The work is partially supported by Wageningen University Fund (WUF), China Scholarship Council Program for Graduate Students, and a NWO-VIDI grant 12378.

1. Plant Growing and Testing Conditions
<ol>
	<li>Grow and maintain plants in controlled greenhouses or climate chambers within the temperature range of 18-22 &#176;C and under natural light regime or with a 16 hr/8 hr day/night regime. Remove axillary branches in order to make the plants more manageable.</li>
	<li>For potato, maintain in vitro plantlets in sterile plastic jars containing Murashige-Skoog (MS) medium (20 g/L sucrose, 5 g/L MS salts with vitamins, 8 g/L agar, pH 5.8) under controlled conditions in climate chambers at 18 &#176;C with a 16 hr/8 hr day/night regime for two weeks and then transfer them into pots of sterilized soil in regulated greenhouse compartments.</li>
</ol>
2. Agroinfiltration
<ol>
	<li>Plant material: 
	For Nicotiana benthamiana, use around 4-5 week old seed-grown plants. For potato, use around 4-5 week old transplants from in vitro. Choose young, healthy and fully developed leaves for infiltrations.</li>
	<li>Agrobacterium culture:
	<ol>
		<li>Prepare the YEB medium in advance (Table 1). Fill the 50 ml tubes with 10 ml YEB medium supplemented with 1 &#956;l acetosyringone (200 mM), 100 &#956;l MES buffer (2-(N-morpholino)-ethane sulfonic acid, 1 M) and the appropriate antibiotics. Pipette 20 &#956;l glycerol stock of the desired strains (Table 2) (containing the gene of interest) into the YEB. Initiate cultures for all the Agrobacterium strains in this assay at the same time. Incubate cells by shaking for 1-2 days at 28 &#176;C and 200 rpm to an OD600 of approximately 1.0.</li>
		<li>Harvest cells by centrifugation at 3,000 x g for 10 min. Pour off the supernatant and resuspend the pellet in freshly made MMA medium (Table 3) to an OD600 of 0.3. Gently vortex cells to resuspend them. For coinfiltration of two bacterial strains, mix the culture in a 1:1 ratio.</li>
		<li>Leave the culture on the bench at room temperature for 1-6 hr before infiltrations. In the meantime, label the plants to be infiltrated and the date of the experiment.</li>
	</ol>
	</li>
	<li>Infiltrations: 
	Use a 1 ml&#160;needleless syringe to infiltrate the Agrobacterium suspensions. Carefully and slowly inject leaf panels with the suspensions from the syringe (for health and safety reasons eye protection should be worn during this process). Infiltrate at least three plants for each strain. Use three leaves per plant to serve as triplicates. 
	Note: Normally, 1 ml of Agrobacterium suspension s could be enough for 3 plants of N. benthamiana. For potato, more suspension is needed because the leaves of Solanum species are more difficult to infiltrate. Required volumes of suspensions depend on the Solanum species. Avoid cross-contamination by changing gloves or sterilizing gloves with ethanol between infiltrations and by not watering the plants until the day after the inoculation.</li>
	<li>Scoring: 
	Score macroscopic responses about 3 days after infiltration when the cell death phenotype is clear (Figure 1). If the cell death phenotype is quantitative, use the given criteria (Figure 2). Briefly, the scales for macroscopic scoring of cell death mainly depend on the cell death percentages of the infiltrated area. Percentages of cell death are depicted on a scale from 0% (no symptoms) to 100% (confluent cell death). Intermediate responses range from weak responses such as chlorosis to increasing levels of cell death. If desired, the macroscopic scoring can also be quantitatively assessed using modern photo-imaging equipment.</li>
</ol>
3. PVX Agroinfection
<ol>
	<li>Plant material: 
	For N. benthamiana, use around 2-3&#160;week&#160;old seed-grown plants. For potato, use around 2-3&#160;week&#160;old transplants from in vitro. For large-scale tests, use slightly older (4-5 weeks) plants, as these plants have more and larger leaves to accommodate higher numbers of Agrobacterium inoculation spots.</li>
	<li>Agrobacterium culturing:
	<ol>
		<li>Prepare the YEB medium in advance. Fill the 10 ml tubes with 3 ml YEB supplemented with the appropriate antibiotics. Pipette 20 &#956;l glycerol stock of the desired strains (Table 2) (containing the gene of interest) into the YEB. Initiate cultures for all the Agrobacterium strains in this assay at the same time. Incubate cells by shaking for 1-2 days at 28 &#176;C and 200 rpm to an OD600 of approximately 1.0.</li>
		<li>Pipette about 300 &#956;l of each Agrobacterium strain and spread them onto LB solid agar medium plates supplemented with the appropriate antibiotics and incubate cells at 28 &#176;C for 1-2 days.</li>
	</ol>
	</li>
	<li>Infections: 
	Use a spatula to collect the Agrobacterium culture in the plate. Dip a wooden toothpick in the Agrobacterium culture on the spatula and pierce the leaves to inoculate large amount of bacteria. Inoculate at least three plants for each strain. Use three leaves per plant to serve as triplicates. In each leaf, make multiple inoculation sites for each strain.</li>
	<li>Scoring: 
	Score macroscopic responses about two weeks after inoculation (Figure 3). For high-throughput screens, record the qualitative responses (yes/no) for each inoculation spot. Then calculate the percentage of responding sites and compare them with controls.</li>
</ol>

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The authors declare that they have no competing financial interests.

Transient assays like agroinfiltration and agroinfection are efficient methods that are vital to modern molecular plant pathology research. Despite some limitations, these methods meet the demand for efficient and robust high-throughput functional analysis in plants.
The agroinfiltration system is a widely used functional assay in a range of plant species. Agroinfiltration facilitates the delivery of several transgenes into the same cell with simultaneous expression of interacting proteins. This is advantageous for recapitulating R-AVR relationships, by coinfiltrating Agrobacterium strains that express Avr genes with strains that express the matching R genes. Also, for known R-AVR pairs, such coinfiltrations can be used as positive controls. Including such controls is important because in some plant genotypes, the transformation efficiency can be below the threshold to detect responses. Including negative controls, e.g. an Agrobacterium strain containing a vector without a gene insert, is also essential to determine whether a certain plant genotype raises nonspecific defense responses to the Agrobacterium. This feature occurs at a certain frequency in potato germplasm, and not all Solanum species are well suitable for this Agrobacterium-based expression system. Generally, the agroinfiltration assay works very well in N. benthamiana and most potato genotypes. In addition to effectoromics, there are various other potential applications for the agroinfiltration technique, such as production of proteins from transgenes and protein localization in plant cells by confocal microscopy.
PVX agroinfection is a highly sensitive screening system and typically more suitable for high-throughput screenings. Since the Agrobacterium is now only locally present, nonspecific responses to this bacterium are now not very disturbing, as the PVX virus takes over further spread of the transgene. However, plants may be resistant to PVX, or mount extreme resistance (ER) responses, and in that case the agroinfection method is not suitable. Another limitation of the PVX agroinfection method is the insert size of the gene of interest. Observed phenotypes of responses may vary from an intense black necrosis surrounding the wound to faint necrosis near the inoculation spot. In both N. benthamiana and Solanum species, PVX agroinfection is recognized as more sensitive than agroinfiltration.
Keeping into account that the genetic background of the diverse tested plant genotypes can have some restrictions (see above), we generally obtain similar conclusions by PVX agroinfection and agroinfiltration. These results are also comparable as obtained in other assays, such as protein infiltrations29 and ELISA3. Considering the advantages and limitations of both systems, we recommend using both methods to either complement each other or confirm independent results.

