The work was supported by the National Science Foundation (IOS-1901566) to S. Xiao. The authors would like to thank F. Coker and C. Hooks for the maintenance of the plant growth facility, and Jorge Zamora for technical help associated with fabrication of the inoculation box and chamber.

1. Making a standard inoculation box with a removable top lid mounted with a mesh
<ol>
	<li>Purchase a roll of 50 &#181;m nylon membrane mesh or 48 &#181;m stainless steel mesh (recommended) from stores. Make sure to order enough for cutting into multiple pieces of 14 in x 26 in for replacement of worn mesh.</li>
	<li>Purchase one 1/4 in x 2 ft x 4 ft medium density fiberboard or plywood, and cut two 24-1/2 in x 10 in pieces and two 12 in x 10 in pieces for making an inoculation box.Use 8 corner clamps for supporting the four sides. 
	NOTE: If facing the broad side of the enclosure, the left 12 in x 10 in side is attached permanently and the right 12 in x 10 in side is a removable door held in place by four magnetic catches (Figure 1A,B).</li>
	<li>Place four 2-in corner braces two in each inside corner of the left side, an inch from the top and an inch from the bottom, mark and drill a hole. Use screws to permanently attach the left short side to two long sides (front and back). The inner distance between the two long sides should be 12 in and not 11-1/2 in.</li>
	<li>With the corner clamps still in place and proper alignment of the right door, mark and drill the location of the 4 magnetic catches to hold the right detachable door in place. Install a button knob to the door. The top of the door catches under the top lid (Figure 1A,B).</li>
	<li>To make a removable top lid with a mesh, cut the long wooden square dowel into 2 sets of 26 in and 12-3/4 in. Glue and nail the four pieces in such a way that it will fit around the box - the inside dimensions of the frame being a little bigger than the outside dimensions of the box, as the screen mesh will be attached to this frame.</li>
	<li>Cut the screen mesh to a little less than 14 in x 26 in. Do not allow the screen to protrude past the frame so as to prevent sharp edges. Have assistance to stretch the screen on the frame. Hold the mesh with the frame by clamps (Figure 1C) and use a stapler gun with the right staples to secure the screen to the frame.</li>
	<li>Cut two sets of shorter wooden square dowel (21 in and 9 in), and use screws to tightly sandwich the mesh along the frame using the 4 shorter pieces of wooden square dowel (Figure 1C, D).</li>
</ol>
2. Making an inoculation chamber
<ol>
	<li>Purchase clear acrylic sheets and hardware items from appropriate stores. Cut a 7 in x 20 in opening window on the front side acrylic sheet of the box as shown in Figure 2C.</li>
	<li>Assemble the left and right sides, front (with window) and rear acrylic sheets into a box using 8 corner clamps as shown in Figure 2A. Ensure that the inner measurements of the chamber are equal to the dimensions of the top sheet. 
	NOTE: The right side is there for support and will not be attached to the front or rear sheets. It is &#189; in shorter than the other sheets on purpose. This right side will become a &#34;draw down&#34; &#34;flap&#34; door held in place by a right angle&#160;hinge at the bottom and two magnetic catches at the top as shown in Figure 2C,D.</li>
	<li>Use corner braces to secure the panels together (Figure 2B,C). Hold the braces in place, mark with a sharpie marker, center punch and then drill the hole. 
	NOTE: Drilling acrylic is different from drilling anything else, do not force or the side of the hole opposite the drill may crack. Use rivets or screws. Do not overtighten the screws or the acrylic may crack with time.</li>
	<li>Turn the enclosure on its rear side and remove the 4 corner clamps and position the top sheet in place. Mark and drill.</li>
	<li>Install the right angle hinge, made out of a &#190; in angle brace. Use a nylon retaining lock nut as a hinge rod. Do not tighten, leave a gap so the door will rotate on this hinge.</li>
	<li>Mark, center punch, drill and attach the two magnetic door magnets with #6-32x3/4&#34; pan head screws. Then attach the catch plates with #6-32x3/8&#34; flat head screws, see Figure 2C,D.</li>
</ol>
3. Inoculating plants in flats
<ol>
	<li>Prepare fresh powdery mildew spores as inocula (e.g., Arabidopsis powdery mildew Golovinomyces cichoracearum UCSC1). Grow clean Arabidopsis immune-compromised pad4-1 mutant plants 11 in a growth chamber (22 &#176;C, 65% relative humidity for 6-8 weeks), and inoculate one or half flat of pad4-1 plants to build up sufficient fresh powdery mildew spores (Figure 3A).</li>
	<li>Grow the plants for determining infection phenotypes in standard 11 in x 21 in x 2.5 in flats. 
	NOTE: Arabidopsis plants grown under short-day (8 h light, 16 h dark; 22 &#176;C) for six to eight weeks are good for infection tests with powdery mildew.</li>
	<li>When fresh powdery mildew spores from infected plants at 8-10 days post-inoculation (dpi) are available, move a flat of plants for infection test into the inoculation box inside the inoculation chamber (Figure 1B,2C).</li>
	<li>Cut 15-20 heavily infected Arabidopsis leaves (or 4-6 infected rosettes, depending on the leaf/rosette size and level of inoculation), let the leaves dry if there is condensed water.</li>
	<li>Grab 1-2 leaves or a rosette with a pair of long forceps using one hand and face the leaves upside down. Then, put both hands through the opening of the chamber and gently hit the arm of the forceps with a pair of scissors while slowly moving the leaves/rosette above the mesh mounted on the inoculation box (Figure 3B). Repeat this until all the leaves or rosettes are used. 
	NOTE: Mature spores will dislodge easily from conidiophores of the infected leaves. Try to dislodge spores on the mesh as evenly as possible.</li>
	<li>Use one or two fine fan-blender brushes to gently brush the spores through the mesh (Figure 3C). Ensure to brush the entire surface of the mesh in different directions for four or more times to maximize even distribution of spores onto the plants in the bottom of the inoculation box (Figure 3D). Gently pat the mesh for a few times with the brushes to shake off the spores that are stuck in the mesh.</li>
	<li>Let the spores settle down for half a minute. Then, move out the flat containing the inoculated plants from the inoculation box. Cover the flat with a plastic dome and place it in a growth chamber (22 &#176;C, 65% relative humidity). Disease phenotyping can be performed at 5, or 8 to 12 dpi 12.</li>
</ol>
4. Inoculate plants with smaller inoculation boxes
NOTE: In cases where fewer plants are to be inoculated, the standard inoculation box can still be used. Make sure to place the plants in the middle of the box. Dislodge and brush spores in the area of the mesh that covers the plants to ensure all plants are inoculated while saving inocula. Alternatively, and preferably, smaller inoculation boxes can be used (as described below).
<ol>
	<li>Convert cardboard boxes to an inoculation box. Simply remove the top and bottom sides of the box, tape the corner to firm it if necessary, and mount a suitably sized 50 &#181;m mesh on the top by gluing or stapling (Figure&#160;4A,B).</li>
	<li>Move the flat containing the plants inside the inoculation chamber, place the smaller inoculation box on top of the flat to cover the plants.</li>
	<li>Dislodge and brush spores as described above.</li>
</ol>
5. Inoculate detached leaves in Petri dishes
NOTE: In cases where (i) fresh powdery mildew spores are very limited and/or (ii) plants must be kept clean while disease phenotypes and/or protein subcellular localization in infected cells need to be assessed, detached leaves can be used for infection in MS-agar plates.
<ol>
	<li>Make a mini inoculation box that is only slightly bigger than a Petri dish.</li>
	<li>Prepare &#189;-strength Murashige and Skoog (MS) medium agar (1.5%) plates. Add thiabendazole (40 mg/L) to reduce mold contamination if required. Store the plates at 4 &#176;C in a fridge until use.</li>
	<li>Cut one or two fully expanded leaves from the base of the petiole for each individual plant to be tested. Insert the petiole of the detached leaves into agar medium at an angle of ~30&#176;. 
	NOTE: Each plate can contain 20-40 leaves depending on the size of the leaves and the plate used (Figure 4C).</li>
	<li>Inoculate the leaves inside the inoculation chamber as described above. Move the plate with the lid to a growth chamber.</li>
	<li>Cut a small section of a leaf and check the subcellular localization of proteins at specific time-points.</li>
	<li>For assessing infection phenotypes, transfer the leaves to a fresh aseptic MS-agar plate at 4 or 5 dpi. Cut the base of the petiole before inserting the leaves into the fresh agar medium. Assess the disease phenotypes at 8 or 9 dpi.</li>
</ol>

