A modified transient gene expression protocol for subcellular protein localization analysis in rice

Yiming Wang,Jingni Wu,Sang Gon Kim,Ju Soon Yoo,Ravi Gupta,제병일,Jong‑Seong Jeon,Ki‑Hong Jung,Yu‑Jin Kim,KyuYoungKang,김선태 한국식물생명공학회 2020 Plant biotechnology reports Vol.14 No.1

Transient protein expression is a useful technique for investigating the protein localization and functional analysis of stress responses in rice plants. Currently, available methods for in planta transient expression analysis include Agrobacteriummediated transformation, protoplast transformation using polyethylene glycol or electroporation, and biolistic bombardment expression which have several disadvantages and are not well suited for the rice. Therefore, development of a method for rapid and efficient analysis of protein expression, subcellular localization, pathogen effector screening, and protein–protein interaction in rice is required. We developed a protocol for in planta gene expression analysis in sliced rice sheath cells by modifying and optimizing the biolistic particle bombardment technique. By obtaining thin sections (~400 μm) of rice sheath cells, auto-fluorescence from chlorophyll was eliminated. This system was validated through the localization of marker genes specifically expressed in nuclei, plasma membranes, and tonoplast. In addition, high transformation efficiency of 30% was achieved. Therefore, this protocol provides a new and rapid method for transient gene expression assay in rice. Protein secretion was examined in rice sheath cells using predicted secretory proteins from rice blast fungus, indicating that this method is applicable to plant–microbe interaction studies. The transient expression protocol established here is well optimized for protein localization, secretion, and host–pathogen protein interaction studies in rice. A typical experiment can be completed in three days.

Self-Adaptive Power Flow Optimization Method for Multi-Terminal SNOP Considering the Difference of Risk of Node Voltage beyond Limit

Yiming Xu,Zhiqiang Wang,Zheng Fang,Sichao Xun,Guirong Hu,Yusheng Shen,Hongyi Xiao,Dongning Wu 대한전기학회 2019 Journal of Electrical Engineering & Technology Vol.14 No.5

Soft Normally Open Point (SNOP) is a power electronic device used widely in power distribution network. It can solve many problems by adjusting reactive power compensation and active power transmission. The uncertainty and fluctuation of distributed generation (DG) may cause many problems to the distribution network. In order to balance the safety and economy in power flow optimization better, a self-adaptive power flow optimization method for active distribution network with multi-terminal SNOP considering difference of the risk of node voltage beyond limit is put forward. It takes both weighted voltage deviation and the network loss into objective function to construct the optimal operation model and mainly has two improvements: one is considering initial voltage deviation and real-time penetration of the DG to establish the self-adaptive weight model of the sum of voltage deviation in the objective function; the other is putting forward the weighting strategy for node voltage deviation which decided by the electrical distance, DG position and correlation between source output and load. Then, taking three improved IEEE33 node feeders as an example to verify the validity of the strategy. The results validate that the self-adaptive strategy can reduce the loss and improve the regulation of voltage better and is useful in distribution network under different penetration

Overexpression of a Pathogenesis-Related Protein 10 Enhances Biotic and Abiotic Stress Tolerance in Rice

Wu, Jingni,Kim, Sang Gon,Kang, Kyu Young,Kim, Ju-Gon,Park, Sang-Ryeol,Gupta, Ravi,Kim, Yong Hwan,Wang, Yiming,Kim, Sun Tae The Korean Society of Plant Pathology 2016 Plant Pathology Journal Vol.32 No.6

Pathogenesis-related proteins play multiple roles in plant development and biotic and abiotic stress tolerance. Here, we characterize a rice defense related gene named "jasmonic acid inducible pathogenesis-related class 10" (JIOsPR10) to gain an insight into its functional properties. Semi-quantitative RT-PCR analysis showed up-regulation of JIOsPR10 under salt and drought stress conditions. Constitutive over-expression JIOsPR10 in rice promoted shoot and root development in transgenic plants, however, their productivity was unaltered. Further experiments exhibited that the transgenic plants showed reduced susceptibility to rice blast fungus, and enhanced salt and drought stress tolerance as compared to the wild type. A comparative proteomic profiling of wild type and transgenic plants showed that overexpression of JIOsPR10 led to the differential modulation of several proteins mainly related with oxidative stresses, carbohydrate metabolism, and plant defense. Taken together, our findings suggest that JIOsPR10 plays important roles in biotic and abiotic stresses tolerance probably by activation of stress related proteins.

Proteasome Inhibitors Affect Appressorium Formation and Pathogenicity of the Rice Blast Fungus, Magnaporthe oryzae

Yiming Wang,김상곤,Jingni Wu,Seok Yu,강규영,김선태 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.3

Previously, we identified the 20S proteasome α-subunit of Magnaporthe oryzae (M. oryzae) induced during appressorium formation, and detected an increase in multiple protein ubiquitination during the early appressorium formation process (Kim et al., 2004). In this study, we further attempted to determine whether the proteasome is involved in the appressorium formation of M. oryzae both in vitro and in planta, using proteasome inhibitors. A significant increase in 20S proteasome during fungal germination and appressorium formation was observed using Western blot analysis with 20S proteasome antibody, demonstrating that proteasomemediated protein degradation was involved in appressorium formation. Pharmacological analysis using proteasome inhibitors, MG-132, proteasome inhibitor I (PI) and proteasome inhibitor II (PII) revealed that germination and appressorium formation were delayed for 4 to 6 h on rice leaf wax-coated plates. Similarly, the treatment of proteasome inhibitors with fungal conidia on the rice leaf surface delayed appressorium formation and host infection processes as well. Additionally, fungal pathogenicity was strongly reduced at 4 days’ postfungal infection. These data indicated that the fungal 20S proteasome might be involved in the pathogenicity of M. oryzae by the suppression of germination and appressorium formation.

Synthesis and Structure of a Cadmium Selenite-Sulfate Cd4(SeO3)2(SO4)2

Yiming Xie*,Jihuai Wu 대한화학회 2007 Bulletin of the Korean Chemical Society Vol.28 No.8

Expression Analysis of Oryza sativa Ascorbate Peroxidase 1 (OsAPx1) in Response to Different Phytohormones and Pathogens

Yiming Wang(왕이밍),Jingni Wu(우징니),Young Whan Choi(최영환),Tae Hwan Jun(전태환),Soon Wook Kwon(권순욱),In Soo Choi(최인수),Yong Chul Kim(김용철),Ravi Gupta(라비굽타),Sun Tae Kim(김선태) 한국생명과학회 2015 생명과학회지 Vol.25 No.10

본 논문에서 벼 ascorbate peroxidase (OsAPx1) 유전자의 발현 분석을 Northern과 Western 분석을 통하여 유묘에서는 뿌리, 정단분열조직(shoot apical meristem, SAM), 잎 보다는 잎집에서 더 많이 발현되는 것을 확인하였다. 성숙된 조직에서는 OsAPx1 유전자가 잎을 제외하고는 뿌리, 줄기, 꽃에서 강하게 발현되었다. 또한 이 OsAPx1 유전자는 벼 곰팡이 병원균인 벼 도열병 및 세균성 병원균인 흰빛잎마름병에도 반응하였고 특히 흥미있게도 OsAPx1 유전자는 식물호르몬에 대해서 서로 다르게 발현 양상을 보였다. 이 유전자는 자스몬산(JA)에 대해서는 강한 발현을 보였지만 반대로 살리실산(SA) 및 ABA와 같이 처리된 세포에서는 강한 발현 억제를 보였다. 이는 이 유전자가 JA에는 반응하지만 SA와 ABA하고는 서로 길항작용을 하는 것으로 보인다. 근연관계분석을 통하여 OsAPx1유전자가 애기장대의 AtAPx1 와 거의 유사하여 AtAPx1 결손 라인을 가지고 표현형 조사를 실시하였다. 그 결과, 외부에서 H₂O₂를 처리하였을 때에 O₂<SUP>-</SUP> 와 H₂O₂의 축적이 wild type과 비교하여 AtAPx1 결손 라인에서는 현저히 높았다. 따라서 본 연구를, 통하여 OsAPx1 유전자는 벼에서 산화 환원 균형 유지를 통하여 다양한 세포 분화발달 및 병원균 방어에도 관여하며 이 유전자의 발현은 JA의 신호전달에 의해서 매개되는 것으로 예상이 된다. We have isolated and characterized an ascorbate peroxidase (APx) gene, OsAPx1 from rice. Northern and Western blot analyses indicated that at young seedling stage, OsAPx1 mRNA was expressed highly in root, shoot apical meristem (SAM) and leaf sheath than leaf. In mature plant, OsAPx1 gene expressed highly in root, stem and flower but weakly in leaf. OsAPx1 gene and protein expression level was induced in leaves inoculated with Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Phytohormones treatment showed that OsAPx1 was up-regulated by jasmonic acid (JA), but was down regulated by ABA and SA co-treatments with JA, resulting that they have antagonistic effect on pathogen responsive OsAPx1 expression. Phylogenetic analysis illustrated that Arabidopsis AtAPx1 has a close relationship with OsAPx1. In AtAPx1 knock out lines, the accumulation of O₂<SUP>-</SUP> and H₂O₂ are all highly detected than wild type, revealing that the high concentration of exogenous H₂O₂ cause the intercellular superoxide anion and hydrogen peroxide accumulation in AtAPx1 knockout plant. These results suggested that OsAPx1 gene may be associated with the pathogen defense cascades as the mediator for balancing redox state by acting ROS scavenger and is associated with response to the pathogen defense via Jasmonic acid signaling pathway.

A secreted chitinase‐like protein (OsCLP) supports root growth through calcium signaling in <i>Oryza sativa</i>

Wu, Jingni,Wang, Yiming,Kim, Sang Gon,Jung, Ki‐,Hong,Gupta, Ravi,Kim, Joonyup,Park, Younghoon,Kang, Kyu Young,Kim, Sun Tae Blackwell Publishing Ltd 2017 Physiologia plantarum Vol.161 No.2

<P>Chitinases belong to a conserved protein family and play multiple roles in defense, development and growth regulation in plants. Here, we identified a secreted chitinase‐like protein, OsCLP, which functions in rice growth. A T‐DNA insertion mutant of OsCLP (<I>osclp</I>) showed significant retardation of root and shoot growth. A comparative proteomic analysis was carried out using root tissue of wild‐type and the <I>osclp</I> mutant to understand the OsCLP‐mediated rice growth retardation. Results obtained revealed that proteins related to glycolysis (phosphoglycerate kinase), stress adaption (chaperonin) and calcium signaling (calreticulin and CDPK1) were differentially regulated in <I>osclp</I> roots. Fura‐2 molecular probe staining, which is an intracellular calcium indicator, and inductively coupled plasma‐mass spectrometry (ICP‐MS) analysis suggested that the intracellular calcium content was significantly lower in roots of <I>osclp</I> as compared with the wild‐type. Exogenous application of Ca<SUP>2+</SUP> resulted in successful recovery of both primary and lateral root growth in <I>osclp</I>. Moreover, overexpression of <I>OsCLP</I> resulted in improved growth with modified seed shape and starch structure; however, the overall yield remained unaffected. Taken together, our results highlight the involvement of OsCLP in rice growth by regulating the intracellular calcium concentrations.</P>

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Yiming Wang,Soon Jae Kwon,Jingni Wu,최재영,이용환,Ganesh Kumar Agrawal,Shigeru Tamogami,Randeep Rakwal,Sang Ryeol Park,김범기,정기홍,강규영,김상곤,김선태 한국식물병리학회 2014 Plant Pathology Journal Vol.30 No.4

Rice blast disease caused by Magnaporthe oryzae is oneof the most serious diseases of cultivated rice (Oryzasativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, weutilized the transcriptome analysis approach using a300 K tilling microarray to rice leaves infected withcompatible and incompatible M. oryzae strains. Priorto the microarray experiment, total RNA was validatedby measuring the differential expression of rice defenserelatedmarker genes (chitinase 2, barwin, PBZ1, andPR-10) by RT-PCR, and phytoalexins (sakuranetinand momilactone A) with HPLC. Microarray analysisrevealed that 231 genes were up-regulated (>2 foldchange, p < 0.05) in the incompatible interaction comparedto the compatible one. Highly expressed genes were functionally characterized into metabolic processesand oxidation-reduction categories. The oxidativestress response was induced in both early and laterinfection stages. Biotic stress overview from MapMananalysis revealed that the phytohormone ethylene aswell as signaling molecules jasmonic acid and salicylicacid is important for defense gene regulation. WRKYand Myb transcription factors were also involved in signaltransduction processes. Additionally, receptor-likekinases were more likely associated with the defenseresponse, and their expression patterns were validatedby RT-PCR. Our results suggest that candidate genes,including receptor-like protein kinases, may play a keyrole in disease resistance against M. oryzae attack

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Wang, Yiming,Kwon, Soon Jae,Wu, Jingni,Choi, Jaeyoung,Lee, Yong-Hwan,Agrawal, Ganesh Kumar,Tamogami, Shigeru,Rakwal, Randeep,Park, Sang-Ryeol,Kim, Beom-Gi,Jung, Ki-Hong,Kang, Kyu Young,Kim, Sang Gon,K The Korean Society of Plant Pathology 2014 Plant Pathology Journal Vol.30 No.4

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Controlled Ambipolar Doping and Gate Voltage Dependent Carrier Diffusion Length in Lead Sulfide Nanowires

Yang, Yiming,Li, Jiao,Wu, Hengkui,Oh, Eunsoon,Yu, Dong American Chemical Society 2012 Nano letters Vol.12 No.11

<P>We report a simple, controlled doping method for achieving n-type, intrinsic, and p-type lead sulfide (PbS) nanowires (NWs) grown by chemical vapor deposition without introducing any impurities. A wide range of carrier concentrations is realized by adjusting the ratio between the Pb and S precursors. The field effect electron mobility of n-type PbS NWs is up to 660 cm<SUP>2</SUP>/(V s) at room temperature, in agreement with a long minority carrier diffusion length measured by scanning photocurrent microscopy (SPCM). Interestingly, we have observed a strong dependence of minority carrier diffusion length on gate voltage, which can be understood by considering a carrier concentration dependent recombination lifetime. The demonstrated ambipolar doping of high quality PbS NWs opens up exciting avenues for their applications in photodetectors and photovoltaics.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/nalefd/2012/nalefd.2012.12.issue-11/nl303294k/production/images/medium/nl-2012-03294k_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/nl303294k'>ACS Electronic Supporting Info</A></P>