Effectoromics, a high-throughput functional genomics approach has recently emerged as a powerful tool to identify resistance (R) genes in crop plants and matching avirulence (Avr) genes of pathogens1-4. In contrast to the more time-consuming stable transformation with R genes, the effectoromics strategy is based on transient assays of pathogen gene sequences.
Since the genomics era, genomes of plant pathogens have widely been explored. For example for oomycetes, which include the most devastating plant pathogens, large collections of sequences have been generated and analyzed for genes that play a role during the interaction with the plant5-10. One class of pathogen proteins represents effectors, which manipulate host cell structure and function either to facilitate infection (virulence factors) or to trigger defense responses (avirulence factors)11-13. Expression of Avr genes in plant cells containing R genes usually results in the hypersensitive cell death response (HR)14,15. In planta expression of R and Avr genes can be accomplished using transient expression systems such as Agrobacterium tumefaciens -based transient transformation (agroinfiltration)16. This transient transformation can also be applied in combination with viral expression systems (agroinfection)17,18.
For agroinfiltration, the most commonly used agent is A . tumefaciens, a broad host range pathogen of dicot plants. A . tumefaciens contains a tumor-inducing (Ti) plasmid. Transfer DNA (T-DNA) from a Ti plasmid will translocate into the plant cells after the virulence machinery of the bacterium is activated. This can be triggered in wounded plant cells, by the released low-molecular-weight phenolic compounds and monosaccharaides in a slightly acidic environment19. The virulence gene is activated after the infiltration of Agrobacterium suspensions into leaf panels defined by major veins. Then plant cells in the leaf panels will be transformed and express the transgene(s) contained in the T-DNA region.
Agroinfection is based on wound-inoculated Agrobacterium, which mediates translocation of a virus to plant cells. The virus then further spreads to adjacent plant tissues, in the absence of Agrobacterium. For agroinfection, several plant viruses can be used. RNA viruses are ideal vectors for gene expression because they can multiply to very high levels in infected plants. Among plant RNA viruses, Potato Virus X (PVX) is widely used for effectoromics screens. To facilitate functional tests for an inserted gene, binary vectors that contain the PVX genome flanked by the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, were cloned into the T-DNA of A. tumefaciens20. After the T-DNA is transferred into plant cells, the PVX genome contained in the T-DNA is transcribed from the 35S promoter. Then virus particles spread systemically in the infected plants, resulting in the expression of the inserted gene. This method based on both Agrobacterium and PVX is called PVX agroinfection.
Here we show examples for both the agroinfiltration and PVX agroinfection assays. As host plants we use potato germplasm (Solanum section Petota), for which effectoromics approaches have been pioneered and proven successful3,4. We also use Nicotiana benthamiana, which is renowned as a model plant in Solanaceous plants14,21,22.

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			<td height="27" style="height:27px;width:252px;">Beef extract</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">B4888</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Bacteriological peptone</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0037</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Yeast extract</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0021</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MgSO4</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">208094</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MS salts (without vitamins)</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M0221</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MES</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M1503</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">LB broth powder</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">L3022</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Acetosyringone</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">D134406</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Syringe (1 ml)</td>
			<td style="width:267px;">BD Plastipak</td>
			<td style="width:219px;">300013</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Incubator</td>
			<td style="width:267px;">Infors HT</td>
			<td style="width:219px;">Multitron II</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Centrifuge</td>
			<td style="width:267px;">Heraeus</td>
			<td style="width:219px;">Multifuge 3S-R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Spectrophotometer</td>
			<td style="width:267px;">Eppendorf</td>
			<td style="width:219px;">Biophotometer 6131</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

