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Transcription factor PU.1 is a regulator of macrophage function, however, the specific function of PU.1 in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of two PU.1 genes from ayu (Plecoglossus altivelis; PaPU.1a and PaPU.1b). Sequence comparisons showed that PaPU.1 were most closely related to the PU.1 of rainbow smelt (Osmerus mordax). The PU.1 transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaPU.1a and PaPU.1b proteins were upregulated in MO/MФ, after infection. RNA interference was employed to knockdown PaPU.1a and PaPU.1b to investigate their function in MO/MФ. The expression of inflammatory cytokines was regulated by PaPU.1a, but not PaPU.1b, in ayu MO/MФ upon V. anguillarum infection. Both PaPU.1a and PaPU.1b knockdown lowered the phagocytic activity of MO/MФ. Furthermore, PaPU.1b knockdown attenuated MO/MФ bacterial killing capability. Our results indicate that two PaPU.1 genes differentially modulate the immune response in ayu MO/MФ against bacterial infection.

Plasmopara viticola, the causal organism of grapevine downy mildew, secretes a vast array of effectors to manipulate host immunity. Previously, several cell death‐inducing PvRXLR effectors have been identified, but their functions and host targets are poorly understood. Here, we investigated the role of PvRXLR111, a cell death‐inducing RXLR effector, in manipulating plant immunity. When coexpressed with other PvRXLR effectors, PvRXLR111‐induced cell death was prevented. Transient expression of PvRXLR111 in Nicotiana benthamiana suppressed bacterial flagellin peptide flg22‐elicited immune responses and enhanced Phytophthora capsici infection. PvRXLR111 induction in Arabidopsis increased susceptibility to Hyaloperonospora arabidopsidis. PvRXLR111 expression in Pseudomonas syringae promoted bacterial colonization. By immunoprecipitation‐mass spectrometry analysis, yeast two‐hybrid, pull‐down, and bimolecular fluorescence complementation assays, it was shown that PvRXLR111 interacted with Vitis vinifera putative WRKY transcription factor 40 (VvWRKY40), which increased VvWRKY40 stability. Transient expression of VvWRKY40 in N. benthamiana inhibited flg22‐induced reactive oxygen species burst and enhanced P. capsici infection and silencing NbWRKY40 attenuated P. capsici colonization. These results suggest VvWRKY40 functions as a negative regulator in plant immunity and that PvRXLR111 suppresses host immunity by stabilizing VvWRKY40., PvRXLR111 from Plasmopara viticola suppresses PTI to promote pathogen virulence by stabilizing VvWRKY40, which functions as a negative regulator in plant immunity.

Fruit aroma is mainly contributed by free and glycosidically bound aroma compounds, in which glycosidically bound form can be converted into free form during storage and processing, thereby enhancing the overall aroma property. In recent years, the bound aroma precursors have been widely used as flavor additives in the food industry to enhance, balance and recover the flavor of products. This review summarizes the fruit-derived aroma glycosides in different aspects including chemical structures, enzymatic hydrolysis, biosynthesis and occurrence. Aroma glycosides structurally involve an aroma compound (aglycone) and a sugar moiety (glycone). They can be hydrolyzed to release free volatiles by endo- and/or exo-glucosidase, while their biosynthesis refers to glycosylation process using glycosyltransferases (GTs). So far, aroma glycosides have been found and studied in multiple fruits such as grapes, mangoes, lychees and so on. Additionally, their importance in flavor perception, their utilization in food flavor enhancement and other industrial applications are also discussed. Aroma glycosides can enhance flavor perception via hydrolyzation by β-glucosidase in human saliva. Moreover, they are able to impart product flavor by controlling the liberation of active volatiles in industrial applications. This review provides fundamental information for the future investigation on the fruit-derived aroma glycosides.

Significance Fungal disease is a major threat to agriculture and consequently human nutrition. Many pathogenic fungi penetrate through stomata. This study reveals that chitin oligosaccharide (CTOS) from fungal cell wall induces stomatal closure through its receptor CERK1, Ca2+, and S-type anion channel SLAC1, which would prevent fungal penetration. It is also shown that conversion of CTOS to chitosan oligosaccharide (CSOS) is a possible fungal strategy to circumvent stomatal immunity. At higher concentration, guard cells perceive CSOS, resulting in guard cell death, which potentially contributes to plant defense halting fungal infection through stomata. The finding of active guard cell death represents a conceptual advance in understanding of stomatal immunity and would guide future research on guard cell–microbe interaction and crop protection., Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor β (PaIL-6Rβ) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rβ knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rβ knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.

Polycomb group (PcG) proteins within polycomb repressive complex 1 (PRC1) and PRC2 are significant epigenetic regulation factors involved in controlling plant developmental transitions. PRC2 catalyzes H3K27 trimethylation (H3K27me3), and PRC1 recognizes the site of H3K27me3 and catalyzes H2AK119 monoubiquitination to maintain transcription repression. PRC2 components are conserved from plants to animals and have been investigated for a long time. However, the compositions and functions of PRC1 vary between animals and plants. Plant PRC1 has only been confirmed in recent years. Very little research has been conducted on PcG in grapevine (Vitis vinifera ‘Cabernet Sauvignon’). Here, we cloned the full-length coding sequence of the VvRING1a gene, which is homologous with the Arabidopsis thaliana PRC1 core component AtRING1a. We then analyzed the structure, subcellular localization, and expression pattern. The tissue-specific expression analysis showed that VvRING1a is expressed in various tissues, including in roots, stems, leaves, flowers, buds, and fruits. This study will be a useful reference for further study of VvRING1a.

Bacillus spp. have been widely reported with the ability to control plant diseases. In this work, we analyzed the whole genome of LJBS06, which was isolated from grapevine rhizosphere soil. In view of physiological and biochemical characteristics, genome data, and phylogenetic analysis of 16S rRNA, LJBS06 was affiliated with Bacillus stercoris. LJBS06 showed antagonistic activities against a variety of plant pathogens. The inhibition rate of Magnaporthe oryzae was up to 75.05% and the inhibition rates of Colletotrichum gloeosporioides, Coniothyrium diplodiella, and Botrytis cinerea were all above 50% in the plate assays. The genome of LJBS06 had a 4,154,362-bp circular chromosome, with an average GC content of 43.96%, containing an 82,935-bp plasmid with a GC content of 35.18%. The circular chromosome of LJBS06 contained 4231 protein-coding genes, 30 rRNA genes, and 87 tRNA genes, including genes related to the synthesis of plant defense-related enzymes and the promotion of plant growth. Meanwhile, 11 gene clusters involved in biosynthesis of secondary metabolites were present in the genome of LJBS06. In conclusion, our findings indicated that LJBS06 strain had the necessary genetic machinery to control plant pathogens and provided insights for future studies of the biocontrol mechanisms of B. stercoris LJBS06.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily, contributing to inflammation, apoptosis, and differentiation. However, the function of OPG in the host immune system of teleosts remains unclear. Here, we cloned the cDNA of the LcOPG gene from large yellow croaker. LcOPG mRNA was expressed in all analyzed tissues and was upregulated by Vibrio alginolyticus infection in immune tissues and monocytes/macrophages (MO/MФ). Subsequently, the LcOPG protein was expressed and purified using a prokaryotic expression system. Recombinant LcOPG protein (rLcOPG) treatment suppressed V. alginolyticus-induced pro-inflammatory cytokine and enhanced V. alginolyticus-induced anti-inflammatory cytokine mRNA expression. Furthermore, rLcOPG decreased V. alginolyticus-induced MO/MФ apoptosis. Therefore, the results indicate that LcOPG might play a role in the immune response of V. alginolyticus-infected large yellow croaker.

