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.

Biotic Stress-Responsive Rice Proteome: An Overview

Yiming Wang,김상곤,김선태,Ganesh Kumar Agrawal,Randeep Rakwal,강규영 한국식물학회 2011 Journal of Plant Biology Vol.54 No.4

Biotic stresses affect the plant growth, seed quality, and crop yield. The monocot model rice crop plant is no exception and is affected by a variety of biotic stress factors. High-throughput proteomics approaches are being applied in rice for the past several years to exploit better understanding the biotic stresses-responsive regulatory mechanisms. A large number of proteins responsive to biotic stresses, including pathogens and herbivores, have been cataloged. Cataloged proteins mainly belong to functional categories into metabolism, energy, defense mechanisms, and signaling. To date, majority of these proteins have not been functionally characterized yet,except the pathogen-related 10 protein, PBZ1. This review will briefly summarize and discuss: (1) the proteomicsbased investigation of biotic stress-responsive proteins in rice and (2) increasing importance of proteomics approach in defense biology and engineering the next-generation rice/crop plants.

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

Wang, Yiming,Kim, Sang-Gon,Wu, Jingni,Yu, Seok,Kang, Kyu-Young,Kim, Sun-Tae The Korean Society of Plant Pathology 2011 Plant Pathology Journal Vol.27 No.3

Previously, we identified the 20S proteasome ${\alpha}$-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 proteasome-mediated 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.

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

<i>Magnaporthe oryzae</i>-Secreted Protein MSP1 Induces Cell Death and Elicits Defense Responses in Rice

Wang, Yiming,Wu, Jingni,Kim, Sang Gon,Tsuda, Kenichi,Gupta, Ravi,Park, Sook-Young,Kim, Sun Tae,Kang, Kyu Young APS Press 2016 Molecular plant-microbe interactions Vol.29 No.4

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.

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.

Molecular mechanism of poisoning to honeybees (Apis mellifera L.) after ingestion of Camellia oleifera nectar

Wang Yang,Liu Xiaohua,Wang Yiming,Jin Mingxia,Xie Guai,Zhong Qiuping,Yu Ailin 한국응용곤충학회 2024 Journal of Asia-Pacific Entomology Vol.27 No.1

To explore the molecular mechanism of honeybees (Apis mellifera L.) poisoning death by ingestion of Camellia oleifera nectar, feeding experiments were designed to investigate the poisoning effects. Observed the develop ment process of poisoning symptoms, and collected bees at key experimental nodes for transcriptome sequencing analysis. The results showed that the first poisoning symptom was observed within 0.5 h after ingestion of Camellia oleifera nectar, with death occurring as early as 2 h post-ingestion and all honey bees dying within 4 h. There were four stages in the development of poisoning symptoms: normal flying, crawling (abdominal swelling, inability to fly normally), fluttering (inability to crawl normally), and death. Transcriptome sequencing analyzed the causes of poisoning death indicated that honey bee poisoning occurred with the transduction of dietary restriction signals into cells, resulting in the down-regulated expression gene elements that ensure cell health and inhibit apoptosis, including intracellular NAD+-dependent protein deacetylase sirtuin 1, insulin-like growth factor 1, and heat shock proteins. At the same time, the expression of cysteinyl aspartate specific proteinase 7, which protects programmed cell apoptosis, was also downregulated, resulting in the rapid and disorderly deaths of a large number of cells in individuals in a short period of time, and eventually leading to bees fatality.

Label-free quantitative secretome analysis of <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> highlights the involvement of a novel cysteine protease in its pathogenicity

Wang, Yiming,Gupta, Ravi,Song, Wei,Huh, Hyun-Hye,Lee, So Eui,Wu, Jingni,Agrawal, Ganesh Kumar,Rakwal, Randeep,Kang, Kyu Young,Park, Sang-Ryeol,Kim, Sun Tae Elsevier 2017 Journal of proteomics Vol.169 No.-

<P><B>Abstract</B></P> <P>Bacterial blight, caused by <I>Xanthomonas oryzae</I> pv. <I>oryzae</I> (<I>Xoo</I>), is one of the most devastating diseases resulting in a huge loss of the total rice productivity. The initial interaction between rice and <I>Xoo</I> takes place in the host apoplast and is mediated primarily by secretion of various proteins from both partners. Yet, such secretory proteins remain to be largely identified and characterized. This study employed a label-free quantitative proteomics approach and identified 404 and 323 <I>Xoo</I>-secreted proteins from <I>in vitro</I> suspension-cultured cells and <I>in planta</I> systems, respectively. Gene Ontology analysis showed their involvement primarily in catalytic, transporter, and ATPase activities. Of a particular interest was a <I>Xoo</I> cysteine protease (<I>Xo</I>CP), which showed dramatic increase in its protein abundance <I>in planta</I> upon <I>Xoo</I> interaction with a susceptible rice cultivar. Knock-out mutants of <I>XoCP</I> showed reduced pathogenicity on rice, highlighting its potential involvement in <I>Xoo</I> virulence. Besides, a parallel analysis of <I>in planta</I> rice-secreted proteins resulted in identification of 186 secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. Identified secretory proteins were exploited to shed light on their possible role in the rice-<I>Xoo</I> interaction, and that further deepen our understanding of such interaction.</P> <P><B>Biological significance</B></P> <P> <I>Xanthomonas oryzae</I> pv. <I>oryzae</I> (<I>Xoo</I>), causative agent of bacterial blight disease, results in a huge loss of the total rice productivity. Using a label-free quantitative proteomics approach, we identified 727 <I>Xoo</I>- and 186 rice-secreted proteins. Functional annotation showed <I>Xoo</I> secreted proteins were mainly associated with the catalytic, transporter, and ATPase activities while the rice secreted proteins were mainly associated with the catalytic, antioxidant, and electron carrier activities. A novel <I>Xoo</I> cysteine protease (<I>Xo</I>CP) was identified, showing dramatic increase in its protein abundance <I>in planta</I> upon <I>Xoo</I> interaction with a susceptible rice cultivar. Knock-out mutants of <I>XoCP</I> showed reduced pathogenicity on rice, highlighting its potential involvement in <I>Xoo</I> virulence.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A label-free quantitative proteomics approach was utilized for identification of <I>Xoo</I> secreted proteins. </LI> <LI> A total of 404 and 323 <I>Xoo</I>-secreted proteins were identified and quantified from <I>in vitro</I> and <I>in planta</I> systems respectively. </LI> <LI> A novel cysteine protease (<I>Xo</I>CP) was identified to be involved in <I>Xoo</I> pathogenicity. </LI> <LI> Moreover, 186 secretory proteins were also identified from <I>in-planta</I> secretome. </LI> <LI> Rice secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Comparative Secretome Investigation of <i>Magnaporthe oryzae</i> Proteins Responsive to Nitrogen Starvation

Wang, Yiming,Wu, Jingni,Park, Zee Yong,Kim, Sang Gon,Rakwal, Randeep,Agrawal, Ganesh Kumar,Kim, Sun Tae,Kang, Kyu Young American Chemical Society 2011 Journal of proteome research Vol.10 No.7

<P><I>Magnaporthe oryzae</I> is a fungal pathogen that causes blast disease in rice. During its early infection process, during which starvation of nutrients, including nitrogen, prevails before establishment of successful infection, the fungally secreted proteins play an important role in the pathogenicity and stress response. In this study, <I>M. oryzae</I>-secreted proteins were investigated in an N-deficient minimal medium using two-dimensional gel electrophoresis (2-DGE) coupled with mass spectrometry analysis (MALDI-TOF-MS and μLC–ESI–MS/MS). The 2-DGE analysis of secreted proteins detected 89 differentially expressed protein spots (14 downregulated and 75 upregulated) responsive to N starvation. Eighty five of the protein spots were identified by mass spectrometry analyses. Identified proteins were mainly cell wall hydrolase enzymes (22.4%), protein and lipid hydrolases (24.7%), reactive oxygen species detoxifying proteins (22.4%), and proteins with unknown function (14.1%), suggesting early production of prerequisite proteins for successful infection of the host. SignalP analysis predicted the presence of signal peptides in 67% of the identified proteins, suggesting that in addition to the classical Golgi/endoplasmic reticulum secretory pathway, <I>M. oryzae</I> might possess other, as yet undefined, secretory pathways. Those nonclassical or leaderless secretion proteins accounted for 25.9% of the total identified proteins by TatP and SecretomeP predictions. Semiquantitative reverse transcriptase polymerase chain reaction of seven randomly selected N-responsive secreted proteins also revealed a good correlation between RNA and protein levels. Taken together, the establishment of the <I>M. oryzae</I> secretome that is responsive to N starvation provides the first evidence of the secretion of 60 unreported and 25 previously known proteins. This developed protein inventory could be exploited to improve our understanding of the secretory mechanisms of <I>M. oryzae</I> and its invasive growth process in rice tissue.</P><P>Nutrient uptake, especially nitrogen, is important for the fungal growth, development, and host infection process. Using rice blast fungus cultured in complete, minimal, and nitrogen starvation medium, we performed comparative proteomics to investigate differentially regulated secreted proteins in response to different culture conditions. Differentially regulated protein expression was identified and confirmed at the mRNA level. This study provides insight into the protein secretion of <I>Magnaporthe oryzae</I> in response to nitrogen starvation.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2011/jprobs.2011.10.issue-7/pr200202m/production/images/medium/pr-2011-00202m_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr200202m'>ACS Electronic Supporting Info</A></P>