Name: bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem
Uploaded: 2015-10-01T13:00:00
Description: Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

The following text describes a stepwise protocol to perform optimized Psm ES4326 syringe infiltration assay in Arabidopsis. Major procedures of this assay are represented in a simplified flowchart (Figure 1).
1. Plant Growth Conditions
<ol>
	<li>Sow seeds
	<ol>
		<li>Prepare 2 pots (4 in&#160;diameter, 3.75 in&#160;tall) loosely filled with soil and water pots by soaking them from the bottom O/N before draining the excess water.</li>
		<li>Sow 50-100 Arabidopsis see...

With decreasing available farmland and increasing population, researchers around the world are challenged with pressing needs for crop improvement. The yield can be greatly influenced by various biotic and abiotic stresses. Among them, pathogen infection is one of the leading causes of crop yield reduction, responsible for approximately 12% losses in the U.S. alone45. To resolve this issue, massive research has been conducted in the model Arabidopsis - P. syringae pathosystem to comprehensively charac...

Due to their sessile nature, plants are constantly threatened by a plethora of pathogens exhibiting various lifestyles and nutritional strategies1. To a first approximation, biotrophic pathogens maintain their host alive to retrieve nutrients, while necrotrophic pathogens actively secret toxins and enzymes to kill host tissue and feed on the dead cells1. Another group of pathogens, termed hemibiotrophs, begins their infection course with the biotrophic stage and shifts to the necrotrophic stage upon reaching a certain threshold of pathogen accumulation2. In order to effectively defend themselves against these microorganisms, plants have evolved a complicated innate immune system equipped with multiple surveillance mechanisms to detect the pathogen attack and trigger localized programmed cell death3 as well as Systemic Acquired Resistance (SAR)4. Current research is focused on characterizing the essential signaling components and cross-talks within the plant immune system5.

