This work was supported by grants from the &#34;Shuguang Program&#34; of Shanghai Education Development Foundation and Shanghai Municipal Education Commission, the National Key Research and Development Program of China National Key R&amp;D Program of China (2018YFD0201500), the National Natural Science Foundation of China (no. 31571696 and 31660510), the Thousand Talents Program for Young Professionals of China, and the Science and Technology Commission of Shanghai Municipality (18DZ2260500).

NOTE: All steps of the procedure should be conducted at room temperature (RT).
CAUTION: Deposit all media containing pathogenic microbes, as well as the plants and plant tissue used in the assay, into the appropriate waste containers and autoclave before discarding.
1. Preparation of plasmid constructs containing putative effectors
<ol>
	<li>Select putative secreted effectors that are highly expressed during infection, as determined by RNA sequencing (RNA-Seq), and further by quantitative real time PCR (qRT-PCR) technique.</li>
	<li>Determine the signal peptide cleavage site of each effector using a software tool such as SignalP18.</li>
	<li>Design primers and perform PCR to amplify the gene encoding the effector of interest using high fidelity DNA polymerase. Perform agarose gel electrophoresis to check the PCR product, then clone the amplicon into Gateway entry vector pQBV3 using the ligation-free cloning kit (Table of Materials), and subsequently into the destination expression vector pEG100 using the LR recombination reaction19.</li>
	<li>Transform 3&#8722;5 &#181;L of LR reaction mixture into 100 &#181;L of competent E. coli cells (e.g., TOP10, DH5&#945;), spread the transformed bacterial cells on Luria-Bertani (LB) agar medium supplemented with 50 &#181;g/mL kanamycin, and incubate at 37 &#176;C for 16&#8722;20 h. 
	NOTE: Positive transformants can be identified by colony PCR20 and further analyzed by plasmid miniprep and sequencing.</li>
	<li>Introduce 50&#8722;100 ng of recombinant plasmids carrying effector protein into 100 &#181;L of chemically competent A. tumefaciens cells (e.g., GV3101 or C58C1), mix gently, and then freeze in liquid nitrogen for 1 min. Thaw the cells in a 37 &#176;C water bath for 5 min.</li>
	<li>Add 500 &#181;L of LB broth and incubate at 30 &#176;C for 4&#8722;6 h with gentle shaking, and then transfer and spread all the agrobacteria cells on LB agar medium supplemented with 50 &#181;g/mL kanamycin and 50 &#181;g/mL rifampicin at 30 &#176;C for 2 days. 
	NOTE: Positive clones can be verified by colony PCR.</li>
	<li>Use a single transformed colony for agro-infiltration experiments unless otherwise directed. 
	NOTE: In the present research, none of the tested effector genes contained predicted introns; each of the genes was directly amplified from genomic DNA of a Phytophthora sojae isolate. A Gateway plant expression vector without any tag is recommended, but not necessary.</li>
</ol>
2. Preparation of N. benthamiana 16c plants
<ol>
	<li>Prepare potting soil mixes consisting of (by volume) 50% peat moss, 30% perlite, and 20% vermiculite, and autoclave at 120 &#176;C for 20 min.</li>
	<li>Soak autoclaved soil mixes with plant fertilizer solution (1 g/L) and sub-package them into smaller pots (80 mm x 80 mm x 75 mm) stored in a larger tray (540 mm x 285 mm x 60 mm).</li>
	<li>Sow one or two seeds of N. benthamiana 16c onto the soil surface of each pot. Cover the tray with a plastic dome and allow seeds to germinate.</li>
	<li>Place the tray under light and temperature-controlled growth chambers with a temperature of 23&#8722;25 &#176;C, 50&#8722;60% relative humidity, and a long-day photoperiod (14 h light/10 h dark, with illumination at 130&#8722;150 &#181;E&#183;m-2s-1).</li>
	<li>Take the plastic dome off after the seeds germinate (3&#8722;4 days) and allow the seedlings to grow under the same conditions used for the germination step.</li>
	<li>Add an appropriate amount of water, keeping soil moist but not soaking, every 2&#8722;3 days; add fertilizer every 10 days to promote further growth. Maintain N. benthamiana 16c plants under normal conditions until they are ready for use; at this stage the plant should have at least five fully developed true leaves and no visible axillary or flower buds, and the leaves should have a healthy green appearance).</li>
	<li>Use 3&#8722;4 week-old leaves of N. benthamiana 16c for local RNA silencing assays, and young N. benthamiana 16c plants (10&#8722;14 days old) for systemic RNA silencing assays. 
	NOTE: Plant growth conditions and facilities vary across laboratories; choose healthy, well-developed and fully expanded leaves for infiltration.</li>
</ol>
3. Preparation of Agrobacterium culture for infiltration
<ol>
	<li>Pick a positive colony from the LB plate and inoculate the cells into glass tube containing 5 mL of LB medium supplemented with 50 &#181;g/mL kanamycin and 50 &#181;g/mL rifampicin. Grow the cells at 30 &#176;C with shaking at 200 rpm for 24&#8722;48 h.</li>
	<li>Transfer 100 &#181;L of culture into 5 mL of LB medium supplemented with same antibiotics, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 5.6) and 20 &#181;M acetosyringone (AS). Grow bacteria at 30 &#176;C with shaking at 200 rpm for 16&#8722;20 h.</li>
	<li>Centrifuge cells at 4,000 x g for 10 min. Pour off the supernatant and resuspend the pellet in 2 mL of 10 mM MgCl2 buffer.</li>
	<li>Repeat step 3.3 to ensure the complete removal of antibiotics.</li>
	<li>Determine the density of the Agrobacterium culture by measuring the optical density at 600 nm (OD600). Adjust to an OD600 of 1.5&#8722;2.0 with 10 mM MgCl2 buffer.</li>
	<li>Add 10 mM MES (pH 5.6) and 150 &#181;M AS to the final suspension cultures and incubate the cells at RT for at least 3 h without shaking. 
	NOTE: Do not leave the final suspension cultures overnight.</li>
</ol>
4. Co-infiltration N. benthamiana leaves
<ol>
	<li>Mix equal volumes of an Agrobacterium culture containing 35S-GFP with an Agrobacterium culture containing 35S- cucumber mosaic virus suppressor 2b (CMV2b), putative effector, or empty vector (EV).</li>
	<li>Carefully and slowly infiltrate the mixed agrobacterium suspensions on the abaxial sides of N. benthamiana 16c leaves using a 1 mL needleless syringe.</li>
	<li>Remove the remaining bacterial suspension from the leaves with soft tissue wipers and circle the margins of the infiltrated patches with a maker pen.</li>
	<li>Leave the infiltrated plants in the growth chamber under the same growth conditions. 
	NOTE: For safety and health reasons, protective eye goggles and a mask should be worn during infiltration. To prevent cross-contamination, gloves should be changed or sterilized with ethanol between infiltrations. Normally at least 1 mL of agrobacterium suspensions per leaf is needed for infiltration. For systemic silencing, at least two leaves will be required for infiltration.</li>
</ol>
5. GFP imaging analysis
<ol>
	<li>Visually detect GFP fluorescence in newly grown leaves of whole plants 2 weeks post-infiltration (for systemic RNA silencing) or infiltrated patches of leaves 3&#8722;4 dpi using a long-wave ultraviolet (UV) lamp without leaves collection.</li>
	<li>Photograph collected plants and/or detached leaves with a digital camera fitted with both UV and yellow filters. 
	NOTE: For assays of local silencing suppression, investigate infiltrated patches of N. benthamiana 16c leaves, whereas for systemic silencing suppression activity, investigate newly grown leaves. RNA silencing suppression activity may vary across individual effectors. Therefore, observe the patches or leaves over a few days starting at 3 dpi. Use CMV2b and EV as positive and negative controls, respectively.</li>
</ol>
6. Northern blot analysis of GFP mRNA levels in infiltrated leaves
<ol>
	<li>Isolation of total RNA from leaf tissue using the RNA isolation reagent (Table of Materials)
	<ol>
		<li>Collect leaf tissues from infiltrated N. benthamiana 16c patches at 4&#8722;7 dpi, pulverize in liquid nitrogen to a fine powder, and transfer the powder to a sterile 2 mL tube.</li>
		<li>Add the RNA isolation reagent (1 mL/100 mg tissue) immediately to the sampled tube in a hood, shake vigorously to homogenate, and incubate at RT for 5 min.</li>
		<li>Add chloroform (200 &#181;L/1 mL RNA isolation reagent) to each tube in a hood, shake vigorously for 15 s, and incubate at RT for 5 min. Centrifuge the homogenate at 12,000 x g for 15 min at 4 &#176;C.</li>
		<li>Transfer the supernatant into a new RNase-free tube and discard the pellet. Add 0.7 volume of isopropanol to the supernatant, gently invert several times, and incubate the mixture at RT for 10 min.</li>
		<li>Precipitate the RNA pellet by centrifugation at 12,000 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant, wash the pellet with 70% ethanol, and air-dry the pellet in a hood. Dissolve the RNA in diethyl pyrocarbonate (DEPC)-treated water by incubating in a 65 &#176;C water bath for 10&#8722;20 min.</li>
	</ol>
	</li>
	<li>Northern blot analysis of GFP mRNA level in the infiltrated leaves
	<ol>
		<li>Prepare a 1.2% formaldehyde denaturing agarose gel in 1x MOPS running buffer.</li>
		<li>Mix RNA 1:1 with RNA loading dye and denature by incubation at 65 &#176;C for 10 min, immediately chill the denatured samples on ice for 1 min.</li>
		<li>Load the samples on the gel and electrophorese at 100 V for 50 min until the RNA is well separated.</li>
		<li>Rinse the gel in 20x saline-sodium citrate (SSC) buffer to remove formaldehyde and transfer the gel to nylon membrane by capillary transfer in 20x SSC buffer overnight. Soak the membrane in 2x SSC and fix the RNA to the membrane by exposing the wet membrane to UV cross linking.</li>
		<li>Add the appropriate amount of hybridization buffer (10 mL per 100 cm2 membrane; Table of Materials) in a hybridization tube and agitate at 60 &#176;C in a hybridization oven.</li>
		<li>Put the membrane into the hybridization tube and incubate for 60 min at 60 &#176;C. Dilute a 5&#39;DIG-labeled DNA probe (final concentration, 50 ng/mL) into the hybridization solution containing prewarmed hybridization buffer.</li>
		<li>Discard the prehybridization buffer and immediately replace with prewarmed hybridization solution containing the DIG-labeled probe. Incubate the blot with probe at 60 &#176;C overnight, with gentle agitation.</li>
		<li>After hybridization, wash the membrane twice in buffers of increasing stringency at 60 &#176;C for 20 min each. Gently wipe the membrane to remove extra washing buffer and add reagents as suggested in the chemiluminescent hybridization and detection kit (Table of Materials).</li>
		<li>Detect hybridized signals by chemiluminescent reaction combined with the imaging system.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

RNA silencing is a key defense mechanism employed by plants to combat viral, bacterial, oomycete, and fungal pathogens. In turn, these microbes have evolved silencing suppressor proteins to counteract antiviral silencing, and these RSSs interfere with different steps of the RNA silencing pathway22,23. Several screening assays have been developed to identify RSSs10.
Here, we describe an improved protocol for screening effector proteins secreted by P. sojae into the host cell upon infection for their ability to suppress RNA silencing in host. This modified assay is based on a viral co-infiltration assay but differs in several important ways. First, both bacteria and N.benthamiana 16c plants should be vigorous and healthy; this is very important for stable expression of effectors from the bacteria and the use of fully developed N.benthamiana 16c ensures experimental reproducibility and reliability. We often use only two or three fully developed leaves per plant at the vegetative growth stage, the old and newly emerged leaves are unsuitable. Secondly, OD600 of the bacterial culture must be adjusted to an optimal value, usually 0.75&#8722;1.0 for Agrobacterium. A lower OD600 (even as low as 0.2) can be used to screen VSRs but not PSRs24. Third, the ideal time point for investigating green fluorescence must be optimized for each effector. In virus, leaves co-injected with cultures containing GFP plus putative VSRs exhibit a marked increase in green fluorescence in the infiltrated area at 3 dpi, and the signal remains high until 9 dpi22, but it was only exhibited at 4 or 5 dpi for PSRs in our present study. Therefore, it is also important to further confirm the RNA silencing activity by quantify the accumulation of GFP mRNA. Finally, it is essential to use the appropriate control. VSRs are not always the ideal positive controls for identifying effector proteins because these proteins have much stronger suppressive activity than PSRs. This could result in failure to detect weak suppressors of RNA silencing.
In this report, we demonstrate the use of our modified assay to screen for suppressors of RNA silencing in Phytophthora and Puccinia graminis pathogens. Thus, our method represents a useful tool for characterizing potential effectors that encode RSSs, which promote disease susceptibility by inhibiting small RNA accumulation15,16,17. Therefore, we believe that our protocol could be used broadly to screen effectors secreted by plant pathogens. Future work will focus on identifying more RSSs in other pathogens, and on elucidating the role of RNA silencing in plant defense against invading microbes.

Over the past two decades, acceleration in genome sequencing of microorganisms that cause plant diseases has led to an ever increasing knowledge gap between sequence information and the biological functions of encoded proteins1. Among the molecules revealed by sequencing projects are effector molecules that suppress innate immunity and facilitate host colonization; these factors are secreted by destructive plant pathogens, including bacteria, nematodes, and filamentous microbes. To respond to these threats, host plants have evolved novel receptors that recognize these effectors, enabling restoration of the immune response. Hence, effectors are subject to various selective pressures, leading to diversification of effector repertoires among pathogen lineages2. In recent years, putative effectors from plant pathogens have been shown to disrupt plant innate immunity by impeding host cellular processes to benefit the microbes in a variety of ways, including dysregulation of signaling pathways, transcription, intracellular transport, cytoskeleton stability, vesicle trafficking, and RNA silencing3,4,5. However, the vast majority of pathogen effectors, particularly those from filamentous pathogens, have remained enigmatic.
RNA silencing is a homology-mediated gene inactivation machinery that is conserved among eukaryotes. The process is triggered by long double-stranded RNA (dsRNA) and targets the homologous single-stranded RNA (ssRNA) in a sequence-specific manner, and it manipulates a wide range of biological processes, including antiviral defense6. To surmount innate immune responses of the host, some viruses have evolved to offset RNA silencing, including the ability to replicate inside intracellular compartments or escape from the silencing reorganized signal. Nevertheless, the most general strategy by which viruses protect their genomes against RNA silencing-dependent loss of gene function is to encode specific proteins that suppress RNA silencing7,8. Several mechanistically different approaches have been established to screen and characterize viral suppressors of RNA silencing (VSRs), including the co-infiltration of Agrobacterium tumefaciens cultures, transgenic plants expressing putative suppressors, grafting and cell culture9,10,11,12,13.
Each of these assays has advantages and disadvantages, and identifies VSRs in its own distinct manner. One of the most common approaches is based on the co-infiltration of individual A. tmuefaciens cultures harboring the potential viral protein and a reporter gene (typically green fluorescent protein [GFP]) on the Nicotiana benthamiana 16c plants constitutively expressing GFP under the control of the cauliflower mosaic virus 35S promoter. In the absence of an active viral silencing suppressor, GFP is identified as exogenous by the host cells and is silenced within 3 days post-infiltration (dpi). By contrast, if the viral protein possesses suppression activity, the expression level of GFP remains steady beyond 3&#8722;9 dpi9. This co-infiltration assay is simple and fast; however, it is neither highly stable nor sensitive. Nonetheless, the assay has identified numerous VSRs with diverse protein sequences and structures in many RNA viruses7,8.
Recently, several effector proteins that can inhibit the cellular RNA silencing activity have been characterized from bacterial, oomycete, and fungal plant pathogens14,15,16. These findings imply that RNA silencing suppression is a common strategy for facilitating infection that is used by pathogens in most kingdoms. In theory, many, if not all, of the effectors might encode RNA silencing suppressors (RSSs); to date, however, only a few have been identified, mainly due to the shortage of the reliable and general screening strategy. Moreover, suppressors of RNA silencing have not been investigated in the vast majority of plant pathogens17.
In this report, we present an optimized and general protocol for identifying plant pathogen effectors that can suppress local and systemic RNA silencing using the agro-infiltration assay. The foremost objective of this study was to emphasize the key aspects of the protocol and describe the steps in detail, thereby providing a screening assay that is suitable for almost all effectors of plant pathogens.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Morpholinoethanesulfonic Acid (MES)</td>
			<td>Biofroxx</td>
			<td>1086GR500</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>2xTaq Master Mix</td>
			<td>Vazyme Biotech</td>
			<td>P112-AA</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propanesulfonic acid (MOPS)</td>
			<td>Amresco</td>
			<td>0264C507-1KG</td>
			<td>MOPS Buffer</td>
		</tr>
		<tr>
			<td>Acetosyringone (AS)</td>
			<td>Sigma-Aldrich</td>
			<td>D134406-5G</td>
			<td>Induction of Agrobacterium</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296-1KG</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biofroxx</td>
			<td>1110GR100</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>Amersham Hybond -N+</td>
			<td>GE Healthcare</td>
			<td>RPN303 B</td>
			<td>Nothern blot</td>
		</tr>
		<tr>
			<td>Amersham Imager</td>
			<td>GE Healthcare</td>
			<td>Amersham Imager 600</td>
			<td>Image</td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Bacto Yeast Extraction</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Nikon</td>
			<td>D5100</td>
			<td>Photography</td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imaging System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescent detection module component of dafa kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>89880</td>
			<td>Luminescence detection</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Amresco</td>
			<td>0230-100G</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>ClonExpress II One Step Cloning Kit</td>
			<td>Vazyme Biotech</td>
			<td>C112-01</td>
			<td>Ligation</td>
		</tr>
		<tr>
			<td>DIG Easy Hyb</td>
			<td>Sigma-Aldrich</td>
			<td>11603558001</td>
			<td>Hybridization buffer</td>
		</tr>
		<tr>
			<td>Easypure Plasmid Miniprep kit</td>
			<td>TransGen Biotech</td>
			<td>EM101-02</td>
			<td>Plasmid Extraction</td>
		</tr>
		<tr>
			<td>EasyPure Quick Gel Extraction Kit</td>
			<td>TransGen Biotech</td>
			<td>EG101-02</td>
			<td>Gel Extraction</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Amresco/VWR</td>
			<td>0105-1KG</td>
			<td>MOPS Buffer</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>LiuYi</td>
			<td>DYY6D</td>
			<td>Nucleic acid electrophoresis.</td>
		</tr>
		<tr>
			<td>FastDigest EcoRV</td>
			<td>Thermo Fisher Scientific</td>
			<td>FD0304</td>
			<td>Vector digestion</td>
		</tr>
		<tr>
			<td>Gel Image System</td>
			<td>Tanon</td>
			<td>Tanon3500</td>
			<td>Image</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Amresco</td>
			<td>0304-5G</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>K1914</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>LR Clonase II enzyme</td>
			<td>Invitrogen</td>
			<td>11791020</td>
			<td>LR reaction</td>
		</tr>
		<tr>
			<td>Nitrocellulose Blotting membrane 0.45um</td>
			<td>GE Healthcare</td>
			<td>10600002</td>
			<td>Northern</td>
		</tr>
		<tr>
			<td>NORTH2south biotin random prime dna labeling kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>17075</td>
			<td>Biotin labeling</td>
		</tr>
		<tr>
			<td>PCR Thermal Cyclers</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Peat moss</td>
			<td>PINDSTRUP</td>
			<td>Dark Gold + clay</td>
			<td>Plants</td>
		</tr>
		<tr>
			<td>Peters Water-Soluble Fertilizer</td>
			<td>ICE</td>
			<td>Peter Professional 20-20-20</td>
			<td>Fertilizer</td>
		</tr>
		<tr>
			<td>Phanta Max Super-Fidelity DNA Polymerase</td>
			<td>Vazyme Biotech</td>
			<td>P505-d1</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>MP Biomedicals</td>
			<td>219549005</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>RNA Gel Loading Dye (2X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0641</td>
			<td>RNA Gel Loading Dye</td>
		</tr>
		<tr>
			<td>Sodium Acetate Hydrate</td>
			<td>Amresco/VWR</td>
			<td>0530-1KG</td>
			<td>MOPS Buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Amresco</td>
			<td>0241-10KG</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Tri-Sodium citrate</td>
			<td>Amresco</td>
			<td>0101-1KG</td>
			<td>SSC Buffer</td>
		</tr>
		<tr>
			<td>Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>UV lamp</td>
			<td>Analytik Jena</td>
			<td>UVP B-100AP</td>
			<td>Observation</td>
		</tr>
		<tr>
			<td>UVP Hybrilinker Oven</td>
			<td>Analytik Jena</td>
			<td>OV2000</td>
			<td>Northern</td>
		</tr>
	</tbody>
</table>

screening and identification of rna silencing suppressors from secreted effectors of plant pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Above, we describe the step-by-step procedure for an improved screening assay for assessing the RSS activity of P. sojae RxLR effectors. Altogether, the experiment takes 5&#8722;6 weeks. Subsequently, the RSSs identified by the assay can be further characterized in terms of function and molecular mechanism. As an example of our approach, we used the P. sojae RxLR effecto Phytophthora suppressor of RNA silencing 1 (PSR1), which is secreted and delivered into host cells through haustoria during infection.
To confirm that PSR1 has RSS activity and is thus suitable for this method, each transformed agrobacterium strain was mixed with a strain harboring 35S-GFP and was infiltrated into leaves of N. benthamiana 16c. EV and CMV2b were used as negative and positive controls, respectively (Figure 1). GFP expression reached the highest level in leaves infiltrated with all mixtures at 2&#8722;3 dpi. Green fluorescence intensity remained strong in patches co-infiltrated with 35S-GFP plus CMV2b during a 6&#8722;9-day period of observation21. Co-infiltration of GFP with PSR1 also resulted in stronger GFP fluorescence during a 3.5&#8722;4.5 day period of observations, as demonstrated by elevated fluorescence in the infiltrated patches. However, the fluorescence intensity in patches infiltrated with 35S-GFP and EV weakened at 3 dpi. At 4&#8722;5 dpi, GFP fluorescence was hardly detectable (Figure 1).
Northern blot revealed that GFP mRNA accumulated higher in the leaves expressing 35S-GFP plus 35S-CMV2b or 35S-GFP plus 35S-PSR1 than in the leaves expressing 35S-GFP plus EV (Figure 2). Thus, transcriptional expression of PSR1 as well as CMV2b contributed to the stabilization of GFP mRNA, which resulted in elevated GFP fluorescence.
We also used the agro-infiltration assay to evaluate the spread of the silencing signal in the leaves of 2-week-old N. benthamiana 16c seedlings. For this purpose, we usually selected 3&#8722;4 larger leaves for whole leaves injection. At 14 dpi, more than 98% of the EV exhibited obvious no GFP signaling in systemic leaves, whereas both PSR1 and CMV2b efficiently inhibited the systemic spread of the silencing signal by observing GFP fluorescence in about 80% of co-infiltrated plants, and in the remaining 20% of infiltrated plants with only a few red veins appeared in newly emerged leaves (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60697/60697fig1.jpg" /> 
Figure 1: Phytophthora sojae RxLR effector PSR1 suppresses local RNA silencing in Nicotiana benthamiana (N. benthamiana) 16c plants. Fully developed leaves of 3&#8722;4-week-old N. benthamiana 16c plants (left panel) were co-infiltrated in patches with Agrobacterium mixtures carrying 35S-GFP and the constructs indicated above each image. GFP fluorescence of the infiltrated area was imaged under natural light (middle panel) and long-wave UV light (right panel) at 4 dpi. The experiment was repeated twice with similar results. Red arrows represent infiltrated leaves. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60697/60697fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60697/60697fig2.jpg" /> 
Figure 2: Accumulation of GFP mRNA in infiltrated N. benthamiana 16c leaves. CK and EV represent N. benthamiana 16c leaves alone and 16c leaves co-infiltrated with 35S-GFP plus EV. Samples from EV and CMV2b were used as a negative and positive controls, respectively. In addition, rRNA was used as a loading control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60697/60697fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60697/60697fig3.jpg" /> 
Figure 3: P. sojae RxLR effector PSR1 suppresses systemic RNA silencing in N. benthamiana 16c plants. Three or four leaves of 2-week-old N. benthamiana 16c seedlings (left panel) were transiently co-infiltrated by Agrobacterium harboring 35S-GFP and either EV or vector expressing PSR1 or CMV2b from the 35S promoter. GFP fluorescence of newly grown leaves was imaged under natural light (upper panel) and long-wave UV light (lower panel) at 14 dpi. This experiment was repeated twice with similar results. Red arrows indicated infiltrated leaves. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60697/60697fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens at JoVE.com

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

screening-identification-rna-silencing-suppressors-from-secreted

Nanyang Technological University

Research

JoVE Journal

Genetics

6.2K Views.  Shanghai Normal University. Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

Video: Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

Summary

Explore More Videos

Chapters in this video

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Identification of Circular RNAs using RNA Sequencing

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands