Stir-fried Semen Armeniacae Amarum Suppresses Aristolochic Acid I-Induced Nephrotoxicity and DNA Adducts.

OBJECTIVE: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma. METHODS: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously. RESULTS: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01). CONCLUSIONS: Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.

[Effects of fucoidan inducing impairment of human osteosarcoma cell 143B and its mechanism].

Objective: To investigate the effects of fucoidan inducing impairment of human osteosarcoma cell 143B, as well its mechanisms. Methods: After 143B cells were treated with different concentrations of FUC (0, 0.5, 1, 10, 100, 400, 800 µg/ml) for 48 h, the cell viability and dehydrogenase (LDH) level were detected by MTT assay and chemical colorimetry with six multiple wells for each concentration. Based on MTT results, we determined the value of IC50 was 244.5 µg/ml. The follow-up experiments were divided into control group (without FUC), FUC (10 µg/ml)-treated group, FUC (100 µg/ml)-treated group, FUC (400 µg/ml)-treated group and positive group (resveratrol, 40 µmol/L). There were four multiple wells for each concentration, and each experiment was repeated at least three times. Flow cytometry was performed to detect cell apoptosis and intracellular reactive oxygen species (ROS) level; acridine orange (AO) staining and lyso-tracker red staining were used to observe the autophagolysosome formation; chemical colorimetric analysis was performed to determine malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); Western blot was used to detect protein expressions of nuclear factor E2-associated factor 2 (Nrf2), heme oxygenase 1 (HO-1) and autophagy-associated proteins including microtubule-associated light chain protein 3 (LC-3), Atg7, Beclin-1 and p62. Results: Compared with control group, the cell viability was decreased significantly in FUC (100～400 µg/ml)-treated groups (P＜0.01); LDH levels in the supernatant (P＜0.05 or P＜0.01), the percentage of cell apoptosis (P＜0.01), intracellular ROS level and MDA content (P＜0.01) were increased remarkably; protein expressions of Atg7 and Beclin-1 were upregulated (P＜0.05 or P＜0.01); the conversion from LC-3I to LC-3II was significant (P＜0.01) together with elevation of autophagolysosome formation (P＜0.05 or P＜0.01); while the activities of SOD and GSH-Px and protein expressions of Nrf2, HO-1 and p62 were decreased remarkably (P＜0.05 or P＜0.01). Conclusion: FUC (100～400 µg/ml) treatment induces oxidative damage and autophagic death in osteosarcoma 143B cells.

Origin, Evolution, Breeding, and Omics of Chayote, an Important Cucurbitaceae Vegetable Crop.

Chayote (Sechium edule), a member of the Cucurbitaceae family, is cultivated throughout tropical and subtropical regions of the world and utilized in pharmaceutical, cosmetic and food industries because it is an excellent source of minerals, dietary fibers, protein, vitamins, carotenoids, polysaccharides, phenolic and flavonoid compounds, and other nutrients. Chayote extracts process various medicinal properties, such as anti-cardiovascular, antidiabetic, antiobesity, antiulcer, and anticancer properties. With the rapid advancements of molecular biology and sequencing technology, studies on chayote have been carried out. Research advances, including molecular makers, breeding, genomic research, chemical composition, and pests and diseases, regarding chayote are reviewed in this paper. Future exploration and application trends are briefly described. This review provides a reference for basic and applied research on chayote, an important Cucurbitaceae vegetable crop.

Effects of diurnal temperature range on mortality in Hefei city, China.

Although several studies indicated an association between diurnal temperature range (DTR) and mortality, the results about modifiers are inconsistent, and few studies were conducted in developing inland country. This study aims to evaluate the effects of DTR on cause-specific mortality and whether season, gender, or age might modify any association in Hefei city, China, during 2007-2016. Quasi-Poisson generalized linear regression models combined with a distributed lag non-linear model (DLNM) were applied to evaluate the relationships between DTR and non-accidental, cardiovascular, and respiratory mortality. We observed a J-shaped relationship between DTR and cause-specific mortality. With a DTR of 8.3 °C as the reference, the cumulative effects of extremely high DTR were significantly higher for all types of mortality than effects of lower or moderate DTR in full year. When stratified by season, extremely high DTR in spring had a greater impact on all cause-specific mortality than other three seasons. Male and the elderly (≥ 65 years) were consistently more susceptible to extremely high DTR effect than female and the youth (< 65 years) for non-accidental and cardiovascular mortality. To the contrary, female and the youth were more susceptible to extremely high DTR effect than male and the elderly for respiratory morality. The study suggests that extremely high DTR is a potential trigger for non-accidental mortality in Hefei city, China. Our findings also highlight the importance of protecting susceptible groups from extremely high DTR especially in the spring.

AvrXa7-Xa7 mediated defense in rice can be suppressed by transcriptional activator-like effectors TAL6 and TAL11a from Xanthomonas oryzae pv. oryzicola.

The closely related plant pathogens Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae cause bacterial leaf streak (BLS) and bacterial leaf blight (BLB), respectively, in rice. Unlike X. oryzae pv. oryzae, endogenous avirulence-resistance (avr-R) gene interactions have not been identified in the X. oryzae pv. oryzicola-rice pathosystem, though both X. oryzae pv. oryzicola and X. oryzae pv. oryzae possess transcriptional activator-like effectors (TALE), which are known to modulate R or S genes in rice. In this report, avrXa7, avrXa10, and avrXa27 from X. oryzae pv. oryzae were transferred into YNB0-17 and RS105, hypovirulent and hypervirulent strains, respectively, of X. oryzae pv. oryzicola. When YNB0-17 containing avrXa7, avrXa10, or avrXa27 was inoculated to rice, hypersensitive responses (HR) were elicited in rice cultivars containing the R genes Xa7, Xa10, and Xa27, respectively. By contrast, RS105 expressing avrXa27 elicited an HR in a rice cultivar containing Xa27 but the expression of avrXa7 and avrXa10 in RS105 did not result in HR in rice cultivars containing Xa7 and Xa10, correspondingly. Southern blot analysis demonstrated that YNB0-17 possesses only approximately nine putative tale genes, whereas the hypervirulent RS105 contains at least 20. Although YNB0-17 contains an intact type III secretion system (T3SS), its genome is lacking the T3SS effector genes avrRxo1 and xopO, which are present in RS105. The introduction of avrRxo1 and xopO into YNB0-17 did not suppress avrXa7- or avrXa10-triggered immunity in rice. However, the transference of individual tale genes from RS105 into YNB0-17 led to the identification of tal6 and tal11a that suppressed avrXa7-Xa7-mediated defense. Thus, YNB0-17 may be a useful recipient for discovering such suppressors. This is the first report that co-evolutionally generated tale genes in X. oryzae pv. oryzicola suppress gene-for-gene defense against BLB, which may explain the lack of BLS-resistant cultivars.

Synthesis and cytotoxicity of novel 20-O-linked homocamptothecin ester derivatives as potent topoisomerase I inhibitors.

In an attempt to improve the antitumor activity of homocamptothecins (hCPTs), a series of novel 20-O-linked hCPT ester derivatives were first designed and synthesized based on a synthetic route, by which hCPTs are acylated with different substituted phenoxyacetic acid ester derivatives. Most of the derivatives were assayed for in vitro cytotoxicity against six human cancer cell lines KB, KB/VCR, A549, HCT-8, Bel7402, and A2780, and most of the assayed compounds exhibited good antiproliferative activity on these tumor cell lines especially on KB.

TCTP overexpression is associated with the development and progression of glioma.

Upregulation of translationally controlled tumor protein (TCTP) has been reported in a variety of malignant tumors. However, the impact of TCTP in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of TCTP in glioma patients. Western blot analysis was used to characterize the expression patterns of TCTP in 45 glioma and 22 normal brain tissues. Immunohistochemistry on a tissue microarray containing 127 cases of glioma was performed to analyze the association between TCTP expression and clinicopathological features. Compared with normal brain tissues, TCTP expression was significantly higher in glioma tissues (p <0.001). In addition, high TCTP expression in glioma was significantly associated with advanced pathological grade (p = 0.018). Kaplan-Meier analysis showed that patients with glioma and higher TCTP expression tend to have shorter overall survival time (p <0.001). In multivariate analysis, TCTP expression was proved to be an independent prognostic factor for patients with glioma (p <0.001). In conclusion, this study confirmed the overexpression of TCTP and its association with tumor progression in glioma. It also provided the first evidence that TCTP expression in glioma was an independent prognostic factor of patients, which might be a potential diagnostic and therapeutic target of glioma.

[Effect of long-term power frequency electromagnetic field exposure on proliferation and apoptosis of SRA01/04 cells].

OBJECTIVE: To investigate the effect of long-term power frequency electromagnetic field (50 Hz) exposure on the proliferation and apoptosis of human lens epithelial cells (SRA01/04 cells). METHODS: SRA01/04 cells in the exponential growth phase were exposed or sham-exposed to power frequency electromagnetic field (50 Hz, 2.3 mT) for 2 hours per day, 5 days every week. After 11 weeks of exposure, the cells were collected; the cell morphology was observed under a microscope, the cell viability was measured by MTT assay, the cell cycle and apoptosis were examined by flow cytometry, and the protein expression levels of cyclin D and proliferating cell nuclear antigen (PCNA) were determined by western blot. RESULTS: Compared with the sham-exposed SRA01/04 cells, most exposed cells became rounded and more stereoscopic, and heterochromatin gathered near the nuclear membrane in some exposed cells. The MTT assay showed that the viability of exposed cells was significantly increased compared with that of the sham-exposed cells (P < 0.05). Long-term power frequency electromagnetic field exposure led to significantly increased number of cells in S phase (P < 0.05), and the proliferation index was significantly higher in the exposed cells than in the sham-exposed cells (P < 0.05). There was no significant difference in apoptotic rate between the exposed cells and sham-exposed cells (P > 0.05). The exposed cells had significantly higher protein expression levels of cyclin D and PCNA than the sham-exposed cells (P < 0.05). CONCLUSION: Long-term power frequency electromagnetic field exposure can promote cellular proliferation and change cell cycle in SRA01/04 cells, but it has no marked effect on the apoptosis of SRA01/04 cells.

A highly-conserved single-stranded DNA-binding protein in Xanthomonas functions as a harpin-like protein to trigger plant immunity.

Harpins are produced by gram-negative phytopathogenic bacteria and typically elicit hypersensitive response (HR) in non-host plants. The characterization of harpins in Xanthomonas species is largely unexplored. Here we demonstrate that Xanthomonas produce a highly conserved single-stranded DNA-binding protein (SSB(X)) that elicits HR in tobacco as by harpin Hpa1. SSB(X), like Hpa1, is an acidic, glycine-rich, heat-stable protein that lacks cysteine residues. SSB(X)-triggered HR in tobacco, as by Hpa1, is characterized by the oxidative burst, the expression of HR markers (HIN1, HSR203J), pathogenesis-related genes, and callose deposition. Both SSB(X)- and Hpa1-induced HRs can be inhibited by general metabolism inhibitors actinomycin D, cycloheximide, and lanthanum chloride. Furthermore, those HRs activate the expression of BAK1 and BIK1 genes that are essential for induction of mitogen-activated protein kinase (MAPK) and salicylic acid pathways. Once applied to plants, SSB(X) induces resistance to the fungal pathogen Alternaria alternata and enhances plant growth. When ssb(X)was deleted in X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak in rice, the resulting ssb(Xoc)mutant was reduced in virulence and bacterial growth in planta, but retained its ability to trigger HR in tobacco. Interestingly, ssb(Xoc)contains an imperfect PIP-box (plant-inducible promoter) and the expression of ssb(Xoc)is regulated by HrpX, which belongs to the AraC family of transcriptional activators. Immunoblotting evidence showed that SSB(x) secretion requires a functional type-III secretion system as Hpa1 does. This is the first report demonstrating that Xanthomonas produce a highly-conserved SSB(X) that functions as a harpin-like protein for plant immunity.

Overexpression of keratin 17 is associated with poor prognosis in epithelial ovarian cancer.

The aim of this study was to investigate the association between keratin 17 (K17) expression and the clinicopathological features of patients with epithelial ovarian cancer (EOC). K17 expression was detected by real-time quantitative RT-PCR in EOC and adjacent noncancerous tissues. In addition, K17 expression was analyzed by immunohistochemistry in 104 clinicopathologically characterized EOC cases. The expression levels of K17 mRNA and protein in EOC tissues were both significantly higher than those in noncancerous tissues. In addition, positive expression of K17 correlated with the clinical stage (p=0.001). Furthermore, Kaplan-Meier survival analysis showed that a high expression level of K17 resulted in a significantly poor prognosis of EOC patients. Multivariate analysis revealed that EOC expression level was an independent prognostic parameter for the overall survival rate of EOC patients. Our data are the first to suggest that increased K17 expression in EOC is significantly associated with aggressive progression and poor prognosis. K17 may be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of EOC.

Lipoic acid attenuates the expression of adhesion molecules by increasing endothelial nitric-oxide synthase activity.

We tried to study the possible effects of lipoic acid (LA) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. Intercellular adhesion molecule-1 (ICAM-1) expression and endothelial nitric oxide synthase (eNOS) activity were determined after endothelial cells were exposed to high glucose in the absence and presence of LA. Coincubation of endothelial cells with high glucose for 24 h resulted in a significant increase of monocyte-endothelial cell adhesion and the expression of ICAM-1 (P < 0.01). These effects were abolished by LA and LA significantly increased eNOS activities (P < 0.01). These findings suggested that LA may play a role in inhibiting expression of adhesion molecules by increasing eNOS activities.

Neuropeptide Y Gene Promoter -399T/C Polymorphism Increases Risk of Ischemic Stroke.

BACKGROUND: Several genetic factors underlying ischemic stroke have been identified. Variants of Neuropeptide Y (NPY), whose product plays diverse roles in modulating physiological functions, have been associated with an increased risk of ischemic stroke in South Korean individuals. AIMS: We explored the association between a polymorphism in the NPY gene promoter at position -399 and the risk of ischemic stroke in Han Chinese. STUDY DESIGN: Case-control study. METHODS: The polymorphism -399T/C in the promoter of NPY was analysed in 500 patients with ischemic stroke and 500 healthy individuals by amplification and sequencing of this region. Non-conditional logistic regression was used to analyse association between genotypes and the risk of ischemic stroke. RESULTS: Genotype and allele frequencies differed significantly between the ischemic stroke and control groups (P<0.05). Additionally, compared to stroke patients with the TT genotype, those with the CC genotype had a 1.7-times higher risk of ischemic stroke (OR=1.739, 95%CI=1.201-2.520, P=0.003), especially for those who were over 60 years old or male. Individuals with the TC genotype did not have an increased risk of ischemic stroke (P>0.05). CONCLUSION: The -399T/C polymorphism of the NPY gene is associated with ischemic stroke in Han Chinese individuals, and the CC genotype may be a risk factor for ischemic stroke.

Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea.

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I­IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II­III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein.Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7­78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

Protective roles of quercetin in acute myocardial ischemia and reperfusion injury in rats.

Myocardial ischemia and reperfusion (MI/R) is associated with an intense inflammatory reaction, which may lead to myocyte injury. In this study, we investigated the effect of quercetin, an inhibitor of c-Jun N-terminal kinase on ischemia/reperfusion injury in isolated rat hearts. Rat models of MI/R were induced by coronary occlusion followed by reperfusion, treatment of rats with quercetin (1.0 mg/kg, i.v.) induced a significant reduction of infarct volume and improvements in baseline hemodynamic abnormalities (P < 0.05). Quercetin treatment also attenuated the expression of both TNF-alpha (TNF-α) and interleukin-10 (IL-10) and lowered the serum levels of inflammatory cytokine (P < 0.05). These findings suggested that quercetin treatment significantly attenuated MI/R injury primarily through anti-inflammatory effects.

Nitric oxide modulated the expression of DREAM/calsenilin/KChIP3 in inflammatory pain of rats.

Downstream regulatory element antagonistic modulator (DREAM) is a critical transcriptional repressor for pain modulation. The role of nitric oxide (NO) plays in modulating DREAM pain pathway in the periphery is unclear. Therefore, we investigated the role of the NO in modulation of the expression of DREAM in formalin-induced rat inflammatory pain models. Male Sprague-Dawley rats were randomly distributed into four groups: the normal group, formalin test group, Nω-nitro-L-arginine (l-NNA) group, and morphine group. One hundred microliters of 2.5 % formalin was injected into the plantar surface of the right hindpaw of rats. l-NNA (40 nmol/L) and morphine (40 nmol/L) were injected intrathecally in the hindpaw before formalin injection. The nociceptive behavioral reaction was recorded. After the formalin test, the expression of DREAM mRNA and protein in the spinal cord of the four groups were measured. The nociceptive reaction induced by injection of formalin exhibited two phases. Morphine and l-NNA significantly decreased pain scores of the second phase. The expression of DREAM was significantly increased in the rat spinal cord after formalin-induced pain. Morphine significantly upregulated the expression of DREAM, and the formalin-induced upregulation was significantly attenuated by l-NNA. NO may play an important role in the DREAM pathway modulation of inflammatory pain.

Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae.

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.

EcpA, an extracellular protease, is a specific virulence factor required by Xanthomonas oryzae pv. oryzicola but not by X. oryzae pv. oryzae in rice.

