<ol><li> 처음에 우리는 추출을위한 2ml 나사 캡 튜브를 준비해야합니다 400ul 추출 버퍼를 추가 (1 - 프로파놀 : H2O : 집중 HCL 2:1:0.002 권 / 권 / 권) 및 해당 내부 표준 10 μl가 (10ng/μl ) 유리병 수 있습니다. 액체 질소로 동결 후 세라믹 비즈를 추가합니다. </li><li> 유리병 여전히 액체 질소에 배치하는 동안 (50 200mg 사이) 식물을 추가합니다. 추가 식물의 무게 추가 처리가 신선한 무게를 기반으로 정확한 부량, 나중에 허용하기 전에 예상되어야합니다. </li><li> 모든 액체 질소가 단단히 마개로 병을를 닫기 전에 증발되어 있는지 확인합니다. 뚜껑이 균질 동안 따라서 용제의 유출하고, 추출한 화합물을 방지하기 위해 고무 도장이 있어야합니다. </li><li> 30 초에 대한 6000의 속도 (Precellys 용)에서 FastPrep 또는 Precellys 비드 분쇄기의 균질 화기의 조직을 균질. </li><li> 개별적으로 균질 화기에서 튜브를 제거 조심스럽게 뚜껑을 열고 각 샘플에 dichloromethane의 1ml를 추가합니다. 닫기 튜브 다시 단단히하고 6000 (Precellys) 10 - 15sec에 다시 균질. </li><li> 테이블탑 원심 분리기에 튜브를 전송하고 60sec에 대한 10.000xg에있는 수성 단계에서 유기농 단계를 구분한다. </li><li> 상 분리 후 깨끗한 4ml 유리관으로 개별 튜브에서 로우어 그린 레이어 (유기 위상, 호르몬 및 내부 표준을 포함​​)을 전송합니다. 물의 양도 (상위 단계)를 피하십시오. 또는 에틸 아세테이트 대신 dichloromethane에 사용할 수 있습니다. 이 경우에는 ethyacetate 단계는 상위 레이어 (녹색)하고 지금은 제거하고 신선한 4ml 유리관으로 전달하는 것이 더 쉽습니다 것입니다. 이전처럼, 물의 이동을하지 마십시오. </li><li> 10-25 분 (샘플에 따라)에 대한 다기관을 통해 공기와 용매를 날려, 샘플이 건조하는 경우 (주의) 한 공기 끝에 문의하십시오. overdry 샘플은하지 마세요,이에 대한 화합물의 손실을 발생할 수 있습니다. </li><li> 샘플 건조되면 우리는 carboxylic의 methylation 산성 함유하는 시료의 성분을 시작할 수 있습니다. 첫째, d​​iethyether / 메탄올 혼합 (9시 1분 권 / 권) 100 μl은 2M trimethylsilyldiazomethane 솔루션 (헥산, 시그마 알드리치 년)의 4μl 다음 각 유리병에 추가됩니다. 튜브는 즉시 테플론 늘어선 실리콘 심장 장착되어 오픈 상단 나사 마개로 닫혀 있습니다. 부드럽게 흔들 25 30 분에 대한 RT에서 알을 품다. </li><li> 9 단계를 마친 후, 원하는 컬렉션 온도에서 가열 블록 설정하고 전원을 켜십시오. methylated 화합물의 휘발성들은 크기뿐만 아니라 = O, - OH, NH 및 같은 다른 더 북극 그룹에 따라 달라집니다. jasmonic 산성과 살리실산의 메틸 에스테르의 회수 온도 80 주변 ° C 충분, 반면에 예를 들어 C18 지방산과 같은 다른 화합물, jasmonic 산성 - 아미노산 conjugates, coronatine, 12 - 옥소 - phytodienoic 산성 메틸 에스테르 사이 온도를 필요로 180-200 ° C 효과적으로 휘발 수 있습니다. </li><li> 30 분 부화 후 methylation 반응은 원치 않는 보조 반응을 피하기 위해 중단해야합니다. 이것은 각 유리병에 2M 초산 - 솔루션 (헥산)을 4 μl를 추가하여 이루어집니다. 모자와 짧은 vortexing으로 튜브를 닫은 후, 샘플은 다른 30 ​​분 대한 부화 사용할 수 있습니다. </li><li> 이 증기 위상 추출에 사용되는 필터는 수작업과 제시 형태로 상업적으로 사용할 수 없습니다. 그들은 한쪽에서 4cm 길이에 대해 잘 맞는 유리 튜브가 삽입되는에 외부 테플론 튜브 (7~8센티미터 길이 약)로 이루어져 있습니다. 그것이 내부 유리관에 도달하고 때까지 테플론 튜브의 반대쪽에있는 스테인레스 스틸 메쉬 디스크가 튜브 안으로 밀려는 것을에 약 20 - 30mg 흡착제 (SuperQ 100분의 80 (단종) 또는 HayeSep ® Q80/100 중) 추가됩니다. 다른 스테인레스 스틸 메쉬 디스크가 삽입되고 마지막으로 유리 팁되므로 신중하게 흡수에 스테인리스 메쉬 디스크를 누르면, 테플론 튜브로 못살게 굴지입니다. 자세한 사진은 필터는 그림 1에 표시됩니다. <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="그림 1" /> 그림 1. </li><li> 11 단계의 배양 시간 동안 따라서 methylated하고, 휘발성 식물 호르몬의 수집에 대한 필터, 200ul의 dichloromethane 각 2 세척 후 메탄올의 첫 200ul를 사용하여 세탁해야합니다. 필터에 남아있는 용매는 다음 공기 날리게됩니다. 필터는 현재 기상 위상 추출을 통해 휘발성 호르몬의 트래핑에 사용될 수 있습니다. </li><li> 부화 후 30 분 4ml 유리 뚜껑의 고무 septa은 메스로 절단됩니다. 청소 필터가 800ml/min 약 흐름 속도로 개별 진공 라인에 연결되어있는 증기 위상 추출하여 식물 호르몬을 수집합니다. 필터는 다음 컷 septa 통해 유리병에 삽입됩니다. 작은 pipet 팁 또한 공기 유입구의 역할을 삽입됩니다. 절차의 초기 부분에서 삽입 필터와 튜브 개최해야후 필터와 또한 GC / MS를 오염시킬 필터 팁, 연락하려 유리병 내부의 액체 중 하나를 피하기 위해 직립 위치에있다. 모든 액체가 필터를 통해 증발 때까지 튜브 그런 다음 수동으로 개최하고 있습니다. 다음 첨부된 필터와 튜브는 가열 블록 및 3min에 대한 수집 열 증발 화합물에 표시됩니다. 시스템뿐만 아니라 설치의 주요 구성 요소는 그림 1에 묘사된 있습니다. 이 마지막 증기 위상 추출 단계 후에도 부착된 필터와 튜브는 랙에 위치하고 진정 사용할 수 있습니다. </li><li> 필터 다시 실내 온도에 도달하면 그들은 삽입과 함께 장착되어 1.5 ML의 GC - 약병에 dichloromethane의 150ml로 eluted 있습니다. 유리병이 뒤덮힌 및 GC / MS로 분석하는 필터에서 남은 용매를 날려 변경할 수 있습니다. </li></ol> 분할 / splitless 인젝터 화학 이온화 모드 (CI)에 운영하는 질량 분석기로 인터페이스 분석을 위해 사용해야합니다 (° C, 사출 볼륨 1μ이 splitless 모드 250))가 장착된 가스 크로마토 그래프. 화합물이 (30m X 0.25mm X 025μm) HP - 1MS 칼럼에서 분리 ° C 주사 후 1min 40에서 개최하고 온도가 15 프로그램되어 ° 헬륨과 함께 250 ° C (10 분)을,에 C / 분 캐리어로 가스 (일정한 흐름 0.7ml/min). MS 기반의 분석을위한 모두,이 quadropole (예 : 애질런트 HP5973)와 이온 트랩 기반 (예 : 베리안 토성 2200) 질량 분석계를 사용할 수 있습니다. CI 가스 중 이소 부탄 (가스 탱크에 제공) 또는 메탄올은 (액체 저수지의 증기를 사용하는)를 사용할 수 있듯이, 그러나 각각의 경우에 이온화 반응의 조각이 선택한 이온 개수 (SIC) 프로그램하기 전에 확인하여야한다 만들어집니다. 우리는 CI의 시약으로 베리안 GC 3900/Saturn MS 2200 시스템과 메탄올에 대한 다음과 같은 프로그램을 사용했을 : 5.00 - 11.30min 이온 (M +1) + 153-158 (메틸 살리실산 153, 메틸 2 H 5 살리실산을); 11.30 - 13.30min 이온 (M +1) + 207-228 (메틸 jasmonate 207과 225, 메틸 dihydrojasmonate 209 및 227), 13.30 - 16.30min 이온 (M +1) + 237-300 (메틸 아브 산 261, 메틸이 H 6 - 아브 산 267 (압시 신산은 주로 [MH 2 O 1] + 이온을 생산하고). 모든 화합물은 본격적인 상용 표준과 비교하여 확인한다. JA, SA 및 ABA의 부량가 정상 영역을 연관하여 이루어집니다 각 내부 표준의 최대 면적 (추출 이온)와 (은 이온을 추출) 또한 사용되는 식물 재료의 신선한 무게에 따라 달라집니다.