The cell wall acts as one of the first barriers of the plant against various biotic stressors. Previous studies have shown that alterations in wall polysaccharides may influence crop disease resistance. In the grapevine family, several native species (e.g., Chinese wild grapevine) show a naturally higher resistance to microbial pathogens than cultivated species (e.g., Vitis vinifera), and this trait could be inherited through breeding. Despite the importance of the cell wall in plant immunity, there are currently no comprehensive cell wall profiles of grapevine leaves displaying differing resistance phenotypes, due to the complex nature of the cell wall and the limitations of analytical techniques available. In this study, the cutting-edge comprehensive carbohydrate microarray technology was applied to profile uninfected leaves of the susceptible cultivar (Vitis vinifera cv. “Cabernet Sauvignon”), a resistant cultivar (Vitis amurensis cv. “Shuanghong”) and a hybrid offspring cross displaying moderate resistance. The microarray approach uses monoclonal antibodies, which recognize polysaccharides epitopes, and found that epitope abundances of highly esterified homogalacturonan (HG), xyloglucan (with XXXG motif), (galacto)(gluco)mannan and arabinogalactan protein (AGP) appeared to be positively correlated with the high resistance of Vitis amurensis cv. “Shuanghong” to mildew. The quantification work by gas chromatography did not reveal any significant differences for the monosaccharide constituents, suggesting that polysaccharide structural alterations may contribute more crucially to the resistance observed, this is again supported by the contact infrared spectroscopy of cell wall residues, revealing chemical functional group changes (e.g., esterification of pectin). The identification of certain wall polysaccharides that showed alterations could be further correlated with resistance to mildew. Data from the use of the hybrid material in this study have preliminarily suggested that these traits could be inherited and may be applied as potential structural biomarkers in future breeding work.

Endevours on the enhancement of nitrate removal efficiency during methane oxidation coupled with denitrification (AME-D) has always overlooked the role of membrane employed. It would be highly beneficial to enrich the biomass content and to manage biofilm on the membrane, in the utilization of methane and denitrification. In this study, an innovative and scalable double-layer membrane (DLM) was designed and prepared for a membrane biofilm reactor (MBfR), to simultaneously enhance nitrate removal flux and methane utilization efficiency during aerobic methane oxidation coupled with the denitrification (AME-D) process. The DLM allowed quick bacterial attachment and biomass accumulation for biofilm growth, which would be then self-regulated for well distribution of functional microbes on/within the DLM. Upon a high biofilm density of over 70 g-VSS m−2 achieved on the DLM, the methane utilization efficiency of the MBfR was enhanced significantly to over 1.3 times than the control MBfR with conventional polypropylene membrane. The MBfR employed DLM also demonstrated the maximum nitrate removal flux of 740 mg-NO3−-N m−2 d−1 that was approximately 1.64 times of that in control MBfR at continuous-mode operation. This DLM indeed favored the enrichment of Type II aerobic methanotrophs of Methylocystaceae, and methanol-utilization denitrifiers of Rhodocyclaceae that preferentially utilize methanol as the cross-feeding intermediates to promote the methane utilization efficiency, and thus to enhance the nitrate removal flux. These results raised from new designed DLM confirmed the importance of membrane surface properties on the effectiveness of MBfR, and offered great potential to address challenging problems of MBfRs during engineering application.

The oomycete pathogen Hyaloperonospora arabidopsidis delivers diverse effector proteins into host plant cells to suppress the plant's innate immunity. In this study, we investigate the mechanism of action of a conserved RxLR effector, HaRxLL470, in suppressing plant immunity. Genomic, molecular and biochemical analyses were performed to investigate the function of HaRxLL470 and the mechanism of the interaction between HaRxLL470 and the target host protein during H. arabidopsidis infection. We report that HaRxLL470 enhances plant susceptibility to H. arabidopsidis isolate Noco2 by interacting with the host photomorphogenesis regulator protein HY5. Our results demonstrate that HY5 is not only an important component in the regulation of light signalling, but also positively regulates host plant immunity against H. arabidopsidis by transcriptional activation of defense-related genes. We show that the interaction between HaRxLL470 and HY5 compromises the function of HY5 as a transcription factor by attenuating its DNA-binding activity. The present study demonstrates that HY5 positively regulates host plant defense against H. arabidopsidis whereas HaRxLL470, a conserved RxLR effector across oomycete pathogens, enhances pathogenicity by interacting with HY5 and suppressing transcriptional activation of defense-related genes.

The mirid bug, Cyrtorhinus lividipennis Reuter, is an important predator of rice planthoppers in Asia. In a previous study, C. lividipennis fed on gramineous weeds with brown planthopper (BPH) eggs had reduced development compared to those fed on rice with BPH eggs. In the current study, the concentrations of selected amino acids (AAs) were higher in rice than five gramineous species, which might explain the enhanced growth of C. lividipennis on rice. When C. lividipennis was fed on AA-deprived artificial diets, the Wnt/β-catenin pathway was inhibited. Furthermore, C. lividipennis females silenced for expression of Frizzled 2 (Fz2) showed a significant reduction in the Wnt/β-catenin and target of rapamycin (TOR) pathways. Silencing Fz2 led to decreased expression of the vitellogenin gene (Vg), lower Vg accumulation in oocytes, reduced soluble protein in ovaries and fat bodies, reduced titers of juvenile hormone, prolonged preoviposition periods, and lower predation capacity, body weight, and egg numbers as controlled to controls. Fz2 silencing resulted in undeveloped ovaries and the inhibition of oocyte growth in the ovarioles, resulting in decreased numbers of offspring and reduced hatching rates. The silencing of Fz2 also resulted in aberrant embryos with undeveloped eyespots and organs, suggesting that Fz2 is an essential gene for embryonic development, oogenesis, and egg maturation. In summary, this study established a potential link between Wnt and TOR pathways, which interact synergistically to regulate C. lividipennis reproduction in response to AA signals. These results provide valuable new information that can be applied to large-scale rearing of C. lividipennis predators, which is important for reducing planthopper damage in rice fields.

Adrenocorticotropic hormone (ACTH), a bioactive peptide of the family of melanocortins, is generated from pro-opiomelanocortin (POMC). So far, the research on the specific functions of ACTH in the immune system of teleosts is limited. We determined two complementary DNA (cDNA) sequences of POMC in ayu (Plecoglossus altivelis), termed PaPOMC-A and PaPOMC-B. PaPOMCs transcripts occurred in all examined tissues, and their expression in immune tissues changed following experimental infection with Vibrio anguillarum. PaACTH-B, but not PaACTH-A, suppressed the phagocytosis of monocytes/macrophages (MO/MФ). Two isoforms of PaACTH increased the bactericidal capacity of MO/MФ. PaACTH-A increased anti-inflammatory cytokine expression, while PaACTH-B decreased pro-inflammatory cytokine expression in MO/MФ. Compared with PaACTH-B treatment, the PaACTH-A treatment improved survival rate and reduced the bacterial load in V. anguillarum-infected ayu through interleukin (IL)-10. Our results indicate that the two PaACTH isoforms exert different effects in the host defense against bacterial infection.

