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Cell Wall Degrading Enzyme Induced Rice Innate Immune Responses Are Suppressed by the Type 3 Secretion System Effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae  [PDF]
Dipanwita Sinha, Mahesh Kumar Gupta, Hitendra Kumar Patel, Ashish Ranjan, Ramesh V. Sonti
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075867
Abstract: Innate immune responses are induced in plants and animals through perception of Damage Associated Molecular Patterns. These immune responses are suppressed by pathogens during infection. A number of studies have focussed on identifying functions of plant pathogenic bacteria that are involved in suppression of Pathogen Associated Molecular Pattern induced immune responses. In comparison, there is very little information on functions used by plant pathogens to suppress Damage Associated Molecular Pattern induced immune responses. Xanthomonas oryzae pv. oryzae, a gram negative bacterial pathogen of rice, secretes hydrolytic enzymes such as LipA (Lipase/Esterase) that damage rice cell walls and induce innate immune responses. Here, we show that Agrobacterium mediated transient transfer of the gene for XopN, a X. oryzae pv. oryzae type 3 secretion (T3S) system effector, results in suppression of rice innate immune responses induced by LipA. A xopN- mutant of X. oryzae pv. oryzae retains the ability to suppress these innate immune responses indicating the presence of other functionally redundant proteins. In transient transfer assays, we have assessed the ability of 15 other X. oryzae pv. oryzae T3S secreted effectors to suppress rice innate immune responses. Amongst these proteins, XopQ, XopX and XopZ are suppressors of LipA induced innate immune responses. A mutation in any one of the xopN, xopQ, xopX or xopZ genes causes partial virulence deficiency while a xopN- xopX- double mutant exhibits a greater virulence deficiency. A xopN- xopQ- xopX- xopZ- quadruple mutant of X. oryzae pv. oryzae induces callose deposition, an innate immune response, similar to a X. oryzae pv. oryzae T3S- mutant in rice leaves. Overall, these results indicate that multiple T3S secreted proteins of X. oryzae pv. oryzae can suppress cell wall damage induced rice innate immune responses.
Xanthomonas oryzae pv. oryzae Type III Effector XopN Targets OsVOZ2 and a Putative Thiamine Synthase as a Virulence Factor in Rice  [PDF]
Hoon Cheong, Chi-Yeol Kim, Jong-Seong Jeon, Byoung-Moo Lee, Jae Sun Moon, Ingyu Hwang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0073346
Abstract: Xanthomonas oryzae pv. oryzae (Xoo) is spread systemically through the xylem tissue and causes bacterial blight in rice. We evaluated the roles of Xanthomonas outer proteins (Xop) in the Xoo strain KXO85 in a Japonica-type rice cultivar, Dongjin. Five xop gene knockout mutants (xopQKXO85, xopXKXO85, xopP1KXO85, xopP2KXO85, and xopNKXO85) were generated by EZ-Tn5 mutagenesis, and their virulence was assessed in 3-month-old rice leaves. Among these mutants, the xopNKXO85 mutant appeared to be less virulent than the wild-type KXO85; however, the difference was not statistically significant. In contrast, the xopNKXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85. These observations indicate that the roles of Xop in Xoo virulence are dependent on leaf stage. We chose the xopN gene for further characterization because the xopNKXO85 mutant showed the greatest influence on virulence. We confirmed that XopNKXO85 is translocated into rice cells, and its gene expression is positively regulated by HrpX. Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP), were identified as targets of XopNKXO85 by yeast two-hybrid screening. Interactions between XopNKXO85 and OsVOZ2 and OsXNP were further confirmed in planta by bimolecular fluorescence complementation and in vivo pull-down assays. To investigate the roles of OsVOZ2 in interactions between rice and Xoo, we evaluated the virulence of the wild-type KXO85 and xopNKXO85 mutant in the OsVOZ2 mutant line PFG_3A-07565 of Dongjin. The wild-type KXO85 and xopNKXO85 mutant were significantly less virulent in the mutant rice line. These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.
Cultivation characteristics and flavonoid contents of wormwood (Artemisia montana Pamp.)  [PDF]
Yong Joo Kim, Jeong-Hoon Lee, Sun-Ju Kim
Journal of Agricultural Chemistry and Environment (JACEN) , 2013, DOI: 10.4236/jacen.2013.24017
Abstract: The aim of this study was to establish the optimum harvesting time and the content of flavonoids in the leaves, stems, and roots of Artemisia montana Pamp. A. montana was monitored from June to October in 2012. The yield of A. montana at high density (30 × 10 cm) was higher than that of A. montana at low density (30 × 20 and 30 cm). Yield in terms of dry weight was increased with an extended growth period and development stage. High yield achieved at 2580 and 2757 kg?10 a?1 in September and October, respectively. Among the leaves, stems, and underground plant organs, jaceosidin and eupatilin were mainly detected in the leaves, and the highest levels were observed in June, at values of 66.6 and 158.2 mg?100 g?1, respectively. In contrast, apigenin was the major compound detected in the underground plant organs, with levels ranging from 21.2 to 29.5 mg?100 g?1 until September. Therefore, optimal harvest times were between September and October, generating a high yield and adding economic value although a higher level of total flavonoids was observed in crops harvested in June.
Chlamydia pneumoniae CopD Translocator Protein Plays a Critical Role in Type III Secretion (T3S) and Infection  [PDF]
David C. Bulir, Daniel A. Waltho, Christopher B. Stone, Kenneth A. Mwawasi, Jordan C. Nelson, James B. Mahony
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0099315
Abstract: Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120–125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1:1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.
Quantitative Resistance of Potato to Pectobacterium atrosepticum and Phytophthora infestans: Integrating PAMP-Triggered Response and Pathogen Growth  [PDF]
Alexander Kr?ner, Ga?lle Hamelin, Didier Andrivon, Florence Val
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023331
Abstract: While the mechanisms underlying quantitative resistance of plants to pathogens are still not fully elucidated, the Pathogen-Associated Molecular Patterns (PAMPs)-triggered response model suggests that such resistance depends on a dynamic interplay between the plant and the pathogen. In this model, the pathogens themselves or elicitors they produce would induce general defense pathways, which in turn limit pathogen growth and host colonisation. It therefore suggests that quantitative resistance is directly linked to a common set of general host defense mechanisms, but experimental evidence is still inconclusive. We tested the PAMP-triggered model using two pathogens (Pectobacterium atrosepticum and Phytophthora infestans) differing by their infectious processes and five potato cultivars spanning a range of resistance levels to each pathogen. Phenylalanine ammonia-lyase (PAL) activity, used as a defense marker, and accumulation of phenolics were measured in tuber slices challenged with lipopolysaccharides from P. atrosepticum or a concentrated culture filtrate from P. infestans. PAL activity increased following treatment with the filtrate but not with lipopolysaccharides, and varied among cultivars. It was positively related to tuber resistance to P. atrosepticum, but negatively related to tuber resistance to P. infestans. It was also positively related to the accumulation of total phenolics. Chlorogenic acid, the main phenolic accumulated, inhibited growth of both pathogens in vitro, showing that PAL induction caused active defense against each of them. Tuber slices in which PAL activity had been induced before inoculation showed increased resistance to P. atrosepticum, but not to P. infestans. Our results show that inducing a general defense mechanism does not necessarily result in quantitative resistance. As such, they invalidate the hypothesis that the PAMP-triggered model alone can explain quantitative resistance. We thus designed a more complex model integrating physiological host response and a key pathogen life history trait, pathogen growth, to explain the differences between the two pathosystems.
Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells  [PDF]
Amy E. L. Stone,Silvia Giugliano,Gretja Schnell,Linling Cheng,Katelyn F. Leahy,Lucy Golden-Mason,Michael Gale Jr,Hugo R. Rosen
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003316
Abstract: Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.
In vitro assessment of the protection from oxidative stress by various fractions of Artemisia incisa Pamp.  [PDF]
Aziz-Ur-Rehman,Gulzar Sehrish,Abbasi Muhammad A.,Shahzadi Tayyaba
Journal of the Serbian Chemical Society , 2011, DOI: 10.2298/jsc110102116a
Abstract: The methanolic extract of Artemisia incisa Pamp. was dissolved in distilled water and successively partitioned with n-hexane, chloroform, ethyl acetate and n-butanol. The antioxidant potential of all these fractions and remaining aqueous fraction was evaluated by four methods, i.e., the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), the total antioxidant activity, the ferric reducing antioxidant power (FRAP) assay and the ferric thiocyanate assay. In addition, the total phenolics was determined. The obtained results revealed that among the studied fractions the ethyl acetate soluble fraction showed the most potent DPPH-radical scavenging activity with an IC50 value of 5.3 ± 0.71 μg mL-1, which is even more effective than the standard antioxidant butylated hydroxytoluene (BHT) (IC50 value 0f 12.1± 0.92 μg mL-1). The ethyl acetate fraction also showed the highest FRAP value (3677.13 ± 27.1 μg TE mL-1), inhibition of lipid peroxidation (60.93 ± 0.84 % at 500 μg mL-1) and total phenolic content (95.5 ± 0.05 μg GAE g-1) as compared to other fractions. However, the remaining aqueous fraction was found to posses the highest antioxidant activity of all the fractions.