<ol>
	<li>Huckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bi+ology+of+the+plant-powdery+mildew+interaction.">Cell bi ology of the plant-powdery mildew interaction.</a> Current Opinion in Plant Biology. 14 (6), 738-746 (2011).</li><li>Dean, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+top+10+fungal+pathogens+in+molecular+plant+pathology.">The top 10 fungal pathogens in molecular plant pathology.</a> Molecular Plant Pathology. 13 (4), 414-430 (2012).</li><li>Sakurai, H., Hirata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+the+relation+between+the+penetration+hypha+and+haustorium+of+barley+powdery+mildew+and+host+cell.+V.+Influence+of+water+spray+on+the+pathogen+and+host+tissue.">Some observations on the relation between the penetration hypha and haustorium of barley powdery mildew and host cell. V. Influence of water spray on the pathogen and host tissue.</a> Annual Phytopathology Society of Japan. 24, 239-245 (1959).</li><li>Shomari, S. H., Kennedy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+Oidium+anacardii+on+cashew+(Anacardium+occidentale)+in+southern+Tanzania.">Survival of Oidium anacardii on cashew (Anacardium occidentale) in southern Tanzania.</a> Plant Pathology. 48 (4), 505-513 (1999).</li><li>Sitterly, W. R., Spencer, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Powdery Mildews. , 369 (1978).</li><li>Reuveni, M., Agapov, V., Reuveni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+systemic+resistance+to+powdery+mildew+and+growth+increase+in+cucumber+by+phosphates.">Induction of systemic resistance to powdery mildew and growth increase in cucumber by phosphates.</a> Biological Agriculture &amp; Horticulture. 9 (4), 305-315 (1993).</li><li>Reifschneider, F. J. B., Boiteux, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vacuum-operated+settling+tower+for+inoculation+of+powdery+mildew+fungi.">A vacuum-operated settling tower for inoculation of powdery mildew fungi.</a> Phytopathology. 78 (11), 1463-1465 (1988).</li><li>Chowdhury, A., Bremer, G. B., Salt, D. W., Miller, P., Ford, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+delivering+Blumeria+graminis+f.+sp+hordei+spores+for+laboratory+experiments.">A novel method of delivering Blumeria graminis f. sp hordei spores for laboratory experiments.</a> Crop Protection. 22 (7), 917-922 (2003).</li><li>Xiao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+mildew+resistance+in+Arabidopsis+thaliana+mediated+by+RPW8.">Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8.</a> Science. 291 (5501), 118-120 (2001).</li><li>Xiao, S., Ellwood, S., Findlay, K., Oliver, R. P., Turner, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+three+loci+controlling+resistance+of+Arabidopsis+thaliana+accession+Ms-0+to+two+powdery+mildew+diseases.">Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms-0 to two powdery mildew diseases.</a> The Plant Journal. 12 (4), 757-768 (1997).</li><li>Reuber, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+defense+gene+induction+defects+with+powdery+mildew+susceptibility+in+Arabidopsis+enhanced+disease+susceptibility+mutants.">Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants.</a> The Plant Journal. 16 (4), 473-485 (1998).</li><li>Xiao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atypical+resistance+gene,+RPW8,+recruits+components+of+basal+defence+for+powdery+mildew+resistance+in+Arabidopsis.">The atypical resistance gene, RPW8, recruits components of basal defence for powdery mildew resistance in Arabidopsis.</a> The Plant Journal. 42 (1), 95-110 (2005).</li></ol>

The authors have nothing to disclose.

Our meshed-box-based inoculation method has several advantages over other inoculation methods. First, it can achieve even distribution of spores if operated properly, as demonstrated in Figure 5. Second, the use of ~50 &#181;m mesh, plus spore-dislodging by gentle shaking of infected leaves can reduce plant infection by thrips or other plant-infecting insects that are present in source plants. Third, the use of different-sized inoculation boxes for inoculating plants or detached leaves insid...

