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

Jingni Wu,김상곤,강규영,김주곤,박상렬,Ravi Gupta,김용환,Yiming Wang,김선태 한국식물병리학회 2016 Plant Pathology Journal Vol.32 No.6

Pathogenesis-related proteins play multiple roles inplant development and biotic and abiotic stress tolerance. Here, we characterize a rice defense related genenamed “jasmonic acid inducible pathogenesis-relatedclass 10” (JIOsPR10) to gain an insight into its functionalproperties. Semi-quantitative RT-PCR analysisshowed up-regulation of JIOsPR10 under salt anddrought stress conditions. Constitutive over-expressionJIOsPR10 in rice promoted shoot and root developmentin transgenic plants, however, their productivitywas unaltered. Further experiments exhibited that thetransgenic plants showed reduced susceptibility to riceblast fungus, and enhanced salt and drought stresstolerance as compared to the wild type. A comparativeproteomic profiling of wild type and transgenicplants showed that overexpression of JIOsPR10 led tothe differential modulation of several proteins mainlyrelated with oxidative stresses, carbohydrate metabolism,and plant defense. Taken together, our findingssuggest that JIOsPR10 plays important roles in bioticand abiotic stresses tolerance probably by activationof stress related proteins.

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>

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.

Effect of Phytohormones and Chemical Inhibitors on Pathogenesis-related Genes Identified by Differential Hybridization in Rice Suspension Culture Cells

김상곤,Jingni Wu,Ethan E White,Young Whan Choi,Keun-Ki Kim,In Soo Choi,Sun-Hyung Kim,강규영,김선태,Yiming Wang,김용철 한국식물병리학회 2010 Plant Pathology Journal Vol.26 No.4

In order to study disease resistance mechanisms in rice against the rice blast fungus Magnaporthe grisea, we screened fungal elicitor-responsive genes from rice suspension-cultured cells treated with fungal elicitors employing differential hybridization (DH). By DH screening,31 distinct rice clones were isolated and a majority of them were full-length cDNAs encoding pathogenesisrelated (PR) genes. Sixteen of the 31 genes were upregulated at 4, 8, and 12 h following fungal elicitor treatment. To elucidate the effect of signal molecules and biotic elicitors on the regulation of rice defense genes, we further characterized the transcriptional expression patterns of representative isolated PR genes;OsGlu1, OsGlu2, OsTLP, OsRLK, and OsPR-10, following treatment with fungal elicitor, phytohormones, cycloheximide,and inhibitors of protein phosphorylation. Jasmonic acid (JA) induced transcriptional expression of OsGlu1, OsTLP, and OsRLK, but not of OsGlu2 and OsPR-10 at any of the tested time points. Salicylic acid (SA) and abscisic acid weakly induced the expression of OsTLP and OsRLK. SA showed an antagonistic effect with fungal elicitor and JA. Cycloheximide suppressed all these genes upon elicitor treatment, except for OsGlu2. Staurosporine only induced the expression of OsRLK. Application of calyculin A strongly induced OsRLK expression, but suppressed the expression of OsGlu2. Our study yielded a number of PR genes that play a role in defense mechanisms against the rice blast fungus, as well as contribute towards the elucidation of crosstalk between phytohormones and other modifications during defense signaling

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.

<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

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>

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.

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.

Physiological and proteomic analysis of young rice leaves grown under nitrogen-starvation conditions

Kim, Sang-Gon,Wang, Yiming,Wu, Jingni,Kang, Kyu-Young,Kim, Sun-Tae The Korean Society of Plant Biotechnology 2011 Plant biotechnology reports Vol.5 No.4

Rice grown in anaerobic waterlogged soil accumulates ammonium as a major source of nitrogen (N). We have compared the physiological symptoms of rice seedlings subjected to N-starvation stress with those receiving sufficient N, based on measurements of shoot/root length and weight and an analysis of protein expression patterns. N starvation marginally increased root growth but notably decreased shoot biomass. N uptake was reduced by >50% in the roots and shoots of N-starved seedlings. To better understand the mechanism of N starvation in rice, we performed a comparative proteome analysis of proteins isolated from rice leaves. Twenty-five differentially expressed proteins were analyzed by matrixassisted laser desorption/ionization time-of-flight (TOF) mass spectrometry and electron spray ionization quadrupole TOF. Functional analysis of the N-starvation response proteins suggested their involvement in protein synthesis and fate, metabolism, and defense. These results indicate that these proteins may play important roles in regulating the plant's complex adaptation responses for N use during N starvation. The proteins may be useful for further characterization of protein function in plant N nutrition.