Virus-encoded tumor necrosis factor receptors (vTNFRs) facilitate viral escape from the host immune response during viral propagation. Cyprinid Herpesvirus-2 (CyHV-2) is a double-stranded DNA virus of alloherpesviridae family that causes great economic losses in the aquaculture industry. The present study identified and characterized a novel TNFR homolog termed ORF4 in CyHV-2. ORF4 was identified as a secreted protein and a homolog of herpesvirus entry mediator (HVEM). ORF4 localized to the cytoplasm in infected GiCF cells. ORF4 overexpression enhanced viral propagation, while downregulation of ORF4 via siRNA decreased viral propagation. ORF4 overexpression promoted GiCF proliferation, and its downregulation suppressed CyHV-2-induced apoptosis. GST-pulldown and LC-MS/MS assays identified 44 conditional binding proteins that interact with ORF4 protein, while the GST pulldown test did not support the idea that ORF4 interact with histone H3.3. Taken together, our results contribute to our understanding of the vTNFR function in alloherpesviridae pathogenesis and host immune regulation.

Here, we present a draft genome of the tapeworm Dipylidium caninum (family Dipylidiidae) and compare it with other cestode genomes. This draft genome of D. caninum is 110 Mb in size, has a repeat content of ~13.4% and is predicted to encode ~10,000 protein-coding genes. We inferred excretory/secretory molecules (representing the secretome), other key groups of proteins (including peptidases, kinases, phosphatases, GTPases, receptors, transporters and ion-channels) and predicted potential intervention targets for future evaluation. Using 144 shared single-copy orthologous sequences, we investigated the genetic relationships of cestodes for which nuclear genomes are available. This study provides first insights into the molecular biology of D. caninum and a new resource for comparative genomic and genetic explorations of this and other flatworms.

Downy mildew, caused by Plasmopara viticola, is a major disease that affects grapevines, and a few resistance (R) genes have been identified thus far. In order to identify R genes, we investigated F1 progeny from a cross between the downy mildew-resistant Vitis amurensis ‘Shuang Hong’ and the susceptible Vitis vinifera ‘Cabernet Sauvignon’. The P. viticola-resistance of the progeny varied continuously and was segregated as a quantitative trait. Genotyping-by-sequencing was used to construct linkage maps. The integrated map spanned 1898.09 cM and included 5603 single nucleotide polymorphisms on 19 linkage groups (LGs). Linkage analysis identified three quantitative trait loci (QTLs) for P. viticola resistance: 22 (Rpv22) on LG 02, Rpv23 on LG15, and Rpv24 on LG18. The phenotypic variance contributed by these three QTLs ranged from 15.9 to 30.0%. qRT-PCR analysis of R-gene expression in these QTLs revealed a CC-NBS-LRR disease resistance gene RPP8, two LRR receptor-like serine/threonine-protein kinases, a serine/threonine-protein kinase BLUS1, a glutathione peroxidase 8, an ethylene-responsive transcription factor ERF038, and a transcription factor bZIP11 were induced by P. viticola, and these genes may play important role in P. viticola response.

While microbial spore production and germination of bacteria have been widely studied for their applications in animal husbandry, aquatic products, medicine, and food, few studies have investigated their use for the crack-healing of concrete. To effectively heal the cracks in concrete, studies suggest that the rate of sporulation and the germination of bacteria should be sufficiently high. This study investigates the effects of different carbon sources, nitrogen sources, Mn2+ concentrations, and external culture conditions on the sporulation rate and analyzes the effects of the pH value, heat activation, germinants, various cations, and nutrients on the germination of spores. Bacillus cohnii (B. cohnii) is chosen as the bacterium to be mixed in concrete because of its alkalophilic nature. The mineralization activity of spores after germination and the crack-healing capacity of concrete are studied. The optimal culture medium and the optimum external conditions for spore production are obtained. The total cell count and sporulation rate of bacteria obtained on this medium are 3.14 × 109 CFU/mL and 92.6%, respectively, under the optimum external conditions. The optimal pH value for the spore germination of B. cohnii is 9.7. While the cation Na+ strongly stimulates the germination of B. cohnii spores, other cations (such as K+, NH4+, and Ca2+) do not stimulate spore germination. The optimal concentration of Na+ is 200 mM. The germination rate of spores in the control group concrete specimen (room temperature 24°C) was more than 50%, thus suggesting that B. cohnii bacteria can be used in the self-healing of concrete cracks. The mineralization activity test proves that the spores of B. cohnii have a mineralizing function after germination, and the crystals produced by microbial-induced carbonate precipitation (MICP) are of pure calcite. When the crack width of the concrete specimen with spores of B. cohnii is less than 1.2 mm, it can be completely repaired after 28 days of healing.

Plasmopara viticola, the casual oomycete of grapevine downy mildew, could cause yield loss and compromise berry quantity. Previously, we have identified several PvRXLR effectors that could suppress plant immunity to promote infection and disease development. In this study, the role of effector, PvRXLR53, in plant–microbe interaction was investigated. PvRXLR53 has several orthologs in other oomycetes and contains a functional signal peptide. Expression level of PvRXLR53 was already detected upon inoculation, further induced in the early stage after P. viticola inoculation and decreased to low level in the late infection stage in grapevine (Vitis vinifera ‘Cabernet Sauvignon’). PvRXLR53 is localized in both nucleus and cytoplasm. When transiently expressed in Nicotiana benthamiana, PvRXLR53 suppressed oomycete elicitor INF1-triggered programmed cell death and defense gene expression, and Phytophthora capsici-induced reactive oxygen species production (ROS) and eventually resistance to P. capsici. In summary, these findings suggest that P. viticola secretes PvRXLR53 to suppress host immunity from the very early stage of infection.

The protein family with nucleotide binding sites and leucine-rich repeat (NBS-LRR) in plants stimulates immune responses caused by effectors and can mediate resistance to hemi-biotrophs and biotrophs. In our previous study, a Toll-interleukin-1(TIR)-NBS-LRR gene cloned from Vitis amurensis &ldquo, Shuanghong&rdquo, VaRGA1, was induced by Plasmopara viticola and could improve the resistance of tobacco to Phytophthora capsici. In this study, VaRGA1 in &ldquo, was also induced by salicylic acid (SA), but inhibited by jasmonic acid (JA). To investigate whether VaRGA1 confers broad-spectrum resistance to pathogens, we transferred this gene into Arabidopsis and then treated with Hyaloperonospora arabidopsidis (Hpa), Botrytis cinerea (B. cinerea), and Pseudomonas syringae pv. tomato DC3000 (PstDC3000). Results showed that VaRGA1 improved transgenic Arabidopsis thaliana resistance to the biotrophic Hpa and hemi-biotrophic PstDC3000, but decreased resistance to the necrotrophic B. cinerea. Additionally, qPCR assays showed that VaRGA1 plays an important role in disease resistance by activating SA and inhibiting JA signaling pathways. A 1104 bp promoter fragment of VaRGA1 was cloned and analyzed to further elucidate the mechanism of induction of the gene at the transcriptional level. These results preliminarily confirmed the disease resistance function and signal regulation pathway of VaRGA1, and contributed to the identification of R-genes with broad-spectrum resistance function.

Plasmopara viticola, the causal oomycete of grapevine downy mildew disease, secrets a series of RXLR effectors to manipulate host immunity. In this study, we characterized the role of a RXLR effector of P. viticola, PvRXLR159, in plant-microbe interaction. Transcription of PvRXLR159 in P. viticola was induced in the early stage of infection in grapevine (Vitis vinifera 'Thomson Seedless'). Further results revealed that PvRXLR159 contains a functional signal peptide and its C terminus was essential to inhibit cell death by elicitors, INF1 and BAX, in Nicotiana benthamiana. Transient expression of PvRXLR159 suppressed N. benthamiana resistance to a pathogenic oomycete, Phytophthora capsici. Taken together, we propose that PvRXLR159 is induced and secreted by P. viticola to suppress host resistance.

Hematopoietic stem/progenitor cells (HSPCs) generate the entire repertoire of immune cells in vertebrates and play a crucial role during infection. Although two copies of CXC motif chemokine receptor 4 (CXCR4) genes are generally identified in teleosts, the function of teleost CXCR4 genes in HSPCs is less known. In this study, we identified two CXCR4 genes from a teleost, ayu (Plecoglossus altivelis), named PaCXCR4a and PaCXCR4b. PaCXCR4b was constitutively expressed in ayu HSPCs, whereas PaCXCR4a was induced by LPS treatment. The stromal-derived factor-1–binding activity of CXCR4b was significantly higher than that of CXCR4a, whereas the LPS-binding activity of CXCR4a was significantly higher than that of CXCR4b in the teleosts ayu, large yellow croaker (Larimichthys crocea), and tiger puffer (Takifugu rubripes). CXCR4a+ HSPCs were mobilized into blood by LPS, whereas CXCR4b+ HSPCs were mobilized by leukocyte cell–derived chemotaxin-2. PaSDF-1 and PaCXCR4b, but not PaCXCR4a, inhibited HSPC proliferation by regulating reactive oxygen species levels. Compared with PaCXCR4b+ HSPCs, PaCXCR4a+ HSPCs preferentially differentiated into myeloid cells in ayu by maintaining high stem cell leukemia expression. These data suggest that the two copies of CXCR4s achieve a division of labor in the regulation of teleost HSPC homeostasis, supporting the concept that subfunctionalization after gene duplication in teleosts may stabilize the immune system.

포도나무 피어스병에 대한 품종간 저항성을 검정하는 데에 이용되는 병원균 접종법이나 포장저항성 검정법을 대체할 수 있는 방법으로 병원균(Xylella fastidiosa)이 생산하는 배양여액을 이용할 수 있는 검정법을 개발하고자 하였다. 상처를 가한 포도나무 잎에 병원균의 배양여액을 처리한 결과 독성이 발현되었으며 품종간에 차이가 나타났다. 병원균 배양여액의 에틸아세테이트 추출물은 공시한 포도나무에서 배양여액 처리와 동일한 반응을 유도하였다. 병원균 배양여액의 독성과 감수성은 에틸아세테이트 추출물과 동일하였다. 감수성 품종은 높은 비율로 희석된 배양여액에도 감수성을 나타내었으며, 저항성품종은 원액에서도 반응하지 않았다. 감수성 품종은 희석된 배양여액보다 원액에서 감수성이었으나 저항성 품종은 원액에도 저항성을 나타내었다. 본 연구에서 유럽종 포도 품종이 중도 감수성이었으나, 배양여액을 이용한 피어스병해 저항성 검정은 병해에 저항성인 유전자원 선발과, 교배조합의 저항성 실생의 조기 에 선발에 중요한 기술로서, 향후 병해에 저항성인 포도품종 육성에 크게 기여할 것으로 여겨진다.

Downy mildew (DM) resistance is a highly desirable agronomic trait in grape breeding. High variation in Plasmopara viticola resistance was found in Vitis cultivars. Some accessions show high P. viticola resistance even under conditions highly conducive to DM. Here, leaf disc inoculation experiments revealed that Vitis amurensis ‘Zuoshaner’ exhibited DM resistance with necrotic spots, whereas the V. amurensis × V. vinifera hybrid cultivar ‘Zuoyouhong’ was susceptible. Changes in plant hormones accumulation profiles differed between the cultivars. To investigate the genetic mechanisms related to DM resistance, we performed genome-wide sequencing of ‘Zuoshaner’ and ‘Zuoyouhong’ and identified cultivar-specific single-nucleotide polymorphisms, insertions/deletions (indels), structural variations (SVs), and copy number variations (CNVs), identifying 5399 SVs and 191 CNVs specific for ‘Zuoshaner’. Genes affected by these genetic variations were enriched in biological processes, including defense response and response to stress and stimulation, and were associated with sesquiterpenoid and triterpenoid biosynthesis, ABC transporters, and phenylalanine metabolism pathways. Additionally, indels and SVs were detected in six NBS-LRR disease resistance genes, and a CNV was mapped to the Rpv8 locus responsible for downy mildew resistance. These findings further our understanding of the genetic mechanisms underlying grape mildew resistance, and will facilitate genomic marker-assisted breeding for improved V. amurensis cultivars.

Microorganisms in grape skins play vital roles in grapevine health, productivity, wine quality and organoleptic properties. To investigate microbial diversity of muscadine grape skins, 16S and ITS sequences of 30 samples from six muscadine (Muscadinia rotundifolia Michx.) cultivars grown in Guangxi, China, were sequenced using Illumina Novaseq platform. A total of 7,317 bacterial operational taxonomic units (OTUs) and 1,611 fungal OTUs were obtained, and clustered into 38 bacterial and 7 known fungal phyla. The dominant bacterial phyla were Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Actinobacteria, Verrucomicrobia, Acidobacteria, and Patescibacteria, and the dominant genera were Lelliottia, Prevotella_9, Escherichia-Shigella, Lactobacillus, Pseudomonas, Akkermansia, Faecalibacterium, Rahnella, and Acinetobacter. For fungi, the dominant phyla were Ascomycota, Basidiomycota, and Mortierellomycota, and the dominant genera were Acaromyces, Uwebraunia, Penicillium, Zygosporium, Ilyonectria, Aspergillus, Neodevriesia, Strelitziana, Mortierella, and Fusarium. Alpha diversity analysis and Kruskal-Wallis H test demonstrated that microbial diversity and composition were affected by the cultivar. The Pearson correlation analysis of species revealed complex interactions among microbes. PICRUSt2 predicted that the metabolism of carbohydrates, cofactors, vitamins, amino acids, terpenoids, polyketides, lipids and biosynthesis of other secondary metabolites were abundant. These results contribute to understanding the uniqueness of muscadine grapes and the links among microorganisms in grape skins.

