Protective effects of an electrophilic metabolite of docosahexaenoic acid on UVB-induced oxidative cell death, dermatitis, and carcinogenesis.

Docosahexaenoic acid (DHA), a representative omega-3 (ω-3) polyunsaturated fatty acids, undergoes metabolism to produce biologically active electrophilic species. 17-Oxo-DHA is one such reactive metabolite generated from DHA by cyclooxygenase-2 and dehydrogenase in activated macrophages. The present study was aimed to investigate the effects of 17-oxo-DHA on ultraviolet B (UVB)-induced oxidative stress, inflammation, and carcinogenesis in mouse skin. UVB-induced epidermal cell death was ameliorated by topically applied 17-oxo-DHA. Topical application of 17-oxo-DHA onto hairless mouse skin inhibited UVB-induced phosphorylation of the proinflammatory transcription factor, STAT3 on tyrosine 705 (Tyr705). The 17-oxo-DHA treatment also reduced the levels of oxidative stress markers, 4-hydroxynonenal-modified protein, malondialdehyde, and 8-oxo-2'-deoxyguanosine. The protective effects of 17-oxo-DHA against oxidative damage in UVB-irradiated mouse skin were associated with activation of Nrf2. 17-Oxo-DHA enhanced the engulfment of apoptotic JB6 cells by macrophages, which was related to the increased expression of the scavenger receptor CD36. The 17-oxo-DHA-mediated potentiation of efferocytic activity of macrophages was attenuated by the pharmacologic inhibition or knockout of Nrf2. The pretreatment with 17-oxo-DHA reduced the UVB-induced skin carcinogenesis and tumor angiogenesis. It was also confirmed that 17-oxo-DHA treatment significantly inhibited the phosphorylation of the Tyr705 residue of STAT3 and decreased the expression of its target proteins in cutaneous papilloma. In conclusion, 17-oxo-DHA protects against UVB-induced oxidative cell death, dermatitis, and carcinogenesis. These effects were associated with inhibition of STAT3-mediated proinflammatory signaling and also activation of Nrf2 with subsequent upregulation of antioxidant and anti-inflammatory gene expression.

Plasma membrane-localized plant immune receptor targets H+ -ATPase for membrane depolarization to regulate cell death.

The hypersensitive response (HR) is a robust immune response mediated by nucleotide-binding, leucine-rich repeat receptors (NLRs). However, the early molecular event that links activated NLRs to cell death is unclear. Here, we demonstrate that NLRs target plasma membrane H+ -ATPases (PMAs) that generate electrochemical potential, an essential component of living cells, across the plasma membrane. CCA 309, an autoactive N-terminal domain of a coiled-coil NLR (CNL) in pepper, is associated with PMAs. Silencing or overexpression of PMAs reversibly affects cell death induced by CCA 309 in Nicotiana benthamiana. CCA 309-induced extracellular alkalization causes plasma membrane depolarization, followed by cell death. Coimmunoprecipitation analyses suggest that CCA 309 inhibits PMA activation by preoccupying the dephosphorylated penultimate threonine residue of PMA. Moreover, pharmacological experiments using fusicoccin, an irreversible PMA activator, showed that inhibition of PMAs contributes to CNL-type (but not Toll interleukin-1 receptor NLR-type) resistance protein-induced cell death. We suggest PMAs as primary targets of plasma membrane-associated CNLs leading to HR-associated cell death by disturbing the electrochemical gradient across the membrane. These results provide new insight into NLR-mediated cell death in plants, as well as innate immunity in higher eukaryotes.

Reprograming of Tumor-Associated Macrophages in Breast Tumor-Bearing Mice under Chemotherapy by Targeting Heme Oxygenase-1.

Tumor-associated macrophages (TAMs) represent one of the most abundant components of the tumor microenvironment and play important roles in tumor development and progression. TAMs display plasticity and functional heterogeneity as reflected by distinct phenotypic subsets. TAMs with an M1 phenotype have proinflammatory and anti-tumoral properties whereas M2-like TAMs exert anti-inflammatory and pro-tumoral functions. Tumor cell debris generated during chemotherapy can stimulate primary tumor growth and recurrence. According to our previous study, phagocytic engulfment of breast tumor cell debris by TAMs attenuated chemotherapeutic efficacy through the upregulation of heme oxygenase-1 (HO-1). To verify the impact of HO-1 upregulation on the profile of macrophage polarization during cytotoxic therapy, we utilized a syngeneic murine breast cancer (4T1) model in which tumor bearing mice were treated with paclitaxel (PTX). PTX treatment markedly downregulated the surface expression of the M1 marker CD86 in infiltrated TAMs. Notably, there were significantly more cytotoxic CD8+ T cells in tumors of mice treated with PTX plus the HO-1 inhibitor, zinc protophorphyrin IX (ZnPP) than in mice treated with PTX alone. Interestingly, the tumor-inhibiting efficacy of PTX and ZnPP co-treatment was abrogated when macrophages were depleted by clodronate liposomes. Macrophage depletion also decreased the intratumoral CD8+ T cell population and downregulated the expression of Cxcl9 and Cxcl10. The expression of the M1 phenotype marker, CD86 was higher in mice injected with PTX plus ZnPP than that in mice treated with PTX alone. Conversely, the PTX-induced upregulation of the M2 marker gene, Il10 in CD11b+ myeloid cells from 4T1 tumor-bearing mice treated was dramatically reduced by the administration of the HO-1 inhibitor. Genetic ablation of HO-1 abolished the inhibitory effect of 4T1 tumor cell debris on expression of M1 marker genes, Tnf and Il12b, in LPS-stimulated BMDMs. HO-1-deficient BMDMs exposed to tumor cell debris also exhibited a diminished expression of the M2 macrophage marker, CD206. These findings, taken all together, provide strong evidence that HO-1 plays a pivotal role in the transition of tumor-inhibiting M1-like TAMs to tumor-promoting M2-like ones during chemotherapy.

Thiostrepton: A Novel Therapeutic Drug Candidate for Mycobacterium abscessus Infection.

Mycobacterium abscessus is a rapid-growing, multidrug-resistant, non-tuberculous mycobacterial species responsible for a variety of human infections, such as cutaneous and pulmonary infections. M. abscessus infections are very difficult to eradicate due to the natural and acquired multidrug resistance profiles of M. abscessus. Thus, there is an urgent need for the development of effective drugs or regimens against M. abscessus infections. Here, we report the activity of a US Food and Drug Administration approved drug, thiostrepton, against M. abscessus. We found that thiostrepton significantly inhibited the growth of M. abscessus wild-type strains, subspecies, clinical isolates, and drug-resistant mutants in vitro and in macrophages. In addition, treatment of macrophages with thiostrepton significantly decreased proinflammatory cytokine production in a dose-dependent manner, suggesting an inhibitory effect of thiostrepton on inflammation induced during M. abscessus infection. We further showed that thiostrepton exhibits antimicrobial effects in vivo using a zebrafish model of M. abscessus infection.

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins. Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th ß-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th ß-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

HSP70s Enhance a Phytophthora infestans Effector-Induced Cell Death via an MAPK Cascade in Nicotiana benthamiana.

A destructive pathogen, Phytophthora infestans, secretes hundreds of effectors for successful survival in its host plants. The effectors modulate the plant defense system at diverse cellular compartments to take an advantage of pathogen survivals. A few research studies have shown the mode of action of each effector and their interacting proteins in plant cells. Here, we investigated the mode of action of a P. infestans effector, Pi23226, which induces cell death in Nicotiana benthamiana. To identify its host factors, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry, and selected members of heat shock protein 70 (HSP70s) as candidates. These HSP70s, known to function as chaperones, were associated with Pi23226 in planta and accelerated Pi23226-induced cell death. Additionally, they were found to be involved in plant basal defense by suppressing the growth of P. infestans. We also found that specific components of a mitogen-activated protein kinase cascade were involved in Pi23226-induced cell death. Our findings show that HSP70s functions in defense systems by regulating effector-triggered cell death and by suppressing the growth of the pathogen. This suggests that host plants manipulate the ubiquitous proteins to detect pathogen effectors for functioning in the defense system.

New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication.

BACKGROUND: Transposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants. RESULTS: We report two high-quality de novo genomes (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum) for peppers. Dynamic genome rearrangements involving translocations among chromosomes 3, 5, and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs are abundant across the angiosperms and, in most cases, are lineage-specific. CONCLUSIONS: Our study reveals that retroduplication has played key roles for the massive emergence of NLR genes including functional disease-resistance genes in pepper plants.

Global Transcriptome Profiling of Xanthomonas oryzae pv. oryzae under in planta Growth and in vitro Culture Conditions.

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight, is a major threat to rice productivity. Here, we performed RNA-Seq based transcriptomic analysis of Xoo transcripts isolated under in planta growth (on both susceptible and resistant hosts) and in vitro culture conditions. Our in planta extraction method resulted in successful enrichment of Xoo cells and provided RNA samples of high quality. A total of 4,619 differentially expressed genes were identified between in planta and in vitro growth conditions. The majority of the differentially expressed genes identified under in planta growth conditions were related to the nutrient transport, protease activity, stress tolerance, and pathogenicity. Among them, over 1,300 differentially expressed genes were determined to be secretory, including 184 putative type III effectors that may be involved in Xoo pathogenicity. Expression pattern of some of these identified genes were further validated by semi-quantitative RT-PCR. Taken together, these results provide a transcriptome overview of Xoo under in planta and in vitro growth conditions with a focus on its pathogenic processes, deepening our understanding of the behavior and pathogenicity of Xoo.