Previously, 12 protease-deficient mutants of the Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain were recovered from a Tn5-tagged mutant library. In the current study, the Tn5 insertion site in each mutant was mapped. Mutations in genes encoding components of the type II secretion apparatus, cAMP regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme and extracellular protease (ecpA(Xoc)) either partially or completely abolished extracellular protease activity (ECPA) and reduced virulence in rice. Transcription of ecpA(Xoc) was induced in planta in all the mutants except RΔecpA. Complementation of RΔecpA with ecpA(Xoc) in trans restored ECPA, virulence and bacterial growth in planta. Purified EcpA(Xoc) induced chlorosis- and necrosis-like symptoms similar to those induced by the pathogen when injected into rice leaves. Heterologous expression of ecpA(Xoc) conferred ECPA upon the vascular bacterium X. oryzae pv. oryzae (Xoo) and upon non-pathogenic Escherichia coli. Genetic analysis demonstrated that the C-terminal residues of EcpA in Xoo PXO99(A) and Xoc RS105 are different, and a frame shift in ecpA(Xoo) may explain the absence of EcpA activity in Xoo. Collectively, these results suggest that EcpA(Xoc) is a tissue-specific virulence factor for Xoc but not Xoo, although the two pathovars are closely related bacterial pathogens of rice.

Fructose-bisphophate aldolase exhibits functional roles between carbon metabolism and the hrp system in rice pathogen Xanthomonas oryzae pv. oryzicola.

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N(9)-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.

Identification of seven Xanthomonas oryzae pv. oryzicola genes potentially involved in pathogenesis in rice.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, an emerging and destructive disease worldwide. Identification of key virulence factors is a prerequisite for understanding the pathogenesis of Xoc. In this study, a Tn5-tagged mutant library of Xoc strain RS105 was screened on rice, and 27 Tn5 mutants were identified that were either non-pathogenic or showed reduced virulence in rice. Fourteen of the non-pathogenic mutants were also unable to elicit the hypersensitive response (HR) in tobacco and were designated Pth(-)/HR(-) mutants; 13 mutants showed attenuated virulence and were able to induce an HR (Vir(-)/HR(+)). Sequence analysis of the Tn5-tagged genes indicated that the 14 Pth(-)/HR(-) mutants included mutations in hrcC, hrcT, hrcV, hpaP, hrcQ, hrpF, hrpG and hrpX. The 13 Vir(-)/HR(+) mutants included tal-C10c-like (a transcriptional activator-like TAL effector), rpfC (regulator of pathogenicity factors), oxyR (oxidative stress transcriptional regulator), dsbC (disulfide isomerase), opgH (glucan biosynthesis glucosyltransferase H), rfbA (glucose-1-phosphate thymidylyltransferase), amtR (aminotransferase), purF (amidophosphoribosyltransferase), thrC (threonine synthase), trpA (tryptophan synthase alpha subunit) and three genes encoding hypothetical proteins (Xoryp_02235, Xoryp_00885 and Xoryp_22910). Collectively, the 27 Tn5 insertions are located in 21 different open reading frames. Bacterial growth and in planta virulence assays demonstrated that opgH, purF, thrC, trpA, Xoryp_02235, Xoryp_00885 and Xoryp_22910 are candidate virulence genes involved in Xoc pathogenesis. Reduced virulence in 13 mutants was restored to wild-type levels when the cognate gene was introduced in trans. Expression profiles demonstrated that the seven candidate virulence genes were significantly induced in planta, although their roles in Xoc pathogenesis remain unclear.

A novel antimicrobial protein for plant protection consisting of a Xanthomonas oryzae harpin and active domains of cecropin A and melittin.

Discoveries about antimicrobial peptides and plant defence activators have made possible the de novo and rational design of novel peptides for use in crop protection. Here we report a novel chimeric protein, Hcm1, which was made by linking the active domains of cecropin A and melittin to the hypersensitive response (HR)-elicitor Hpa1 of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak. The resulting chimeric protein maintained not only the HR-inducing property of the harpin, but also the antimicrobial activity of the cecropin A-melittin hybrid. Hcm1 was purified from engineered Escherichia coli and evaluated in terms of the minimal inhibitory concentration (MIC) and the 50% effective dose (ED(50)) against important plant pathogenic bacteria and fungi. Importantly, the protein acted as a potential pesticide by inducing disease resistance for viral, bacterial and fungal pathogens. This designed drug can be considered as a lead compound for use in plant protection, either for the development of new broad-spectrum pesticides or for expression in transgenic plants.