<ol>
	<li>Schmelz, E. A., Engelberth, J., Tumlinson, J. H., Alborn, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vapor+phase+extraction+in+metabolic+profiling+of+phytohormones+and+other+metabolites.">The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites.</a> Plant Journal. 39, 790-808 (2004).</li><li>Engelberth, J., Alborn, H. T., Schmelz, E. A., Tumlinson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+signals+prime+plants+against+insect+herbivore+attack.">Airborne signals prime plants against insect herbivore attack.</a> PNAS. 101, 1781-1785 (2004).</li><li>Schmelz, E. A., Engelberth, J., Alborn, H. T., O&amp;acute, D. o. n. n. e. l., Sammons, P., Toshima, M., H, J. H. T. u. m. l. i. n. s. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+phytohormones,+phytotoxins,+and+volatile+organic+compounds+in+plants.">Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants.</a> PNAS. 100, 10552-10557 (2003).</li><li>Engelberth, J., Schmelz, E. A., Alborn, H., Cardoza, Y. J., Huang, J., Tumlinson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+Jasmonic+acid+and+Salicylic+acid+by+Vapor+Phase+Extraction+and+Gas+Chromatography+-+Chemical+Ionization-Mass+Spectrometry.">Simultaneous Quantification of Jasmonic acid and Salicylic acid by Vapor Phase Extraction and Gas Chromatography - Chemical Ionization-Mass Spectrometry.</a> Analytical Biochemistry. 312, 242-250 (2003).</li></ol>