Hypoxia-inducible factor-1α (HIF-1α) plays a critical role in immune and inflammatory responses and is important in controlling a variety of processes in monocytes and macrophages. However, the role of HIF-1α in the teleost immune system remains less known. In this study, we cloned the cDNA sequence of HIF-1α from the ayu (Plecoglossus altivelis, PaHIF-1α). Sequence and phylogenetic tree analysis showed that PaHIF-1α clustered within the fish HIF-1α tree and was closely related to that of Northern pike (Esox lucius). PaHIF-1α was expressed in all tested tissues and expression increased in liver, head kidney, and body kidney upon Vibrio anguillarum infection. PaHIF-1α was found to regulate the expression of cytokines in ayu monocytes/macrophages (MO/MФ). PaHIF-1α mediated hypoxia-induced enhancement of MO/MФ phagocytic and bactericidal activities to enhance host defenses. Compared with the control, intermittent hypoxia further increased the expression of PaHIF-1α mRNA, improved the survival rate, and reduced the bacterial load of V. anguillarum-infected ayu. Therefore, PaHIF-1α may play a predominant role in the modulation of ayu MO/MФ function.

Salinity stress have detrimental effect on teleosts and may increase susceptibility to disease. Betaine is a compound utilized by organisms to regulate osmotic pressure and plays an important role in the immune response and anti-inflammatory functions. Here, the protective effects of betaine against Vibrio anguillarum infection of ayu under conditions of salinity was determined. A total of 60 ayu were cultured at 10 ppt salinity for 14 days, all fish were intraperitoneally injected with 100 μL of 100 or 200 mg/kg of betaine or saline on days 0, 2, 4, and 6. The fish were subsequently infected with V. anguillarum. The results showed that betaine treatment improved the survival rate of V. anguillarum-infected ayu. Betaine treatment decreased the bacterial load and histological damage in the head-kidney, liver, and spleen. Besides, the inflammatory response was decreased by betaine in V. anguillarum-infected ayu tissues and monocytes/macrophages (MO/MФ). Furthermore, in hypertonic-exposed MO/MФ (19 mOsm), betaine treatment enhanced phagocytosis activity and promoted the bacterial killing capability. Finally, we found that betaine mediated the inflammatory response through BGT-1 in V. anguillarum-infected ayu MO/MФ. In conclusion, when exposed to osmotic stress, betaine was able to mediate the host defense in ayu and modulate the antibacterial response through MO/MФ.

Downy mildew (DM), one of the most devastating grape diseases worldwide, is caused by the biotrophic oomycete Plasmopara viticola (Pv). In general, grapevine responds to Pv infection with the accumulation of phytoalexins as part of the innate immune system, and diverse phytoalexins are induced on grapevines with different DM-resistance levels in response to Pv invasion. However, the regulation of phytoalexin biosynthesis during grapevine against Pv is still unclear. Herein, we detected stilbenes by UPLC-ESI-MS/MS and found that resveratrol was accumulated to higher level and earlier in the DM-immune Muscadinia rotundifolia 'Noble' than that in the DM-susceptible Vitis vinifera 'Thompson Seedless' after Pv inoculation. Additionally, a considerable amount of pterostilbene and ε-viniferin was found in 'Noble', while a little was detected in 'Thompson Seedless'. Resveratrol was glycosylated into piceid both in 'Noble' and 'Thompson Seedless' after Pv inoculation. The qPCR analysis of gene expression indicated that the resveratrol-synthesis gene (STS) was induced by Pv inoculation earlier in 'Noble' than that in 'Thompson Seedless', while the pterostilbene-synthesis gene (ROMT) was induced in 'Noble' but not in 'Thompson Seedless' at all. The piceid-synthesis gene (GT) was generally up-regulated in both cultivars. Sequence analysis of STS, ROMT, and GT promoters revealed that they contained cis-regulatory elements responsive to phytohormones and pathogens. Following Pv inoculation, the level of SA, MeJA, and ABA was found to be consistently higher in 'Noble' than those in 'Thompson Seedless'. The results of exogenous hormone elicitation further demonstrated that the accumulation of stilbenes was regulated by phytohormones. The earlier and higher accumulation of phytohormones and consequent induction of stilbene synthesis may play an important role in grapevine defense against downy mildew disease.

The study of multiple copies of chemokine receptor genes in various teleosts has long appealed to investigators seeking to understand the evolution of the immune system. The CXCR CXCR3 gene has two isoforms, CXCR3.1 and CXCR3.2, which are both expressed in macrophages. The distinct roles of teleost CXCR3s have not been identified previously. In this article, we found that CXCR3.1 and CXCR3.2 differentially contributed to macrophage polarization in the teleosts: ayu (Plecoglossus altivelis), grass carp (Ctenopharyngodon idella), and spotted green pufferfish (Tetraodon nigroviridis). In ayu macrophages, the P. altivelis CXCR3.1 (PaCXCR3.1) gene was constitutively expressed, whereas the P. altivelis CXCR3.2 (PaCXCR3.2) gene was induced postinfection with Escherichia coli. Upon E. coli infection, PaCXCR3.1+ and PaCXCR3.2+ macrophages showed an M1 and an M2 phenotype, respectively. CXCL9–11-like proteins mediated M1 and M2 polarization by interacting with the PaCXCR3.1 and PaCXCR3.2 proteins on macrophages, respectively. The transcription factors P. altivelis STAT1 and P. altivelis STAT3 were activated in PaCXCR3.1+ and PaCXCR3.2+ macrophages, respectively. Furthermore, the prognosis of septic ayu adoptively transferred with PaCXCR3.2+ macrophages was improved. Our data reveal a previously unknown mechanism for macrophage polarization, suggesting that redundant genes may regulate crucial functions in the teleost immune system.

Many U-box proteins have been identified and characterized as important factors against environmental stresses such as chilling, heat, salinity and pathogen attack in plant. Our previous research reported the cloning of a novel U-box protein gene VaPUB from Vitis amurensis ‘Zuoshanyi’ grape and suggested a function of it in related to cold stress in the model plant Arabidopsis system. In this study, the role of VaPUB in response to biotic and abiotic stress was further analyzed in the homologous grapevine system by studying the transcript regulation and the protein accumulation in VaPUB transgenic vines. The expression analysis assay shown that VaPUB was significantly up-regulated 6 h after cold treatment and as early as 2 h post inoculation with Plasmopara viticola , a pathogen causing downy mildew disease in grapevine. Over-expressing VaPUB in V. Vinifera ‘Thompson Seedless’ affected the microstructure of leaves. The proteome assay shown that the accumulation of pathogenesis-related protein PR10 and many proteins involved in carbon and energy metabolism, oxidation reaction and protein metabolism were significantly altered in transgenic vines. In comparison with wild type plants, the expression level of PR10 family genes was significantly decreased in VaPUB transgenic vines under P. viticola treatment or cold stress. Results from this study showed that the U-box protein gene PUB quickly responded to both biotic stress and abiotic stress and significantly influenced the accumulation of resistance related proteins in grapevine.