As proposed in the &#8220;Zig-Zag&#8221; model5, the first layer of the plant innate immune response requires the presence of plasma membrane-localized Pattern Recognition Receptors (PRRs) to detect the invasion of a microbe. PRRs are able to recognize Microbe-Associated Molecular Patterns (MAMPs) and establish MAMP-Triggered Immunity (MTI)6. Besides inducing a transcriptional upregulation of genes encoding antimicrobial PR proteins7, MTI leads to a variety of events that arrest pathogen growth, including the production of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), deposition of callose to the cell wall as well as the activation of multiple kinase signaling pathways8.
Until now, several MAMPs have been identified to trigger MTI in Arabidopsis, including bacterial flg229 (a 22 amino acid fragment derived from flagellin), elf1810 (18 amino acids from the bacterial translation elongation factor Tu) and a structural cell wall component peptidoglycans11. To establish a successful infection, some specialized pathogens have evolved the ability to secret virulence effector proteins into the intracellular or intercellular spaces, and consequently repress MTI and trigger Effector-Triggered Susceptibility (ETS)12,13. For instance, virulence effectors can inactivate Mitogen-Activated Protein Kinase (MAPK) phosphorylation cascades of MTI to induce the disease development within the infected tissue14-16. During the dynamic co-evolution between hosts and pathogens, plants also developed the counterattack strategy to recognize the effector proteins and attenuate the pathogen virulence molecules17. This direct or indirect effector recognition is mediated by disease resistance (R) proteins18. Most of them are members of NB-LRR (Nucleotide Binding and Leucine-Rich Repeats) family19. The perception of an avirulent effector by an R protein elicits a stronger and broader immune response characterized as Effector-Triggered Immunity (ETI)20. Besides inducing the expression of defense genes21 and the production of defense metabolites22, ETI often leads to a rapid localized programmed cell death known as Hypersensitive Response (HR) to restrict the pathogen from spreading into the adjacent tissue3.
In addition to the localized programmed cell death23, plants are capable of initiating a long-term and system-wide immune response termed Systemic Acquired Resistance (SAR)4. Upon challenge with a biotrophic pathogen, plant cells trigger the biosynthesis and accumulation of an endogenous phytohormone salicylic acid (SA) and PR proteins in both local and systemic tissues24. Through this process, a heightened state of preparedness is achieved in the uninfected leaves that allows for mounting faster defense responses during a subsequent infection by a broad spectrum of pathogens24. SA and its synthetic analogs such as benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and 2,6-dichloroisonicotinic acid (INA) are capable of chemically inducing Salicylic Acid (SA)-Triggered Immunity (STI) upon external application24. Nonexpressor of Pathogenesis-Related genes 1 (NPR1) is proposed to be one of the SA receptors and functions as a major transcriptional regulator during SA-mediated defense response in both local and systemic tissues21,25,26. It has been conclusively demonstrated that NPR1 is required for SAR establishment and the loss of NPR1 leads to dramatic susceptibility towards Pseudomonas syringae25.
To extensively characterize the molecular contribution of plant components in the plant-pathogen interactions, multiple bioassays have been developed to measure specific defense events, including ROS burst27, callose deposition28, defense genes expression and accumulation of their protein products21. While these individual assays can provide insights into a specific form of the plant immune response, none of them, however, are able to represent the complete defense response on the whole plant level. Conversely, quantification of pathogen growth after infection provides an overall estimation of immune response at the organismal level. Therefore, the development and optimization of a precise and highly standardized pathogen inoculation assay is critical to fuel up the research and discoveries on the Arabidopsis immune responses.
Pseudomonas syringae, a Gram-negative bacterium, was identified as a phytopathogen capable of causing disease in a range of plant hosts including Arabidopsis29. As the model plant-pathogen system, the Arabidopsis - P. syringae interaction has been widely applied to understand the molecular mechanisms underlying plant defense responses29. Until now, over 50 P. syringae pathovars have been identified based on their ability to infect different plant species30 . P. syringae pv. tomato DC3000(Pst DC3000)31and P. syringae pv. maculicola ES4326 (Psm ES4326)32 are the two most widely used and extensively characterized virulent strains. Aside from being recognized by the plant and triggering the MTI response, Pst DC3000 and Psm ES4326 are capable of secreting virulent effector proteins to suppress MTI and trigger ETS to favor the pathogen growth31,33.
To functionally dissect the interaction between Arabidopsis and P. syringae, multiple&#160;pathogen infection methods have been developed based on the pathogen delivery approach. For soil-grown plants, pathogen can be delivered by syringe infiltration, vacuum infiltration, dip inoculation and spray inoculation29,34. Recently, seedling flood-inoculation assay was developed to perform large-scale screens on tissue culture plates-grown young Arabidopsis plants35. Syringe infiltration, as one of the most commonly used approach, manually delivers the pathogen into the apoplast through the natural leaf openings termed stomata29. Through this approach, equal amounts of P. syringae can be infiltrated into the infected leaf and the strength of plant immune response is inversely correlated to the pathogen growth levels. Therefore, quantification of pathogen growth serves as an optimal approach to evaluate the immune function at the whole plant level. In addition, syringe infiltration can distinguish the local and systemic tissue, which can be applicable in characterizing the molecular mechanisms underlying SAR36.
In the following protocol, we describe an optimized syringe infiltration assay with Psm ES4326 to screen Arabidopsis mutants for enhanced disease susceptibility (EDS). This protocol will employ two Arabidopsis genotypes: a wild-type ecotype Columbia-0 (Col-0) plants (control) and npr1-1 loss-of-function&#160;mutants (hypersusceptible) that will be infected with virulent bacterial strain Psm ES432637. The npr1-1 mutant carries a point mutation within the ankyrin-repeat consensus sequence of the NPR1 molecule, which alters the highly conserved histidine to tyrosine and renders the protein non-functional25. In addition, a number of modifications of the syringe infiltration assay are described that allow quantification of defects in the specific layers of immune response, including MTI and STI.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MetroMix 360&#160;</td>
			<td>Grosouth</td>
			<td>SNGMM360</td>
			<td></td>
		</tr>
		<tr>
			<td>Large pots</td>
			<td>Grosouth</td>
			<td>TEKUVCC10TC</td>
			<td></td>
		</tr>
		<tr>
			<td>12x6 Inserts</td>
			<td>Grosouth</td>
			<td>LM1206</td>
			<td></td>
		</tr>
		<tr>
			<td>11x21 Flats with no holes</td>
			<td>Grosouth</td>
			<td>LM1020</td>
			<td></td>
		</tr>
		<tr>
			<td>11x21 Flats with holes</td>
			<td>Grosouth</td>
			<td>LM1020H</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinyl propagation domes</td>
			<td>Grosouth</td>
			<td>CW-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose Peptone</td>
			<td>Fisher Scientific</td>
			<td>DF0122-17-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic Trihydrate&#160;</td>
			<td>MP Biomedicals</td>
			<td>151946</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>A360-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Bio Basic Inc</td>
			<td>SB0494</td>
			<td></td>
		</tr>
		<tr>
			<td>100x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>150x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>R80150</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular plate</td>
			<td>Fisher Scientific</td>
			<td>12-565-450&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 Hexahydrate</td>
			<td>Bio Basic Inc</td>
			<td>MB0328</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bio Basic Inc</td>
			<td>GB0232</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#160;</td>
			<td>Bio Basic Inc</td>
			<td>MN1988</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>NC9992493&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding tubes&#160;</td>
			<td>Denville Scientific</td>
			<td>B1257</td>
			<td></td>
		</tr>
		<tr>
			<td>Caps for grinding tubes</td>
			<td>Denville Scientific</td>
			<td>B1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grinding ball</td>
			<td>Fisher Scientific</td>
			<td>2150</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-556-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Salicylate</td>
			<td>Sigma Aldrich</td>
			<td>s3007-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>flg22 (QRLSTGSRINSAKDDAAGLQIA)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
		</tr>
		<tr>
			<td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td>146308</td>
			<td></td>
		</tr>
		<tr>
			<td>Biophotometer plus</td>
			<td>Eppendorf</td>
			<td>952000006</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerGen High-Throughput Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>02-215-503</td>
			<td></td>
		</tr>
		<tr>
			<td>Accu spin micro centrifuge</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (10-100 &#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.043</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (30-300&#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.060</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (20&#181;l)</td>
			<td>Eppendorf</td>
			<td>3120 000.038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>3552-HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpie permanent marker</td>
			<td>Staples</td>
			<td>507130</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>08-890</td>
			<td></td>
		</tr>
	</tbody>
</table>

bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

The protocol we describe here represents an optimized P. syringae syringe infiltration assay to quantitatively evaluate the immune response in Arabidopsis plants. As illustrated in Figure 1, the syringe infiltration of Psm ES4326 is followed by pathogen extraction and quantification via serial dilutions and colonies enumeration.
As described in Step 3 within the protocol text, Enhanced Disease Susceptibility (EDS) against Psm ES4326 can be a...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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