방법은 크기와 화합물의 화학적 특성이 추출 methylated, 그리고 증발되지 않도록하지 않습니다 제공하는 식물 신호 화합물의 다양한위한 안정적으로 작동하도록 입증되었습니다 여기를 발표했다. 또한, 그것이 본 설명으로 methylation 절차는 derivati​​zation의 유일한 방법은 아니다. Silyllation 및 아세틸화 대체 방법과 프로토콜을하는 것은 쉽게 온라인으로 찾을 수 있습니다. 질...

monitoring plant hormones during stress responses

식물 호르몬 및 관련 신호 화합물은 다양한 환​​경 자극과 스트레스에 식물 반응의 조절에 중요​​한 역할을한다. 가장 심각한 스트레스 중 곤충 herbivory, 병원체 감염과 가뭄 스트레스 있습니다. 이러한 각함으로써 식물의 생존을 보장 그것의 조정 반응하는 것으로 알려져 있습니다 특정 호르몬의 설정 및 / 또는 조합을 강조하십시오. 이러한 반응의 규정에 관련된 주요 호르몬은 jasmonic 산성 (JA), 살리실산 (SA), 그리고 아브 산성 (ABA)입니다. 더 시간적뿐만 아니라 공간적 패션 그들의 풍부한 변화를 모니터링하는 개인 호르몬뿐만 아니라 그것이 필요합니다 이러한 반응 중에 잠재적인 상호 작용의 역할을 이해한다. 이러한 다른 신호 화합물의 쉬운 민감한 및 재현성 부량 위해 우리는 증기 위상 추출 및 가스 크로마 토그래피 / 질량 분석계 (GC / MS) 분석 (1, 2, 3, 4)에 따라 방법을 개발했습니다. 산성 수성하여 식물 조직에서 이러한 화합물을 추출 후 1 프로파놀는 dichloromethane와 혼합 카르복실산 함유 화합물은, methylated 열 아래 volatilized 및 고분자 흡착제에서 수집하고 있습니다. 샘플 약병에 용출 후 analytes는 가스 크로마 토그래피로 구분되며 화학적 이온화 질량 분광법에 의해 감지. 적절한 내부 표준의 사용은 다음 analyte 및 내부 표준의 최대 영역을 관련된하여 간단한 부량 수 있도록합니다.

Watch this Scientific Journal Video about Monitoring Plant Hormones During Stress Responses at JoVE.com

Monitoring Plant Hormones During Stress Responses

간단한 방법이 작은 조직 샘플에서 여러 식물 호르몬의 급격한 추출 및 분석을위한 허용 제공됩니다. 절차는 엄숙한 정화 단계로 증기 위상 추출을 사용합니다. 샘플은 GC / 주로 생산하는 화학 이온화 (M +1)과 MS + 이온에 의해 분석됩니다.

스트레스 응답하는 동안 식물 호르몬을 모니터링

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

monitoring-plant-hormones-during-stress-responses

Research

JoVE Journal

Biology

16.5K 조회수.  University of Texas. 간단한 방법이 작은 조직 샘플에서 여러 식물 호르몬의 급격한 추출 및 분석을위한 허용 제공됩니다. 절차는 엄숙한 정화 단계로 증기 위상 추출을 사용합니다. 샘플은 GC / 주로 생산하는 화학 이온화 (M +1)과 MS + 이온에 의해 분석됩니다.

비디오: Monitoring Plant Hormones During Stress Responses

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

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Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

모니터링 살충제 저항 Bioassays

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Predation 위험, Herbivore 생리적 스트레스와 식물 돼지 미생물 분해를 연결

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Polysom​​e 프로파일에 의한 스트레스 조건에서 번역 개시 분석

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

수경법, • 다목적 시스템은 유해 요소에 영양소 가용성 및 노출에 영양 할당 및 플랜트 응답을 공부하기

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

녹색 형광 단백질 박테리아에서 감염 하는 동안 전달 하는 이펙터를 시각화 하기 위해 시스템을 분하시오

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans의 생리적 스트레스 반응 측정

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

시험관 내 E3 유비퀴틴 리가제 기능 분석

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

CRISPR/Cas9 기술을 이용한 철새 메뚜기의 동형접합 돌연변이 구축

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Plasmodiophora brassicae에 대한 유전자 발현 및 생리적 반응의 변화를 시각화하기 위해 투명화와 형광 현미경을 결합

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...