Powdery mildew is one of the most common and important diseases of numerous food crops and ornamental plants1. Studies of powdery mildew diseases have been very popular, as evidenced by over 10,500 publications as the search result with "powdery mildew" as key word at the Web of Science (as of November 2020). Indeed, powdery mildew (represented by Blumeria graminis) is considered to be one of the top 10 fungal pathogens by the journal of Molecular Plant Pathology2. Quantification of disease susceptibility is a necessary step in characterization of plant genes contributing to disease resistance or susceptibility, or functional identification of candidate effector genes in powdery mildew. However, reliable disease phenotyping is far more challenging with powdery mildew compared to that with most other fungal pathogens, partly because, unlike spores of the latter, spores of powdery mildew species (such as Golovinomyces cichoracearum UCSC1 based on our lab experience) show reduced viability after going through a water-suspension process3,4. Inadequate and/or uneven inoculation of test plants with a particular powdery mildew pathogen may lead to inaccurate phenotyping results.
A number of inoculation methods were reported for powdery mildew studies. These include (i) brushing spores directly from infected leaves to test plants5, (ii) spraying a spore suspension to test plants6, (iii) blowing spores using a vacuum-operated settling tower to plants at the bottom of the tower7, and (iv) spore delivery by the combinatorial use of a nylon mesh membrane and sound-based vibration8. The spore-brushing (or dusting) method is easy to perform but uneven in nature, thus it may not be accurate for quantitative assessment. Spore-spraying is convenient and even, but as stated above may result in poor spore germination4. The latter two (i.e., iii-iv) are much-improved methods capable of achieving even inoculation; however, both are not flexible in adjusting their inoculation capacity in terms of the number of plants to be inoculated in a single event, making either apparatus is not trivial, and their operation is restricted to lab areas where there is a vacuum and/or electricity source.
Our lab has been working with plant-powdery mildew interaction for over 20 years9,10. Over the past decade, we tested a number of inoculation methods and recently developed a simple and yet effective powdery mildew inoculation method. This mesh-based spore-brushing method can ensure even inoculation, and is simple and scalable, thus should be easily adopted by any laboratory working with powdery mildew.

<table><tbody><tr><td>&amp;nbsp;48 &amp;micro;m stainless steel grid mesh screen; Size: 24&quot; X 48&quot;&amp;nbsp;</td><td>Amazon</td><td>NA</td><td>For making the lid of an inoculation box</td></tr><tr><td>#6-32 x &amp;frac34;&quot; machine screws, flat washers and nuts&amp;nbsp;</td><td>Home Depot</td><td>NA</td><td>For making an inoculation chamber</td></tr><tr><td>#6-32 zinc plated nylon lock nut (4-Pack)</td><td>Home Depot</td><td>NA</td><td>For making an inoculation chamber</td></tr><tr><td>#6-32x3/8&amp;rdquo; Phillips flat head machine screws, flat washers and nuts&amp;nbsp;</td><td>Home Depot</td><td>NA</td><td>For securing&amp;nbsp; magnet door catch plates</td></tr><tr><td>#8-32x1/2&quot; machine screws, flat washers and nuts</td><td>Home Depot</td><td>NA</td><td>For securing corner braces and door hinge</td></tr><tr><td>0.250 thick clear extruded acrylic film-masked sheet;&amp;nbsp; Size: 17 &amp;frac12;&quot; X 20&quot;</td><td>Professional Plastics</td><td>SACR.250CEF</td><td>For making an inoculation chamber</td></tr><tr><td>0.250 thick clear extruded acrylic film-masked sheet; Size: 18&quot; X 20&quot;&amp;nbsp;&amp;nbsp;</td><td>Professional Plastics</td><td>&amp;nbsp;SACR.250CEF</td><td>For making an inoculation chamber</td></tr><tr><td>0.250 thick clear extruded acrylic film-masked sheet; Size: 18&quot; X 30&quot;&amp;nbsp;</td><td>Professional Plastics</td><td>SACR.250CEF</td><td>For making an inoculation chamber</td></tr><tr><td>0.250 thick clear extruded acrylic film-masked sheet; Size: 20&quot; X 29 &amp;frac12; &quot;</td><td>Professional Plastics</td><td>SACR.250CEF</td><td>For making an inoculation chamber</td></tr><tr><td>1-5/8&quot; cabinet door magnetic catch white</td><td>Home Depot</td><td>Model #P110-W</td><td>For making an inoculation chamber</td></tr><tr><td>2&quot; steel zinc-plated corner brace (8-Pack)&amp;nbsp;</td><td>Home Depot</td><td>&amp;nbsp;Model #13611&amp;nbsp;</td><td>For making an inoculation box &amp;amp; chamber</td></tr><tr><td>3&quot; Corner Clamp</td><td>Harbor Freight Tools</td><td>SKU 63653, 1852, 60589</td><td>For making inoculation chamber</td></tr><tr><td>3/4&quot;&amp;nbsp; steel zinc plated corner brace (4-Pack)</td><td>Home Depot</td><td>Model #13542</td><td>For making an inoculation box &amp;amp; chamber</td></tr><tr><td>4-7/8&quot; zinc-plated light duty door pull handles</td><td>Home Depot</td><td>Model #15184</td><td>For making an inoculation box</td></tr><tr><td>Fine fan-blender brushes</td><td>Michaels Store</td><td>M10472846&amp;nbsp;</td><td>For inoculation</td></tr><tr><td>Kelleher 3/4&quot; x 3/4&quot; x 36&quot; wood square dowel&amp;nbsp;</td><td>Home Depot</td><td>NA</td><td>For making the lid of an inoculation box</td></tr><tr><td>Medium density fiberboard (1/4&quot; x 2&#039; x 4&#039;);&amp;nbsp;</td><td>Home Depot</td><td>Model# 1508104</td><td>For making an inoculation box</td></tr><tr><td>Round glass coverslips with a 500 &amp;micro;m grid</td><td>ibidi USA Inc.</td><td>10816</td><td>For determining&amp;nbsp; spore density</td></tr></tbody></table>

