Wen-Jen Chen, Tzu-Yen Kuo , Feng-Chia Hsieh, Pi-Yu Chen, Chang-Sheng Wang, Yu-Ling Shih, Ying-Mi Lai, Je-Ruei Liu, Yu-Liang Yang, Ming-Che Shih. (2016) Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis Scientific Reports 6, Article number: 32950 doi:10.1038/srep32950
Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.
Kuen-Jin Tsai, Chih-Yu Lin, Chen-Yun Ting, Ming-Che Shih*(2016) Ethylene-regulated glutamate dehydrogenase fine-tunes metabolism during anoxia-reoxygenation in Arabidopsis. Plant Physiology doi: http://dx.doi.org/10.1104/pp.16.00985.
Ethylene is an essential hormone in plants that is involved in low oxygen and reoxygenation responses. As a key transcription factor in ethylene signaling, ETHYLENE INSENSITIVE 3 (EIN3) activates targets that trigger various responses. However, most of these targets are still poorly characterized. Through analyses of our microarray data and the published Arabidopsis EIN3 ChIP-seq dataset, we inferred the putative targets of EIN3 during anoxia-reoxygenation. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further studies for its role in TCA cycle replenishment. We demonstrated that both GDH1 and GDH2 are induced during anoxia and reoxygenation and that this induction is mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the level of GDH during anoxia-reoxygenation decreased in the ethylene insensitive mutants ein2-5 and ein3eil1. Global metabolite analysis indicated that the deamination activity of GDH might regenerate 2-oxoglutarate that is a co-substrate that facilitates the breakdown of alanine by alanine aminotransferase (AlaAT) when reoxygenation occurs. Moreover, ineffective TCA cycle replenishment, disturbed carbohydrate metabolism, reduced phytosterol biosynthesis, and delayed energy regeneration were found in gdh1gdh2 and ethylene mutants during reoxygenation. Taken together, these data illustrate the essential role of EIN3-regulated GDH activity in metabolic adjustment during anoxia-reoxygenation.
Hsing-Yi Cho, Tuan-Nan Wen, Ying-Tsui Wang, and Ming-Che Shih. (2016) Quantitative Phosphoproteomics of Protein Kinase SnRK1 regulated protein phosphorylation in Arabidopsis under Submergence. J. Exp. Bot. (2016)doi: 10.1093/jxb/erw107
SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of plant stress and energy starvation signaling-related regulatory pathways. We observed in this study that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1K48M) had lower tolerance to submergence than the wild-type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in energy starvation signaling triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3 h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1K48M under submergence. These proteins are involved mainly in sugar synthesis and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under different abiotic stresses, was disrupted in the SnRK1.1K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1.. We further showed that the energy conservation was disrupted in SnRK1.1K48M, mpk6 and PTP1S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signaling factors related to submergence.
Chin-Hsien Tsai, Sheue-Fen Tzeng, Tai-Kuang Chao, Chia-Yun Tsai, Yu-Chih Yang, Ming-Ting Lee, Jiuan-Jiuan Hwang, Yu-Ching Chou, Mong-Hsun Tsai, Tai-Lung Cha and Pei-Wen Hsiao* (2016) Metastatic progression of prostate cancer is mediated by autonomous binding of galectin-4-O-glycans to cancer cells. Cancer Research 76(19); 5756-67, 2016
Metastatic prostate cancer (PCa) continues to pose a difficult therapeutic challenge. PCa progression is associated with aberrant O-glycosylation of cancer cell surface receptors, but the functional impact of such events are uncertain. Here we report spontaneous metastasis of human PCa xenografts which express high levels of galectin-4 along with genetic signatures of EGFR-HER2 signaling and O-glycosylation. Galectin-4 expression in clinical specimens of PCa correlated with poor patient survival. Galectin-4 binding to multiple receptor tyrosine kinases stimulated their autophosphorylation, activated expression of pERK, pAkt, fibronectin and Twist1, and lowered expression of E-cadherin, thereby faciliating epithelial-mesenchyme transition, invasion and metastasis. In vivo investigations established that galectin-4 expression enabled PCa cells to repopulate tumors in orthotopic and heterotopic tissues. Notably, these effects of galectin-4 relied upon O-glycosylation mediated by C1GALT1, a galactosyltransferase implicated in other cancers. Parallel changes in galectin-4 and O-glycosylation triggered aberrant receptor signaling and more aggressive invasive character in PCa cells, which through better survival in the circulation also contributed to the bulk cell progeny of distal tumors. Our findings establish galectin-4 and C1GALT1-mediated glycosylation in a signaling axis that is activated during CaP progression, with implications for therapeutic targeting of advanced metastatic disease.
M. Tsednee, Y.-C. Huang, Y.-R. Chen and K.-C. Yeh* (2016) Identification of metal species by ESI-MS/MS through release of free metals from the corresponding metal-ligand complexes. Scientific Reports 6, 26785; doi: 10.1038/srep26785
Our manuscript describes a new approach for the identification of metal species using ESI-MS/MS through releasing the free metals from the corresponding metal-ligand complexes. The approach has potential for the accurate and simultaneous identifications of different metal species in biological samples. We also showed the applicability of the approach in several biological samples and in different MS instruments. The methodology provides an alternative and quick procedure for the identification of metal species in biological ligands.
Tzu-Yin Liu, Teng-Kuei Huang, Shu-Yi Yang, Yu-Ting Hong, Sheng-Min Huang, Fu-Nien Wang, Su-Fen Chiang, Shang-Yueh Tsai, Wen-Chien Lu and Tzyy-Jen Chiou* (2016) Identification of plant vacuolar transporters mediating phosphate storage Nature Communications (2016) 7: 11095
Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. Here we demonstrate that the SPX-MFS proteins, designated as PHOSPHATE TRANSPORTER 5 family (PHT5), also named Vacuolar Phosphate Transporter (VPT), function as vacuolar Pi transporters. Based on 31P-magnetic resonance spectroscopy analysis, Arabidopsis pht5;1 loss-of-function mutants accumulate less Pi and exhibit a lower vacuolar-to-cytoplasmic Pi ratio than controls. Conversely, overexpression of PHT5 leads to massive Pi sequestration into vacuoles and altered regulation of Pi starvation-responsive genes. Furthermore, we show that heterologous expression of the rice homologue OsSPX-MFS1 mediates Pi influx to yeast vacuoles. Our findings show that a group of Pi transporters in vacuolar membranes regulate cytoplasmic Pi homeostasis and are required for fitness and plant growth.
J.-C. Lo, M. Tsednee, Y.-C. Lo, S.-C. Yang, J.-M. Hu, K. Ishizaki, T. Kohchi, D.-C. Lee and K.-C. Yeh* (2016) Evolutionary analysis of Fe acquisition system in Marchantia polymorpha. New Phytologist doi: 10.1111/nph.13922
Fe is essential for plant growth but toxic in excess. To acquire appropriate Fe, vascular plants have developed two unique strategies in sporophyte generation, the reduction-based Strategy I of non-graminaceous plants for Fe2+ and the chelation-based Strategy II of graminaceous plants for Fe3+. Recent studies have also shown that algae can take up both Fe2+ and Fe3+. However, the mechanism of Fe uptake in bryophytes the earliest diverging branch of land plants and dominant in gametophyte generation, is less clear. In this study, we found that a nonvascular liverwort, Marchantia polymorpha, used Strategy I for Fe acquisition. This system may have been acquired in the common ancestor of land plants and co-opted from the gametophyte to sporophyte generation in the evolution of land plants. This finding fills the gap in knowledge about the differences in Fe acquisition mechanisms in the alga and land plants.
Tien-Jen Lin, Hsin-Ting Lin, Wei-Ting Chang, Pradeep Mitapalli. S, Pei-Wen Hsiao, Shu-Yi Yin* and Ning-Sun Yang*(2015)Shikonin-enhanced cell immunogenicity of tumor vaccine is mediated by the differential effects of DAMP components.Mol Cancer. 2015 Sep 24;14(1):174. doi: 10.1186/s12943-015-0435-9.
The dendritic cell (DC)-based cancer vaccine approaches are being actively evaluated for application to cancer immunotherapy. In our previous study, phytochemical shikonin (SK) has been shown to drastically enhance ICD activity in mouse tumor cells treated ex-vivo, and the resultant tumor cell lysate (TCL) can effectively augment such SK-TCL pulsed DC vaccine activity in vivo in anti-tumor activities. For the first part of my dissertation study, I further investigated the specifics and the multi-functional effects of various damaged associated molecular pattern (DAMP) components of the ICD complex for their participation, roles and potential cross talks in activating DCs, as measured by five different functional assays. Among three DAMPs tested, HSP70 and CRT mediate a key role in SK-TCL-induced DC immunity for both CD4+ and CD8+ T cell proliferations in vitro. HSP70 is the most important component, followed by CRT, then HMGB1 in facilitating DC immunity on suppressing metastasis of mouse 4T1 mammary tumors and prolonging survival in test mice. Only HSP70, but not CRT or HMGB1, is effective for the suppression of both granulocytic and monocytic MDSC populations in vivo. Both HSP70 and HMGB1, but not CRT, are essential in activating the expression of three key ICD molecules-associated receptors on test DCs. Each of the three test ICD proteins can exhibit a distinguishable pattern in stimulating the expression of four key chemokines in test DCs. Together, these findings may help formulate new strategies for future cancer vaccine designs.
V. Shanmugam, Y.-W. Wang, M. Tsednee, K. Karunakaran and K.-C. Yeh* (2015) Glutathione plays an essential role in nitric oxide-mediated iron deficiency signaling and iron deficiency tolerance in Arabidopsis. The Plant Journal, DOI: 10.1111/tpj.13011.
In this study, we found that the zir1 mutant, defective in glutathione production, was found to be more sensitive to Fe deficiency than the wild type, and grew poorly in alkaline soil. Furthermore, the key genes involved in Fe uptake are expressed at low levels in zir1. The expression of these genes was positively correlated to the accumulation of nitric oxide (NO) under Fe deficiency. NO was proposed to be a signaling molecule involved in the up-regulation of Fe-uptake-related genes during Fe deficiency. We found that zir1 failed to accumulate NO under Fe deficiency and demonstrated that the NO mediated induction of Fe uptake genes was dependent on glutathione supply in the zir1 mutant. The results provide direct evidence that glutathione plays an essential role in Fe deficiency tolerance and NO-mediated Fe deficiency signaling.
Pei-Shan Chien, Hong Gil Nam and Yet-Ran Chen* (2015) A salt-regulated peptide derived from the CAP superfamily protein negatively regulates Salt-stress tolerance in Arabidopsis J. Exp. Bot. (2015) doi: 10.1093/jxb/erv263
High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance.
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2017/10/16 10:30 AM
Dr. Cris Argueso (Assistant Professor, Department of Bioagricultural Sciences and Pest Management, Colorado State University, USA)
Cytokinin-Induced Priming of the Plant Immune System
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center 2017/10/16 3:30 PM
Dr. Xianlin Han (Professor of Medicine and Biochemistry at the Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, USA)
Lipidomics sheds light on diabetic neuropathy
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center 2017/10/16 2:00 PM
Dr. Stephen Blanksby (Director, Central Analytical Research Facility (CARF), Institute for Future Environments, Queensland University of Technology, Australia)
Advances in mass spectrometry for the identification of lipid isomers
Auditorium A134, Agricultural Technology Building, Agricultural Biotechnology Research Center