multiple Evolutionary Events Involved in Maintaining Homologs of Resistance to Powdery Mildew 8 in Brassica napus.

The Resistance to Powdery Mildew 8 (RPW8) locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs) in Brassica rapa and three in Brassica oleracea (BoHRs). Brassica napus (Bn) is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs). It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here, we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion, and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants.