Name: agrobacterium tumefaciens and agrobacterium rhizogenes-mediated transformation of potato and the promoter activity of a suberin gene by gus staining
Uploaded: 2019-03-29T13:30:00
Description: Watch this Scientific Journal Video about Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining at JoVE.c

This work was supported by the Ministerio de Innovaci&#243;n y Ciencia (AGL2009-13745, FPI grant to PB), the Ministerio de Econom&#237;a y Competitividad and FEDER funding (AGL2012-36725, AGL2015-67495-C2-1-R), and the University of Girona (PhD grant to SF, and grant SING11/1). The authors are grateful to Dr. Inge Broer (Institute for Land Use, University of Rostock, Rostock, Germany) and Dr. Salom&#233; Prat (Centro Nacional de Biotecnolog&#237;a, Madrid, Spain) for providing the A. rhizogenes and the A. tumefaciens strain, respectively, and Dr. Mar&#231;al Soler and Dr. Anna Plasencia for the help and support received in initiating the A. rhizogenes transformation experiments (Toulouse III Paul Sabatier University&#8212;CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France). The authors thank Sara G&#243;mez (Departament de Biologia, UdG, Girona) for her valuable assistance in carrying out the laboratory work and taking care of plants, and Ferran Fontdecaba and Carla S&#225;nchez who assisted with some of the experiments while they were doing their final degree projects.&#160;

The A. rhizogenes transformation protocol was adapted and modified from Horn et al.7 and the genotype tested was S. tuberosum ssp. tuberosum (cv. D&#233;sir&#233;e). The A. tumefaciens transformation protocol was adapted and modified from Banerjee et al.22 and the genotypes tested were S. tuberosum ssp. tuberosum (cv. D&#233;sir&#233;e) and S. tuberosum ssp. andigena. The main steps of both procedures a...

In potato, the most common system to obtain stable complete transgenic plants uses the transformation by Agrobacterium tumefaciens strains that require organogenesis using exogenous phytohormones. Although the Agrobacterium based protocols has the potential to integrate non-T-DNA vector sequence25, this methodology is still the easiest and less expensive available to transform potato plants. During last years, the interest in A. rhizogenes-mediated transformation has got...

Aside from economic interest, the generation of transgenic plants has its own relevance in research to demonstrate the ultimate function of genes and to better understand plant physiology and development. The most widely used method for plant DNA insertion is Agrobacterium-mediated transformation. Agrobacterium tumefaciens is able to generate crown galls in the infected tissue of many plant species by the action of its tumour-inducing (Ti) plasmid. The plasmid contains a T-DNA region with a set of genes that will be integrated in the plant genome and induce tissue dedifferentiation1,2. The exchange of these genes within the T-DNA by the transgene has allowed the generation of specific plant modifications avoiding phenotypic effects3. To facilitate&#160;the transgene cloning into the T-DNA, the T-DNA region has been excised in an independent plasmid called a binary plasmid, while the rest of the genes of the Ti plasmid (the virulence genes that allow the T-DNA transfer and insertion mechanisms) have been placed in a helper plasmid. For plant biotechnology research, transformation by A. tumefaciens has several advantages: it does not need expensive devices, is able to generate both stable and transient plant transformation, and&#160;low numbers of gene copies are integrated into the chromosome4. However, for most plants, but not Arabidopsis, the generation of stable transformants requires plant regeneration from a single or a few cells using exogenous phytohormones, making this process laborious and time consuming. A. rhizogenes is also able to modify the plant genome, producing hairy roots or adventitious roots at the infection sites due to the expression of rol (root loci) genes encoded in the root-inducing (Ri) plasmid5. Although less studied than A. tumefaciens, A. rhizogenes is also used for obtaining transgenic roots. In this case, the A. rhizogenes contains the original T-DNA in the Ri plasmid and a binary plasmid with a second T-DNA carrying the transgene. When the infection site is in stems or hypocotyls, a composite plant can be obtained, with new hairy transgenic roots emerging from wild type shoots. Alternatively, hairy transformed roots can grow autonomously in vitro in media with carbon source inputs. The use of A. rhizogenes instead of A. tumefaciens to produce transgenic tissue is gaining relevance when the root is the target organ, because plant regeneration is not required and hence it is faster and less costly. Previous studies have demonstrated this methodology appropriated for the phenotypic characterization of root specific genes6,7,8,9.
The potato (Solanum tuberosum) is the fourth most important crop in the world according to the Food and Agriculture Organization of the United Nations (FAO) since the tuber has nutritional relevance for human consumption for being a good source of vitamins and minerals. For that reason, potato has been placed in the spotlight of agricultural biotechnology and also is considered as a good biological model for genetic and developmental studies10,11. Potato transformation significantly contributed to the understanding of molecular mechanisms underlying suberized tissues through the characterization of genes involved in suberin and wax biosynthesis12,13,14,15,16,17, suberin monomer transport18 and transcription regulation19. The suberin feruloyl transferase gene, FHT, is one of these characterized biosynthetic genes; its downregulation gives rise to a strong impairment of the periderm protection, which is correlated with a strong decrease in ferulate esters of suberin and waxes in potato tubers14. Concomitantly, in roots and seeds of Arabidopsis, the knockout of its putative orthologue (ASFT/RWP1) also demonstrated its role in producing alkyl ferulates in suberin20,21. In potato, the FHT transcriptional reporter line and the FHT antibody showed respectively that the promoter activity and the protein are located in the exodermis, the endodermis, the phellogen-derivatives and in wounded tissues15.
In this work, we detail a protocol using A. rhizogenes to produce transgenic hairy roots that are maintained in a wild type shoot, generating composite potato plants, or excised to grow autonomously in vitro. We also provide the protocol using A. tumefaciens to obtain complete transgenic potato plants. As a case study, A. rhizogenes and A. tumefaciens transformed with the same binary vector are used to obtain roots with the FHT promoter driving GUS reporter gene expression. The results are reported and compared.

