Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plants Arabidopsis and Nicotiana benthamiana (N. benthamiana). Like numerous Gram-negative bacterial pathogens of ...Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plants Arabidopsis and Nicotiana benthamiana (N. benthamiana). Like numerous Gram-negative bacterial pathogens of animals and plants, Pst DC3000 exploits the conserved type III secretion system (TTSS) to deliver multiple virulence effectors directly into the host cells. Type III effectors (T3Es) collectively participate in causing disease, by mechanisms that are not well clarity. Elucidating the virulence function of individual effector is fundamental for understanding bacterial infection of plants. Here, we focused on studying one of these effectors, HopAA1-1, and analyzed its potential function and subcellular localization in N. benthamiana. Using an Agrobacterium-mediated transient expression system, we found that HopAA1-1 can trigger domain-dependent cell death in N. benthamiana. The observation using confocal microscopy showed that the YFP-tagged HopAA1-1 localizes to diverse cellular components containing nucleus, cytoplasm and cell membrane, which was demonstrated through immunoblot analysis of membrane fractionation and nuclear separation. Enforced HopAA1-1 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that HopAA1-1-induced cell death in N. benthamiana is suppressed in the nucleus but enhanced in the cytoplasm. Our research is lay a foundation for revealed the molecular pathogenesis of Pseudomonas syringae pv. tomato.展开更多
Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expres...Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expression of these mRNAs is increased in malignant tumors and strongly induced in different cancer cell lines by hypoxia inducible factor (HIF) through active HIF binding sites in promoter region of PFKFB-4 and PFKFB-3 genes. Moreover, the expression and hypoxia responsibility of PFKFB-4 and PFKFB-3 was also shown for pancreatic (Panc1, PSN-1, and MIA PaCa-2) as well as gastric (MKN45 and NUGC3) cancer cells. At the same time, their basal expression level and hypoxia responsiveness vary in the different cells studied: the highest level of PFKFB-4 protein expression was found in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with a stronger response to hypoxia in the pancreatic cancer cell line. Overexpression of different PFKFB in pancreatic and gastric cancer cells under hypoxic condition is correlated with enhanced expression of vascular endothelial growth factor (VEGF) and Glut1 mRNA as well as with increased level of HIF-1α protein. Increased expression of different PFKFB genes was also demonstrated in gastric, lung, breast, and colon cancers as compared to corresponding non-malignant tissue counterparts from the same patients, being more robust in the breast and lung tumors. Moreover, induction of PFKFB-4 mRNA expression in the breast and lung cancers is stronger than PFKFB-3 mRNA. The levels of both PFKFB-4 and PFKFB-3 proteins in non-malignant gastric and colon tissues were more pronounced than in the non-malignant breast and lung tissues. It is interesting to note that Panc1 and PSN-1 cells transfected with dominant/negative PFKFB-3 (dnPFKFB-3) showed a lower level of endogenous PFKFB-3, PFKFB-4, and VEGF mRNA expressions as well as a decreased proliferation rate of these cells. Moreover, a similar effect had dnPFKFB-4. In conclusion, there is strong evidence that PFKFB-4 and PFKFB-3 isoenzymes are induced under hypoxia in pancreatic and other cancer cell lines, are overexpressed in gastric, colon, lung, and breast malignant tumors and undergo changes in their metabolism that contribute to the proliferation and survival of cancer cells. Thus, targeting these PFKFB may therefore present new therapeutic opportunities.展开更多
文摘Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plants Arabidopsis and Nicotiana benthamiana (N. benthamiana). Like numerous Gram-negative bacterial pathogens of animals and plants, Pst DC3000 exploits the conserved type III secretion system (TTSS) to deliver multiple virulence effectors directly into the host cells. Type III effectors (T3Es) collectively participate in causing disease, by mechanisms that are not well clarity. Elucidating the virulence function of individual effector is fundamental for understanding bacterial infection of plants. Here, we focused on studying one of these effectors, HopAA1-1, and analyzed its potential function and subcellular localization in N. benthamiana. Using an Agrobacterium-mediated transient expression system, we found that HopAA1-1 can trigger domain-dependent cell death in N. benthamiana. The observation using confocal microscopy showed that the YFP-tagged HopAA1-1 localizes to diverse cellular components containing nucleus, cytoplasm and cell membrane, which was demonstrated through immunoblot analysis of membrane fractionation and nuclear separation. Enforced HopAA1-1 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that HopAA1-1-induced cell death in N. benthamiana is suppressed in the nucleus but enhanced in the cytoplasm. Our research is lay a foundation for revealed the molecular pathogenesis of Pseudomonas syringae pv. tomato.
文摘Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expression of these mRNAs is increased in malignant tumors and strongly induced in different cancer cell lines by hypoxia inducible factor (HIF) through active HIF binding sites in promoter region of PFKFB-4 and PFKFB-3 genes. Moreover, the expression and hypoxia responsibility of PFKFB-4 and PFKFB-3 was also shown for pancreatic (Panc1, PSN-1, and MIA PaCa-2) as well as gastric (MKN45 and NUGC3) cancer cells. At the same time, their basal expression level and hypoxia responsiveness vary in the different cells studied: the highest level of PFKFB-4 protein expression was found in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with a stronger response to hypoxia in the pancreatic cancer cell line. Overexpression of different PFKFB in pancreatic and gastric cancer cells under hypoxic condition is correlated with enhanced expression of vascular endothelial growth factor (VEGF) and Glut1 mRNA as well as with increased level of HIF-1α protein. Increased expression of different PFKFB genes was also demonstrated in gastric, lung, breast, and colon cancers as compared to corresponding non-malignant tissue counterparts from the same patients, being more robust in the breast and lung tumors. Moreover, induction of PFKFB-4 mRNA expression in the breast and lung cancers is stronger than PFKFB-3 mRNA. The levels of both PFKFB-4 and PFKFB-3 proteins in non-malignant gastric and colon tissues were more pronounced than in the non-malignant breast and lung tissues. It is interesting to note that Panc1 and PSN-1 cells transfected with dominant/negative PFKFB-3 (dnPFKFB-3) showed a lower level of endogenous PFKFB-3, PFKFB-4, and VEGF mRNA expressions as well as a decreased proliferation rate of these cells. Moreover, a similar effect had dnPFKFB-4. In conclusion, there is strong evidence that PFKFB-4 and PFKFB-3 isoenzymes are induced under hypoxia in pancreatic and other cancer cell lines, are overexpressed in gastric, colon, lung, and breast malignant tumors and undergo changes in their metabolism that contribute to the proliferation and survival of cancer cells. Thus, targeting these PFKFB may therefore present new therapeutic opportunities.