Downy mildew is a highly destructive disease in grapevine production. A gene encoding pathogenesis-related (PR) thaumatin-like protein was isolated from the downy mildew-resistant grapevine "Zuoshan-1," a clonal selection from wild Vitis amurensis Rupr. The predicted thaumatin-like protein (VaTLP) has 225 amino acids and it is acidic, with a calculated isoelectric point of 4.8. The full length of the VaTLP gene was transformed into somatic embryogenic calli of V. vinifera 'Thompson Seedless' via Agrobacterium tumefaciens. Real-time RT-PCR confirmed that the VaTLP gene was expressed at a high level in the transgenic grapevines. Improved resistance of the transgenic lines against downy mildew was evaluated using leaf disks and whole plants inoculated with Plasmopara viticola, the pathogen causing grapevine downy mildew disease. Bioassay of the pathogen showed that both hyphae growth and asexual reproduction were inhibited significantly among the transgenic plants. Histological analysis also confirmed this disease resistance by demonstrating the inhibition and malformation of hyphae development in leaf tissue of the transgenic plants. These results indicated that the accumulation of VaTLP could enhance resistance to P. viticola in transgenic 'Thompson Seedless' grapevines.

Summary The grapevine downy mildew pathogen Plasmopara viticola secretes a set of RXLR effectors (PvRXLRs) to overcome host immunity and facilitate infection, but how these effectors function is unclear. Here, the biological function of PvRXLR131 was investigated via heterologous expression. Constitutive expression of PvRXLR131 in Colletotrichum gloeosporioides significantly enhanced its pathogenicity on grapevine leaves. Constitutive expression of PvRXLR131 in Arabidopsis promoted Pseudomonas syringae DC3000 and P. syringae DC3000 (hrcC‐) growth as well as suppressed defence‐related callose deposition. Transient expression of PvRXLR131 in Nicotiana benthamiana leaves could also suppress different elicitor‐triggered cell death and inhibit plant resistance to Phytophthora capsici. Further analysis revealed that PvRXLR131 interacted with host Vitis vinifera BRI1 kinase inhibitor 1 (VvBKI1), and its homologues in N. benthamiana (NbBKI1) and Arabidopsis (AtBKI1). Moreover, bimolecular fluorescence complementation analysis revealed that PvRXLR131 interacted with VvBKI1 in the plasma membrane. Deletion assays showed that the C‐terminus of PvRXLR131 was responsible for the interaction and mutation assays showed that phosphorylation of a conserved tyrosine residue in BKI1s disrupted the interaction. BKI1 was a receptor inhibitor of growth‐ and defence‐related brassinosteroid (BR) and ERECTA (ER) signalling. When silencing of NbBKI1 in N. benthamiana, the virulence function of PvRXLR131 was eliminated, demonstrating that the effector activity is mediated by BKI1. Moreover, PvRXLR131‐transgenic plants displayed BKI1‐overexpression dwarf phenotypes and suppressed BR and ER signalling. These physiological and genetic data clearly demonstrate that BKI1 is a virulence target of PvRXLR131. We propose that P. viticola secretes PvRXLR131 to target BKI1 as a strategy for promoting infection.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases in bananas resulting in significant loss of Cavendish bananas production worldwide. Here we show the agronomic traits and the resistance of 'Guijiao 9' in the field trials from 2012 to 2017. And then we dissect and compare the transcriptome response from these two cultivars (cv. 'Guijiao 9' and cv. Williams) in an attempt to understand the molecular basis that contribute to the enhanced Foc tropical race 4 (Foc-TR4) resistance. 'Guijiao 9' is a Cavendish cultivar with strong resistance to Foc-TR4, which was reflected in a lower disease severity and incidence in glasshouse and field trails, when compared to the susceptible cultivar Williams. Gene expression profiles of 'Guijiao 9' and Williams were captured by performing RNA-Seq analysis on 16 biological samples collected over a six day period post inoculation with Foc-TR4. Transcriptional reprogramming in response to Foc-TR4 was detected in both genotypes but the response was more drastic in 'Guijiao 9' than in Williams. Specific genes involved in plant-pathogen interaction and defense signaling including MAPK, calcium, salicylic acid, jasmonic acid and ethylene pathways were analyzed and compared between 'Guijiao 9' and Williams. Genes associated with defense-related metabolites synthesis such as NB-LRR proteins, calmodulin-binding protein and phenylpropanoids biosynthesis genes were significantly up-regulated in 'Guijiao 9' resistant to Foc-TR4 infection. Taken together, this study highlights the important roles of plant hormone regulation and defense gene activation in mediating resistance in 'Guijiao 9'.

The whole promoter regions of SUTs in Vitis were firstly isolated. SUTs are involved in the adaptation to biotic and abiotic stresses. The vulnerability of Vitis vinifera to abiotic and biotic stresses limits its yields. In contrast, Vitis amurensis displays resistance to environmental stresses, such as microbial pathogens, low temperatures, and drought. Sucrose transporters (SUTs) are important regulators for plant growth and stress tolerance; however, the role that SUTs play in stress resistance in V. amurensis is not known. Using V. amurensis Ruper. ‘Zuoshan-1’ and V. vinifera ‘Chardonnay’, we found that SUC27 was highly expressed in several vegetative organs of Zuoshan-1, SUC12 was weakly expressed or absent in most organs in both the species, and the distribution of SUC11 in source and sink organs was highest in Zuoshan-1. A search for cis-regulatory elements in the promoter sequences of SUTs revealed that they were regulated by light, environmental stresses, physiological correlation, and hormones. The SUTs in Zuoshan-1 mostly show a higher and rapid response than in Chardonnay under the induction by Plasmopara viticola infection, cold, water deficit, and dark conditions. The induction of SUTs was associated with the upregulation of key genes involved in sucrose metabolism and the biosynthesis of plant hormones. These results indicate that stress resistance in Zuoshan-1 is governed by the differential distribution and induction of SUTs by various stimuli, and the subsequent promotion of sucrose metabolism and hormone synthesis.

The volatile aroma compounds in wines produced from Chinese wild/hybrid species were investigated in comparison to wines from European grapes. Volatiles were extracted by headspace solid-phase micro-extraction and identified by gas-chromatography/mass-spectrometry. The identification of analyte was performed by a combination of the linear retention index approach with the comparison of the obtained mass spectra. A total of 98 peaks were tentatively assigned as wine aroma components, and 15 odorants can be found at concentrations above their odor threshold among the odor activity values (OAVs) of 46 compounds evaluated in all of the wines. The "OAVs' aroma wheels" showed that the classes of aromatic series are first fruity, next herbaceous and roasty and no spicy and caramelized notes. Via principal component analysis, all the grape germplasm studied could be divided into four groups: "Jingsheng-1," "Cabernet Gernischt," "Beibinghong," and others, which exhibited distinctive aroma features, respectively. Practical applications Chinese wild grape species have many desirable properties for wine grape breeding such as possessing strong resistant genes to fungal diseases and cold condition, especially after hybridization with European grapes. As an emerging wine country, wines from Chinese wild/hybrid species have achieved much success in the last few years. The chemical content and biological properties have been studied extensively in many but there are a few studies in our knowledge about the wine tastes of these species. In this paper, the key odorants in wines produced from the Chinese grape species of V. amurensis Rupr., V. davidii Foex., and V. quinquangularis Rehd. and its hybrids were characterized in comparison to wines produced from European grapes (V. vinifera). The findings will help to the further understanding of the key aroma components in the different Chinese grapevine germplasm, and make further efforts to enrich the wine types in the marketplace.

