Understanding the Responses of Rice to Environmental Stress Using Proteomics

Singh, Raksha,Jwa, Nam-Soo American Chemical Society 2013 Journal of proteome research Vol.12 No.11

<P>Diverse abiotic and biotic stresses have marked effects on plant growth and productivity. To combat such stresses, plants have evolved complex but not well understood responses. Common effects upon perception of environmental stress are differential expression of the plant proteome and the synthesis of novel regulatory proteins for protection from and acclimation to stress conditions. Plants respond differently in terms of activation of stress-responsive signaling pathways depending upon the type and nature of the stresses to which they are exposed. Progress in proteomics and systems biology approaches has made it possible to identify the novel proteins and their interactions that function in abiotic stress responses. This will enable elucidation of the functions of individual proteins and their roles in signaling networks. Proteomic analysis of the responses to various stress conditions is performed most commonly using 2D gel electrophoresis and high-throughput identification by LC-MS/MS. Because of recent developments in proteomics techniques, numerous proteomics studies of rice under abiotic stress conditions have been performed. In this review, proteomics studies addressing rice responses to the major environmental stressesincluding cold, heat, drought, salt, heavy metals, minerals, UV radiation, and ozone are discussed. Unique or common protein responses to these stress conditions are summarized and interpreted according to their possible physiological responses in each stress. Additionally, proteomics studies on various plant systems under various abiotic stress conditions are compared to provide deeper understanding of specific and common proteome responses in rice and other plant systems, which will further contribute to the identification of abiotic stress tolerance factor at protein level. Functional analysis of stress-responsive proteins will provide new research objectives with the aim of achieving stable crop productivity in the face of the increasing abiotic stress conditions caused by global climate change.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2013/jprobs.2013.12.issue-11/pr400689j/production/images/medium/pr-2013-00689j_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr400689j'>ACS Electronic Supporting Info</A></P>

Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity

Singh, Raksha,Dangol, Sarmina,Chen, Yafei,Choi, Jihyun,Cho, Yoon-Seong,Lee, Jea-Eun,Choi, Mi-Ok,Jwa, Nam-Soo Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.5

Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity

좌남수,Raksha Singh,Sarmina Dangol,Yafei Chen,Jihyun Choi,Yoon-Seong Cho,Jea-Eun Lee,Mi-Ok Choi 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.5

Plant disease resistance occurs as a hypersensitive re-sponse (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant im-mune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

Rice OsACDR1 (Oryza sativa Accelerated Cell Death and Resistance 1) Is a Potential Positive Regulator of Fungal Disease Resistance

김정아,조경원,Raksha Singh,Young-Ho Jung,Seung-Hee Jeong,So-Hee Kim,Jae-eun Lee,Yoon-Seong Cho,Ganesh K. Agrawal,Randeep Rakwal,Shigeru Tamogami,Birgit Kersten,전종성,Gynheung An,Nam-Soo Jwa 한국분자세포생물학회 2009 Molecules and cells Vol.28 No.5

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal patho-gen (Magnaporthe grisea) and showed inhibition of appres-sorial penetration on the leaf surface. In contrast, loss-of-function and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its local-ization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa

Dangol, Sarmina,Singh, Raksha,Chen, Yafei,Jwa, Nam-Soo Korean Society for Molecular and Cellular Biology 2017 Molecules and cells Vol.40 No.11

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

Visualization of Multicolored in vivo Organelle Markers for Co-Localization Studies in Oryza sativa

Sarmina Dangol,Raksha Singh,Yafei Chen,좌남수 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.11

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in mon-ocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our gen-erated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

Establishment of transformation-based indica rice molecular breeding using a new toxoflavin/tfIA selection system

Hye Jeong Kim,Mi Ok Choi,Raksha Singh,Ji Hyoun Choi,Nam Soo Jwa 한국육종학회 2011 한국육종학회 학술발표회 발표요지 Vol.43 No.-

Toxoflavin/tflA selection-based new indica rice transformation

Eun-Hye Kim,Ji-Hyoun Choi,Ji-Hoon Park,Raksha Singh,Nam-Soo Jwa 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Most indica rice varieties show a low efficiency of transformation because of difficulties in callus formation and low-regeneration frequencies in conventional culture such as MS16 or N6 medium. Recently, some improved methods were reported for Agrobacterium-mediated transformation using mature elite indica seeds however, these procedures take a long time (5–7 months) to obtain transgenic plants and still with significantly low efficiency. In this study, we provide additional improvements in the indica rice transformation protocol introducing new selection method by tosoflavin/tflA which was based on bacterial photosensitizer and its degrading enzyme pair. We introduced direct in planta transformation using early stage of germinating rice seeds instead of usual embryogenic callus. Methods that use embryos as a starting material for inoculation with Agrobacterium are also used for comparison with the new protocol using rice seeds. Transformed cells proliferated from rice seeds obtain toxoflavin resistance, and transgenic plants are eventually regenerated from those proliferated tissues. However, we found out that tissue proliferation from indica seeds and shooting and rooting are very sensitive to minor salt nutrients in the media and need to pay attention to use indica rice specific nutrient media. The use of naturally occurring photosensitizers such as toxoflavin as selection agents appears to give rapid and unambiguous selection results owing to their unique phytotoxic mode of action. In particular, the toxoflavin/tflA selection system might be useful for generating transgenic indica rice cultivar where high false-positive backgrounds with current selection marker systems are problematic.