agroinfiltration and pvx agroinfection in potato and nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Figure 1 shows a representative experiment of agroinfiltration with 7 different effectors (E1-E7) in potato and N. benthamiana. Cell death appears in the infiltrated leaf panels about 3 days after infiltration. The extent of the cell death phenotype needs to be compared with the controls. A mixture of the Agrobacterium strain AGL1(pVirG)23 containing pBINplus-R3a and pK7WG2-AVR3a was used as positive control24,25, while AGL1(pVirG) was used as negative control. Figures 1A and&#160;1B show good examples of agroinfiltration in the potato genotype MaR8 (Mastenbroek R8)26 and N. benthamiana, respectively, which are very amenable for agroinfiltration. There is confluent cell death in the leaf panel coinfiltrated with positive control, while no cell death response occurs with negative control or pBINplus-R3a. In MaR8, two effectors AVR3a (E2) and AVR4 (E3) induce a cell death, while the other effectors (E1 and E4-E7) do not. In N. benthamiana, none of the tested effectors induce a cell death response. Figures 1C and&#160;1D show examples of agroinfiltration in the wild potato Solanum berthaultii 483-1 and Solanum rechei 210-5, which are not well amenable for the agroinfiltration technique. In S. berthaultii 483-1, the leaf tissue shows a nonspecific necrosis to negative controls as well as to all tested effectors. In S. rechei 210-5, infiltrated leaf panels show very weak cell death response to positive control.
Figure 2 shows a range of scoring scales that can be used to quantify the response to agroinfiltrated Agrobacterium. Percentages of cell death are depicted on a scale 0-100%. Observed phenotypes range from macroscopically not visible symptoms (0%), through a range of intermediate responses displaying chlorosis and increasing levels of cell death, up to confluent cell death (100%).
Figure 3 shows a representative experiment after PVX agroinfection in potato. Normally, expanding cell death can be found at the sites about two weeks after tooth-pick inoculation. As shown in both figure panels, expanding cell death is present at the sites that were tooth-pick inoculated with pGR106-CRN2 (positive control using the effector crinkler Crn2), which is a general cell death inducing gene from P. infestans27. Apart from minor response to wounding, no expanding cell death is noted at the sites that were tooth-pick inoculated with the pGR106-empty (negative control). In Figure 3A, representing the potato genotype MaR3 (Mastenbroek R3), two pGR106-effectors (E1 and E2) did not induce cell death. In Figure 3B, a positive result of effector screening in the wild Solanum huancabambense 354-1 is presented; cell death response was observed to two pGR106-effectors (E1-E3, representing elicitins)28.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig1highres.jpg" src="/files/ftp_upload/50971/50971fig1.jpg" /> 
Figure 1. Examples of agroinfiltration in potato and N. benthamiana. Plants including (A) a potato genotype MaR8 (Mastenbroek R8), (B) N. benthamiana, (C) Solanum berthaultii 483-1 and (D) Solanum rechei 210-5 were infiltrated with a mixture of the Agrobacterium strain AGL1(pVirG) 23 containing pBINplus-R3a and pK7WG2-AVR3a (positive control), AGL1(pvirG) (negative control), pBINplus-R3a, and seven effectors (E1-E7). <a href="https://www.jove.com/files/ftp_upload/50971/50971fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig2highres.jpg" src="/files/ftp_upload/50971/50971fig2.jpg" /> 
Figure 2. Quantification of agroinfiltration responses.&#160;The photograph shows representative scoring scales for cell death, ranging from 0% (no symptoms) to 100% (confluent cell death). Intermediate responses range from weak responses such as chlorosis to increasing levels of cell death. <a href="https://www.jove.com/files/ftp_upload/50971/50971fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971fig3highres.jpg" src="/files/ftp_upload/50971/50971fig3.jpg" /> 
Figure 3. Examples of PVX agroinfection in potato.&#160;Potato genotype MaR3 (Mastenbroek R3) (A) and Solanum huancabambense 354-1 (B) tooth-pick inoculated with pGR106-CRN2 (positive control), pGR106-empty (negative control) and pGR106-E1-2 (effectors), or pGR106-E1-E3, respectively. <a href="https://www.jove.com/files/ftp_upload/50971/50971fig3highres.jpg" target="_blank">Click here to view larger image</a>.
Table 1. YEB medium
<table border="1">
	<tbody>
		<tr>
			<td>1 L</td>
			<td>distilled H2O</td>
		</tr>
		<tr>
			<td>5 g</td>
			<td>sucrose</td>
		</tr>
		<tr>
			<td>5 g</td>
			<td>beef extract</td>
		</tr>
		<tr>
			<td>5 g</td>
			<td>bacteriological peptone</td>
		</tr>
		<tr>
			<td>1 g</td>
			<td>yeast extract</td>
		</tr>
		<tr>
			<td>2 ml</td>
			<td>MgSO4 (1 M)</td>
		</tr>
	</tbody>
</table>
Table 2. Vectors and strains used for agroinfiltration and PVX agroinfection. Several binary vectors can be used. Vectors in the list below allow high expression of the candidate genes and have worked well in our hands. We prefer using the Agrobacterium strain GV3101(pMP90)29 in N. benthamiana and find AGL130 containing the helper plasmid pVirG (pBBR1MCS-5.virGN54D)23 more suitable in potato31. Additional strains have been analyzed in other groups on various model plants including N. benthamiana but not in potato32.
<img alt="Table 2" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table2highres.jpg" src="/files/ftp_upload/50971/50971table2.jpg" />
GW: gateway version36
Table 3. MMA medium. 
<img alt="Table 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table3highres.jpg" src="/files/ftp_upload/50971/50971table3.jpg" />
Adjust the pH to 5.6.

Watch this Scientific Journal Video about Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana at JoVE.com

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

agroinfiltration-pvx-agroinfection-potato-nicotiana

Research

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Biology

20.2K Views.  Wageningen University. Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Video: Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...