Downy mildew (DM), caused by oomycete Plasmopara viticola (Pv), can lead to severe damage to Vitis vinifera grapevines. Vitis amurensis has generally been regarded as a DM resistant species. However, when V. amurensis ‘Shuanghong’ were inoculated with Pv strains ‘ZJ-1-1’ and ‘JL-7-2’, the former led to obvious DM symptoms (compatible), while the latter did not develop any DM symptoms but exhibited necrosis (incompatible). In order to underlie molecular mechanism in DM resistance, mRNA-seq based expression profiling of ‘Shuanghong’ was compared at 12, 24, 48 and 72 h post inoculation (hpi) with these two strains. Specific genes and their corresponding pathways responsible for incompatible interaction were extracted by comparing with compatible interaction. In the incompatible interaction, 37 resistance (R) genes were more expressed at the early stage of infection (12 hpi). Similarly, genes involved in defense signaling, including MAPK. ROS/NO, SA, JA, ET and ABA pathways, and genes associated with defense-related metabolites synthesis, such as pathogenesis-related genes and phenylpropanoids/stilbenoids/flavonoids biosynthesizing genes, were also activated mainly during the early stages of infection. On the other hand, Ca2+ signaling and primary metabolism, such as photosynthesis and fatty acid synthesis, were more repressed after ‘JL-7-2’ challenge. Further quantification of some key defense-related factors, including phytohormones, phytoalexins and ROS, generally showed much more accumulation during the incompatible interaction, indicating their important roles in DM defense. In addition, a total of 43 and 52 RxLR effectors were detected during ‘JL-7-2’ and ‘ZJ-1-1’ infection processes, respectively.

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor superfamily that can be categorized into αARs and βARs. The specific function of ARs in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of ARs from ayu (Plecoglossus altivelis; PaαAR and PaβAR). Sequence comparisons showed that PaαAR was most closely related to the αAR of the Japanese flounder and Nile tilapia, while PaβAR was most closely related to the βAR of Atlantic salmon. The AR transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaαAR and PaβAR proteins were upregulated in MO/MФ after infection, and PaβAR knockdown resulted in a pro-inflammatory status in ayu MO/MФ upon V. anguillarum infection and lowered the phagocytic activity of MO/MФ. Our results indicate that PaβAR plays the role of an anti-inflammatory mediator in the immune response of ayu against bacterial infection.

Plant pattern recognition receptors (PRRs) are essential for immune responses and establishing symbiosis. Plants detect invaders via the recognition of pathogen-associated molecular patterns (PAMPs) by PRRs. This phenomenon is termed PAMP-triggered immunity (PTI). We investigated disease resistance in Vitis amurensis to identify PRRs that are important for resistance against downy mildew, analyzed the PRRs that were upregulated by incompatible Plasmopara viticola infection, and cloned the full-length cDNA of the VaHAESA gene. We then analyzed the structure, subcellular localization, and relative disease resistance of VaHAESA. VaHAESA and PRR-receptor-like kinase 5 (RLK5) are highly similar, belonging to the leucine-rich repeat (LRR)-RLK family and localizing to the plasma membrane. The expression of PRR genes changed after the inoculation of V. amurensis with compatible and incompatible P. viticola; during early disease development, transiently transformed V. vinifera plants expressing VaHAESA were more resistant to pathogens than those transformed with the empty vector and untransformed controls, potentially due to increased H2O2, NO, and callose levels in the transformants. Furthermore, transgenic Arabidopsis thaliana showed upregulated expression of genes related to the PTI pathway and improved disease resistance. These results show that VaHAESA is a positive regulator of resistance against downy mildew in grapevines.

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate "JL-7-2" genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.

RPW8 genes are atypical broad-spectrum genes that provide resistance to powdery mildew, downy mildew, the cauliflower mosaic virus in Arabidopsis thaliana, and powdery mildew in tobacco. They play important roles in basal plant pathogen defense. They also provide insights into a novel disease resistance mechanism. In this study, we report on homologous RPW8 genes in Vitis pseudoreticulata. Five VpRPW8 genes were cloned; their Open Reading Frame (ORF) sequences ranged from 1994 base pairs to 2478 base pairs. They were comprised of five exons and four introns and shared 78.66% identity. Their proteins had typical conserved RPW8 and NB-LRR (the nucleotide-binding site and the leucine-rich repeats) domains (except VpRPW8-d, which lacked LRR domains). Prokaryotic expression results were consistent with predicted molecular weights. All five RPW8 genes were located in the cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis showed that VpRPW8s in V. pseudoreticulata were induced by Plasmopara viticola, but nearly only VvRPW8-d genes were induced in Vitis vinifera. Furthermore, a VpRPW8 transgenic tobacco system was established. Overexpressed VpRPW8s enhanced resistance to Phytophthora capsici and VpRPW8s conferred varying degrees of resistance to Ph. capsici in Nicotiana benthamiana. Our study presents novel members of the plant RPW8 family and suggests that VpRPW8s are involved in enhanced resistance to P. viticola and Ph. capsici.

In the present study, the complete chloroplast genome of Vitis davidii Foex strain ‘SJTU003’ was assembled and subjected to phylogenetic analysis. This chloroplast genome of ‘SJTU003’ was 161,335 b...

Tissue factor (TF) plays an important role in the host's immune system as the principal initiator of coagulation. However, the precise function of TF in teleosts remains unclear. We determined the cDNA sequence of TF from ayu Plecoglossus altivelis (PaTF). The PaTF transcript was expressed in all tested tissues, and changes in expression were observed in tissues and monocytes/macrophages (MO/MФ) upon infection with Vibrio anguillarum. PaTF was prokaryotically expressed and purified to prepare anti-PaTF antibodies. Western blot analysis revealed that native PaTF was glycosylated in thrombocytes, but not in ayu MO/MФ. Microparticles could transfer PaTF to thrombocytes. PaTF neutralization or knockdown led to anti-inflammatory status in ayu MO/MФ upon V. anguillarum infection. PaTF neutralization reduced the apoptosis of ayu MO/MФ and improve survival rate in V. anguillarum-infected ayu. Our results indicate that PaTF plays a role in ayu immune response against bacterial infection as a pro-inflammatory mediator.

The CC motif chemokine 19 (CCL19) functions in acute inflammation by recruiting lymphocytes and other cells. However, CCL19 has only been investigated in few fish species. In this study, we characterized a CCL19-like molecule (PaCCL19l) in ayu (Plecoglossus altivelis), a teleost fish. Sequence analysis revealed that PaCCL19l was most closely related to Atlantic salmon (Salmon salar) CCL19l1, which belonged to the fish CCL19a.1 subcluster. PaCCL19l was constitutively expressed in the tested ayu tissues and peripheral blood mononuclear cells (PBMCs), with the highest transcript level in PBMCs. Upon infection with Vibrio anguillarum, the expressions of PaCCL19l in the head kidney, liver, spleen, PBMCs, and monocytes/macrophages (MO/MΦ) were dramatically up-regulated. Recombinant PaCCL19l (rPaCCL19l) exhibited a significant effect on the chemotaxis of lymphocytes and MO/MΦ in vitro and in vivo. Meanwhile, rPaCCL19l exerted a high chemotaxic activity for lipopolysaccharide (LPS)-stimulated MO/MΦ (M1-type), but not for cyclic adenosine monophosphate (cAMP)-stimulated MO/MΦ (M2-type). When ayu MO/MΦ was treated with rPaCCL19l along with Vibrio anguillarum infection, the mRNA expression of proinflammatory cytokines (IL-1β, TNFα, IL-6, IL-12b, and IFN-γ) was up-regulated, while that of anti-inflammatory cytokines (IL-10, TGFβ, and IL-22) was down-regulated. Ayu MO/MΦ treated with anti-PaCCL19l IgG gave the opposite result. These results implicated that PaCCL19l is involved in the selective chemotaxis of ayu immune cells and promotes the host at a pro-inflammatory state.