Label-free quantitative secretome analysis of Xanthomonas oryzae pv. oryzae highlights the involvement of a novel cysteine protease in its pathogenicity.

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases resulting in a huge loss of the total rice productivity. The initial interaction between rice and Xoo takes place in the host apoplast and is mediated primarily by secretion of various proteins from both partners. Yet, such secretory proteins remain to be largely identified and characterized. This study employed a label-free quantitative proteomics approach and identified 404 and 323 Xoo-secreted proteins from in vitro suspension-cultured cells and in planta systems, respectively. Gene Ontology analysis showed their involvement primarily in catalytic, transporter, and ATPase activities. Of a particular interest was a Xoo cysteine protease (XoCP), which showed dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence. Besides, a parallel analysis of in planta rice-secreted proteins resulted in identification of 186 secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. Identified secretory proteins were exploited to shed light on their possible role in the rice-Xoo interaction, and that further deepen our understanding of such interaction. BIOLOGICAL SIGNIFICANCE: Xanthomonas oryzae pv. oryzae (Xoo), causative agent of bacterial blight disease, results in a huge loss of the total rice productivity. Using a label-free quantitative proteomics approach, we identified 727 Xoo- and 186 rice-secreted proteins. Functional annotation showed Xoo secreted proteins were mainly associated with the catalytic, transporter, and ATPase activities while the rice secreted proteins were mainly associated with the catalytic, antioxidant, and electron carrier activities. A novel Xoo cysteine protease (XoCP) was identified, showing dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence.

Current Understandings of Plant Nonhost Resistance.

Nonhost resistance, a resistance of plant species against all nonadapted pathogens, is considered the most durable and efficient immune system of plants but yet remains elusive. The underlying mechanism of nonhost resistance has been investigated at multiple levels of plant defense for several decades. In this review, we have comprehensively surveyed the latest literature on nonhost resistance in terms of preinvasion, metabolic defense, pattern-triggered immunity, effector-triggered immunity, defense signaling, and possible application in crop protection. Overall, we summarize the current understanding of nonhost resistance mechanisms. Pre- and postinvasion is not much deviated from the knowledge on host resistance, except for a few specific cases. Further insights on the roles of the pattern recognition receptor gene family, multiple interactions between effectors from nonadapted pathogen and plant factors, and plant secondary metabolites in host range determination could expand our knowledge on nonhost resistance and provide efficient tools for future crop protection using combinational biotechnology approaches. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Comparative Biochemical and Proteomic Analyses of Soybean Seed Cultivars Differing in Protein and Oil Content.

This study develops differential protein profiles of soybean (Glycine max) seeds (cv. Saedanbaek and Daewon) varying in protein (47.9 and 39.2%) and oil (16.3 and 19.7%) content using protamine sulfate (PS) precipitation method coupled with a 2D gel electrophoresis (2DGE) approach. Of 71 detected differential spots between Daewon and Saedanbaek, 48 were successfully identified by MALDI-TOF/TOF. Gene ontology analysis revealed that up-regulated proteins in Saedanbaek were largely associated with nutrient reservoir activity (42.6%), which included mainly seed-storage proteins (SSPs; subunits of glycinin and ß-conglycinin). Similar results were also obtained in two cultivars of wild soybean (G. soja cv. WS22 and WS15) differing in protein content. Western blots confirmed higher accumulation of SSPs in protein-rich Saedanbaek. Findings presented and discussed in this study highlight a possible involvement of the urea cycle for increased accumulation of SSPs and hence the higher protein content in soybean seeds.

Understanding the plant-pathogen interactions in the context of proteomics-generated apoplastic proteins inventory.

The extracellular space between cell wall and plasma membrane acts as the first battle field between plants and pathogens. Bacteria, fungi, and oomycetes that colonize the living plant tissues are encased in this narrow region in the initial step of infection. Therefore, the apoplastic region is believed to be an interface which mediates the first crosstalk between host and pathogen. The secreted proteins and other metabolites, derived from both host and pathogen, interact in this apoplastic region and govern the final relationship between them. Hence, investigation of protein secretion and apoplastic interaction could provide a better understanding of plant-microbe interaction. Here, we are briefly discussing the methods available for the isolation and normalization of the apoplastic proteins, as well as the current state of secretome studies focused on the in-planta interaction between the host and the pathogen.

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: insight into proteins at intracellular and extracellular spaces.

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.