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			M0254.0050
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			Panreac
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			131679
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			NAA
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			Duchefa
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			N0903
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			Panreac
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			131659
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			NaH2PO4
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			Sigma
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			58282
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			NightSea Stereo
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			SFA Moonting Adapter
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			<td style="width:216px;height:21px;">
			Parafilm
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			Anorsa
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			PRFL-001-001
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			Peptone
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			1616
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			Petri dishes (90 x 14)
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			Anorsa
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			200200
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			Liebherr Medline
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			59007
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			Shimadzu
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			<td style="width:101px;height:21px;">
			
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			Square plates (120 x 120)
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			Deltalab
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			200204
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			Streptomycin
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			<td style="width:161px;height:21px;">
			Sigma
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			S6501
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			Sucrose
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			<td style="width:161px;height:22px;">
			Panreac
			</td>
			<td style="width:101px;height:22px;">
			131621
			</td>
			<td nowrap="nowrap" style="width:80px;height:22px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Surgical blades
			</td>
			<td style="width:161px;height:21px;">
			Swann-Morton
			</td>
			<td style="width:101px;height:21px;">
			201
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Surgical needle
			</td>
			<td style="width:161px;height:21px;">
			NIPRO
			</td>
			<td style="width:101px;height:21px;">
			015/0204
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Triptone
			</td>
			<td style="width:161px;height:21px;">
			Lab Conda
			</td>
			<td style="width:101px;height:21px;">
			1612
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Triton
			</td>
			<td style="width:161px;height:21px;">
			Serva
			</td>
			<td style="width:101px;height:21px;">
			37240
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Unimax 1010 shaker
			</td>
			<td style="width:161px;height:21px;">
			Heidolph
			</td>
			<td style="width:101px;height:21px;">
			
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Vacuum
			</td>
			<td style="width:161px;height:21px;">
			Dinko
			</td>
			<td style="width:101px;height:21px;">
			
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:63px;">
			x-GlcA (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt anhydrous)
			</td>
			<td style="width:161px;height:63px;">
			Biosynth
			</td>
			<td style="width:101px;height:63px;">
			B-7398
			</td>
			<td nowrap="nowrap" style="width:80px;height:63px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Yeast extract
			</td>
			<td style="width:161px;height:21px;">
			Lab Conda
			</td>
			<td style="width:101px;height:21px;">
			1702.00
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
		</tr>
		<tr>
			<td style="width:216px;height:21px;">
			Zeatin riboside
			</td>
			<td style="width:161px;height:21px;">
			Sigma
			</td>
			<td style="width:101px;height:21px;">
			1001042850
			</td>
			<td nowrap="nowrap" style="width:80px;height:21px;"></td>
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agrobacterium tumefaciens and agrobacterium rhizogenes-mediated transformation of potato and the promoter activity of a suberin gene by gus staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium rhizogenes-mediated potato transformation 
In this manuscript, the step-by-step procedure set up to obtain transformed root with A. rhizogenes is presented. Figure 1 presents an overview of the procedure, which altogether takes around 5-6 weeks (from injection of A. rhizogenes to obtaining fully developed hairy roots). Then, ...

Watch this Scientific Journal Video about Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining at JoVE.c

