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

JoVE Journal

Biology

20.2K Aufrufe.  Wageningen University. Agroinfiltration und PVX-Agroinfektion sind routinemäßige funktionelle Assays für die transiente ektopische Expression von Genen in Pflanzen. Diese Methoden sind effiziente Assays in Effektoromik-Strategien (schnelle Resistenz und Avirulenz-Gen-Entdeckung) und entscheidend für die moderne Forschung in der molekularen Pflanzenpathologie. Sie erfüllen die Nachfrage nach robuster Hochdurchsatz-Funktionsanalyse in Anlagen.

Serie: 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 ...

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 ...

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 ...

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 ...

Quantifying Cell Death Initiation Through Continuous Imaging

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...

An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and diseases of the CPB are conducted during active feeding stages. Nonetheless, there are fewer studies on resting stages, although these beetles spend most of their life cycle in a state of winter diapause (hibernation). In this work, a method for investigating CPB hibernation under natural conditions was developed and tested, offering an opportunity to collect a sufficient number of individuals in winter. In this article, CPB survival was assessed, and infectious agents at different stages of hibernation were identified. CPB mortality increased during the hibernation, reaching a maximum in April-May. Entomopathogenic fungi (Beauveria, Isaria, and Lecanicillium) and bacteria Bacillus, Sphingobacterium, Peribacillus, Pseudomonas, and Serratia were isolated&#160;from the dead insects. The survival rate of the beetles for the entire hibernation period was 61%. No frozen or desiccated beetles were found, indicating the success of the presented method.

Here we present a method for studying Colorado potato beetle hibernation under the natural conditions of the temperate zone as well as a technique for collecting beetles in winter. This method allows to obtain a desired number of overwintering individuals for various analyses at any stage of hibernation.

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and ...

Potato Resilience Analysis Under Climate-Related Stresses

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Author Spotlight: Unraveling Plant Responses to Abiotic Stresses Using the PlantScreen Robotic Platform

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

Scale Up Culture of Transformed Agrobacterium tumefaciens and Agroinfiltration of Large-Sized Woody Plants

To begin, streak transform Agrobacterium tumefaciens cell culture onto selective antibiotic supplemented Yeast Mannitol Agar plates. Incubate the plates overnight at 28 degrees Celsius in a standing incubator. The next day, inoculate colonies from the culture on 12.5 milliliters of Yeast Mannitol Luria-Bertani broth mix, and incubate on an orbital shaker at 28 degrees Celsius for 16 hours at 250 RPM.Inoculate 12.5 milliliters of the Agrobacterium strain LBA4404 in 112.5 milliliters of Yeast Mannitol broth with antibiotics. Incubate the culture on an orbital shaker at 28 degrees Celsius for 16 hours at 250 RPM. Adjust the overnight culture to an optical density of 0.4 with the culture broth.Then add 20 micromolar of acetosyringone before incubation. Once the incubated culture reaches an optical density of one, centrifuge to pellet the cells. Resuspend the pellet in suspension solution to an optical density of 0.6, and add 200 micromolar acetosyringone before incubation.For agroinfiltration, pick woody cacao plants that have branches with leaves. Add 250 micromolar jasmonic acid to the Agrobacterium suspension. Next, set up a desiccator with a vacuum gauge as a vacuum chamber to measure the pressure inside.Place a jump ring to allow the plant branch to pass through. Place the beaker with Agrobacterium culture inside the desiccator, and place the branch between the desiccator and its lid, with the desired leaves submerged in the Agrobacterium culture. Next, use silicone impression material to seal the gaps between the jump ring, the plant branch, and the desiccator.Once the silicone material has polymerized, close the desiccator, leaving no gaps, then connect it to the vacuum pump. Start the vacuum pump until it reaches minus 0.07 megapascals. When the desired pressure is reached, close the pressure valve, and turn off the pump to maintain the pressure for five minutes.Open the pressure valve gradually and steadily to restore the chamber pressure. After repeating infiltration two more times, take the branch off the cell suspension. Clean the infiltrated leaves with distilled water, then place the plants at 25 degrees Celsius in the dark for 48 hours.After incubation, expose the infiltrated tissue to a 16 hour light and eight hour dark photo period. Infiltrated leaves of the plants appear darker in certain parts, indicative of Agrobacterium penetration. The reporter system used produced betalain accumulation on the leaves, which can be easily observed as bright red pigmentation with the naked eye.Betalain accumulation was observed to vary with the tissue viability.

Efficient Extraction and Purification of GFP-His from &lt;i&gt;N. benthamiana&lt;/i&gt; Leaves

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein  

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

Author Spotlight: Optimizing Apoplast Protein Extraction for Efficient Recovery of Recombinant Proteins from Plant Cells

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...