Colony-stimulating factor 1 receptor (CSF1R) is an important regulator of monocytes/macrophages (MO/MΦ). Although CSF1R gene has been identified and functionally studied in many fish, the precise role of CSF1R in grass carp (Ctenopharyngodon idellus) remains unclear. In this study, we determined the cDNA sequence of CSF1R (CiCSF1R) from a teleost fish, grass carp. Sequence comparison and phylogenetic tree analysis showed that CiCSF1R was most closely related to the CSF1R of zebrafish. The CiCSF1R transcript was mainly expressed in the spleen, head kidney, and head kidney-derived MO/MΦ, and its expression was altered in various tissues upon Aeromonas hydrophila infection. We prepared antibodies for neutralization of CiCSF1R on grass carp MO/MΦ. CiCSF1R neutralization or knockdown led to anti-inflammatory status in MO/MΦ upon A. hydrophila infection. CiCSF1R neutralization or knockdown also decreased the phagocytic activity of MO/MΦ. Flow cytometric analysis showed that more than 85% of grass carp MO/MΦ were CiCSF1R-positive cells. The percentage of CiCSF1R-positive cells in the head kidney of grass carp was above 10%, whereas it was only 5% and 4% in the spleen and liver, respectively. In conclusion, CSF1R is a specific surface marker of grass carp MO/MΦ, and it regulates the functions of MO/MΦ.

Plasmopara viticola is an oomycete pathogen that causes downy mildew, one of the most devastating diseases of grapevine. Currently, the molecular basis of the interaction between this pathogen and the grapevine host is not well understood. To explore the genes involved in P. viticola pathogenicity we performed RNA-Seq analysis of cDNAs derived from downy mildew-infected grapevine leaves for three different P. viticola isolates; two from China (JL-7-2, ZJ-1-1) and one from Australia (CSIRO-L-2). Approximately 30,000 unigenes were predicted for each of the three isolates and from these over 500 potential secreted proteins were identified. Using three prediction methods, a total of 51 PvRXLR effectors were identified to be present in the secretome, with at least 26 shared by two or more P. viticola isolates. Expression profiling, based on RNA-Seq data, indicated that PvRXLRs showed three different expression patterns during infection. Transient expression of selected PvRXLRs in Nicotiana benthamiana demonstrated that all were capable of suppressing programmed cell death triggered by the mouse BAX protein or the PAMP INF1. Additionally, BLASTP and Hidden Markov Model (HMM) searches identified 10 predicted proteins belonging to the CRN (Crinkler) group of oomycete effectors. Pfam domain analysis of the secretome also identified a diverse range of putative apoplastic effectors, the major groups being glycosyl hydrolases, peptidases and protease-inhibitors. This study provides the first detailed analysis of the secretome of grapevine downy mildew and its encoded effector arsenal.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is widespread in fish and plays an important role in the host's innate immune system. In this study, full-length cDNA for LEAP-2 (PaLEAP-2) gene was cloned and sequenced from ayu, Plecoglossus altivelis. PaLEAP-2 mRNA was detected in a wide range of tissues, with the highest level of transcripts found in the liver. Upon induction by Vibrio anguillarum, its expression significantly increased in the liver, kidney, spleen, gill, and heart, but decreased in the intestine. The PaLEAP-2 mature peptide was chemically synthesized; it exhibited selective antimicrobial activity against various bacteria in vitro. PaLEAP-2 at high concentration reduced the bacterial load and improved the survival rate of V. anguillarum-infected ayu. Moreover, it inhibited the expression of mRNAs for TNF-α and IL-1β in V. anguillarum-infected ayu, both at high and low concentrations. PaLEAP-2 induced hydrolysis of the pET-22b plasmid DNA and bacterium genomic DNA. These results suggest that PaLEAP-2 plays a role in ayu immune responses against bacterial infection.

WRKY transcription factors (TFs) play important roles in many plant processes, including responses to biotic and abiotic stresses. In the present study, Muscadinia rotundifolia MrWRKY30 dramatically accumulated in grapevine leaves in response to inoculation of Plasmopara viticola, a pathogen causing grapevine downy mildew disease. Similar responses were also found on grapevines treated with exogenous SA/JA/ET. Ectopic expression of MrWRKY30 in Arabidopsis thaliana "COL0" enhanced its resistance to downy mildew pathogen Peronospora parasitica. Pathogenesis-related (PR) genes, including AtPR1, AtPR4, AtPR5, and AtPDF1.2, were significantly upregulated in transgenic A. thaliana after P. parasitica inoculation. In the mean time, two critical genes in SA and JA signaling pathways, AtEDS5 and AtJAR1, were abundantly expressed as well, indicating that MrWRKY30 may enhance disease resistance of A. thaliana through SA and JA defense system. The transgenic A. thaliana plants also enhanced tolerance to cold stress accompanied with upregulation of AtCBF1, AtCBF3, AtICE1, and AtCOR47. MrWRKY30 might protect A. thaliana from cold damage by activating the AtCBF-mediated signaling pathway to induce the downstream AtCOR47 gene. Interestingly, the transgenic seedlings had a negative effect on salt tolerance. Reverse transcription PCR (RT-PCR) analysis revealed that antioxidant enzyme genes AtAPX (ascorbate peroxidase), AtCAT (catalase), and AtGST (glutathione-S-transferase) were suppressed in transgenic plants, which may lead to reactive oxygen species (ROS)-mediated sensitivity to salt stress.

In this study, eight different submerged aquatic species were screened by batch biochemical methane potential and anaerobic degradability tests to identify a promising/suitable feedstock. Kinetics of the best-screened substrate were studied in a mesophilic semi-continuous experiment. The aquatic species Myriophyllum aquaticum , Egeria densa and Potamogeton perfoliatus showed relatively higher methane yields of over 400 NmL/g-VS (volatile solids). Semi-continuous operation was carried out by feeding E. densa for over 400 days. The achieved results were 33%–53% chemical oxygen demand (COD) reduction and methane yield of 126–231 NmL/g-VS with a short hydraulic retention time (HRT). Additionally, the NH 4 + and PO 4 3− releases from the biomass to water were found to be low (18%–27% and 2.5%–3.9%) throughout the experiment. Hydrolysis was the limiting step in the digestion of E. densa , regardless of changes in HRT (15–45 days). The acid-phase model indicated that the hydrolysis rate constant (k h ) of E. densa was 0.058 one/day, which was one third lower the k h value of food waste, but quite similar to cow manure.