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

The agrobacterium-mediated transformation is an easy method for producing genetically-modified potato plants. In order to understand specific gene functions and their contribution to organ physiology. Transformation by agrobacterium rhizogenes is a robust tool for rapidly assessing gene functioning roots, as similar results can be obtained by agrobacterium tumefaciens transformation.Demonstrating this procedure will be Sandra Fernandez-Pinan, a post-doc, and Jennifer Lopez, a maste5r's student from our lab. Begin by growing a single agrobacterium colony overnight in five milliliters of yeast extract broth, or YEB medium, supplemented with antibiotics in a 50 milliliter centrifuge tube at 28 degrees Celsius, at 200 rotations per minute. For a-tumefaciens transformation, measure the optical density the next morning.When the optical density at 600 nanometers, or OD 600 reaches 0.6 to one, centrifuge one milliliter of the agrobacterium culture and resuspend the pellet in one milliliter of fresh YEB medium without antibiotics. After the second wash, resuspend the pellet in fresh YEB medium without antibiotics, to the appropriate concentration to obtain a final OD 600 of 0.8, and place the bacterial cells on ice. For plant transformation using A.rhizogenes, carefully transfer a donor plant from a 2MS medium culture to a 120 by 120 millimeters square plate, and use a surgical needle to inject three microliters from an A.rhyzogenes culture to different stem internodes as possible.Then immediately transfer the entire plant to a new square plate containing solid MS medium, supplemented with 0.1 millimolar acetosyringone. After placing a second plant onto the same plate, transformed in the same manner, seal the plate with surgical tape, and place the plate vertically inside a growth cabinet for four days. Transfer the plant to a square plate with MS medium supplemented with cefotaxime sodium to kill the A.rhizogenes and place the sealed plate vertically inside a growth cabinet for four days.After 10 to 12 days, new hairy roots will appear. Their transformation can be confirmed by fluorescent stereo microscope. At this time, excise the native roots of each plant, and transfer the plants to a new square plate with MS medium, supplemented with cefotaxime sodium to kill the A.rhizogenes.To obtain a composite plant, let the transgenic hairy roots grow for another three to four weeks in the MS medium, supplemented with cefotaxime sodium. For plant transformation using A.tumefaciens. Cut and place a leaf from a three to four week old plant in a petri dish, and use a scalpel to make one to three transverse cuts from the center of the leaf to the edges without cutting the pieces off of the leaf.and to exclude the petiole. Immediately place the leaf into a petri dish containing 10 milliliters of fresh 2MS liquid medium, abaxial side up, and cover the plate. Quickly add 80 microliters of the A.tumefaciens culture to the liquid medium and gently stir the medium for one minute to homogeneously distribute the bacterial solution.Then carefully seal and cover the plate with aluminum foil for a two-day incubation in a 24-degree Celsius chamber. At the end of the transformation period, transfer the leaves abaxial-side up, to the callous-inducing medium, scraped with tweezers for a one-week incubation in the growth cabinet. At the end of the incubation, transfer the leaves, abaxial-side up, onto tweezer-scraped shoot inducing medium, and incubate the leaves in a growth cabinet.When the emerged chutes are about two centimeters tall, cut three emerging chutes from each callous. Place up to five different chute transformation events into individual culture flasks containing MG medium supplemented with cefotaxime sodium to allow rooting, and label the subset as appropriate for a three to four week incubation in the growth cabinet until the chutes are vigorous. At the end of the incubation, select the most vigorous plant of each event, and cut the apical segments of the chute with three to four internodes from each plant.Place the segments into a culture flask containing 2MS medium supplemented with cefotaxime sodium and grow the plants until they develop vigorous chutes and roots. To perform a beta-glucuronidase or GUS histochemical reporter gene assay, fix the roots from a two to three week in-vitro plant culture with 90%chilled acetone for 20 minutes on ice. At the end of the fixation, wash the roots two times in distilled water and treat the roots with fresh GUS-staining solution under vacuum for 20 minutes.At the end of the vacuum treatment, place the roots at 37 degrees Celsius in the dark for about four hours. When a blue color is visible, wash the roots two times with 70%ethanol, and visualize the staining under a bright field microscope. Transformed hairy roots exhibit a red florescence when illuminated with green light, while negative control untransformed agrobacterium demonstrate no such florescence, indicating the suitability of the DS red transformation marker for identifying transgenic hairy roots.Here, GUS staining in roots of transgenic plants obtained by A.tumefaciens and transgenic hairy roots obtained by A.rhizogenes are shown. As observed, roots transformed with A, tumefaciens and grown in-vitro, demonstrate blue staining in the endodermis, a cell layer between the cortex and the stele. In more developed roots, the blue labeling is patchy in the external layer, corresponding to the exodermis.In transformed hairy roots obtained by A.rhizogenes, and grown in hydroponics, the GUS marker is specifically located within the endodermis in the emergence of lateral roots in the wounded areas and in the exodermis. In addition to being a fast method, the transgenic roots obtained with agrobacterium rhizogenes also carried the DNA from the root-inducing plasmid, which may deregulate some directly-controlled processes. The transgenic hairy roots obtained with agrobacterium rhizogenes can be maintained while they shoot, but alternatively can be on-site until propagated in-vitro for massive transit production.The potato transformation provides a good tool to check the gene function and promoter activation, and also to produce methodological rules in a species of high agronomic interest. The genetically modified organism should be discarded safely after use to avoid environmental contamination. Also, gas solution contains toxic cyanide derivative, so wear protection and discard the solution safely.

agrobacterium-tumefaciens-agrobacterium-rhizogenes-mediated

Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...