Pathogen conservation molecules and PAMP-triggered innate immunity in plants

Dai Jing-Cheng,Huang Jian-Guo,Wang Chun-Lian,Zhao Kai-Jun,

微生物学通报 , 2012,
Abstract: Pathogen associated molecular patterns (PAMPs) recognized by host cell surface localized pattern-recognition receptors (PRRs) are pathogen conservation molecules. So far, a few pairs of characterized PRR/PAMP in plants provided useful models to study the specificity of ligand-binding and likely activation mechanisms. For example, recognition models of FLS2-flagellin, EFR-Tu (EF-Tu), CEBiP/CERK1-chitin and XA21-Ax21 have been extensively studied. The perception between PRR and PAMP triggers immune response (PTI) to resist pathogens. However, to successfully grow and proliferate on their hosts, virulent pathogens had to override the PTI, for which, these pathogens evolved a variety of strategies, such as injecting effector proteins into the plant cell or sequestering their PAMPs. Based on the knowledge on the interaction of PRR-PAMP, researchers are trying to engineer PRRs ( chimeras of PRRs) to develop new strategies in molecular breeding for achieving durability and a broad-spectrum disease resistance. We reviewed the recent findings about recognitions of PRRs-PAMPs, the engineered PRRs, and discussed the future prospects and several issues in researches on PTI.
LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells  [PDF]
Selinda J. Orr,Ashley R. Burg,Tim Chan,Laura Quigley,Gareth W. Jones,Jill W. Ford,Deborah Hodge,Catherine Razzook,Joseph Sarhan,Yava L. Jones,Gillian C. Whittaker,Kimberly C. Boelte,Lyudmila Lyakh,Marco Cardone,Geraldine M. O'Connor,Cuiyan Tan,Hongchuan Li,Stephen K. Anderson,Simon A. Jones,Weiguo Zhang,Philip R. Taylor,Giorgio Trinchieri,Daniel W. McVicar
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003357
Abstract: Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2?/? mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2?/? DCs. Accordingly, Lat2?/? DCs directed reduced Th1 polarization in vitro and Lat2?/? mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.
C-PAmP: Large Scale Analysis and Database Construction Containing High Scoring Computationally Predicted Antimicrobial Peptides for All the Available Plant Species  [PDF]
Anastasia Niarchou, Anastasia Alexandridou, Emmanouil Athanasiadis, George Spyrou
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0079728
Abstract: Background Antimicrobial peptides are a promising alternative to conventional antibiotics. Plants are an important source of such peptides; their pharmacological properties are known since antiquity. Access to relevant information, however, is not straightforward, as there are practically no major repositories of experimentally validated and/or predicted plant antimicrobial peptides. PhytAMP is the only database dedicated to plant peptides with confirmed antimicrobial action, holding 273 entries. Data on such peptides can be otherwise retrieved from generic repositories. Description We present C-PAmP, a database of computationally predicted plant antimicrobial peptides. C-PAmP contains 15,174,905 peptides, 5–100 amino acids long, derived from 33,877 proteins of 2,112 plant species in UniProtKB/Swiss-Prot. Its web interface allows queries based on peptide/protein sequence, protein accession number and species. Users can view the corresponding predicted peptides along with their probability score, their classification according to the Collection of Anti-Microbial Peptides (CAMP), and their PhytAMP id where applicable. Moreover, users can visualise protein regions with a high concentration of predicted antimicrobial peptides. In order to identify potential antimicrobial peptides we used a classification algorithm, based on a modified version of the pseudo amino acid concept. The classifier tested all subsequences ranging from 5 to 100 amino acids of the plant proteins in UniProtKB/Swiss-Prot and stored those classified as antimicrobial with a high probability score (>90%). Its performance measures across a 10-fold cross-validation are more than satisfactory (accuracy: 0.91, sensitivity: 0.93, specificity: 0.90) and it succeeded in classifying 99.5% of the PhytAMP peptides correctly. Conclusions We have compiled a major repository of predicted plant antimicrobial peptides using a highly performing classification algorithm. Our repository is accessible from the web and supports multiple querying options to optimise data retrieval. We hope it will greatly benefit drug design research by significantly limiting the range of plant peptides to be experimentally tested for antimicrobial activity.
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