an easy and flexible inoculation method for accurately assessing powdery mildew-infection phenotypes of arabidopsis and other plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

Here, we present a new powdery mildew spore inoculation method that is easy to prepare, operate and adjust. Figure 1 shows the assembly of the standard inoculation box with emphasis on the make of the removable lid mounted with a 50 &#181;m membrane mesh. Figure 2 shows the assembly of the inoculation chamber. Figure 3 illustrates the key steps of the inoculation process using this system. Figure 4 show...

Watch this Scientific Journal Video about An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants at JoVE.com

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

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Research

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Biology

2.6K Views.  University of Maryland. Echte meeldauw is een belangrijke plantenziekte. Nauwkeurige bepaling van het fenotype van de ziekte vereist zelfs inenting van de ziekteverwekker. Omdat bestaande methoden inefficiënt zijn, hebben we dit protocol ontwikkeld.Onze sporeninentingsmethode kan gemakkelijk zelfs inenting bereiken en is eenvoudig en schaalbaar. Vandaar dat elk laboratorium dat met echte meeldauw werkt, deze methode gemakkelijk kan gebruiken. De onderzoekers kunnen ook gaas over een kartonnen doos monteren e...

Video: An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Immunology and Infection

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Environment

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

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).

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.

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

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the southeastern USA, especially in Florida. Deployment of integrated pest management strategies, such as race-specific resistant cultivars, requires information on the diversity and population density of the pathogen in growers' fields. Despite some progress in developing molecular diagnostic tools to identify pathogen isolates, race determination often requires bioassay approaches.
Race typing was conducted by root-dip inoculation, infested kernel seeding method, and the modified tray-dip method with each of the four watermelon differentials (Black Diamond, Charleston Grey, Calhoun Grey, Plant Introduction 296341-FR). Isolates are assigned a race designation by calculation of disease incidence five weeks after inoculation. If less than 33% of the plants for a particular cultivar were symptomatic, they were categorized as resistant. Those cultivars with incidence greater than 33% were regarded as susceptible. This paper describes three different methods of inoculation to ascertain race, root-dip, infested kernel, and modified tray-dip inoculation, whose applications vary according to the experimental design.

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the ...

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

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

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

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

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

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

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