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><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

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

Environment

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Immunology and Infection

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

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

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

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

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

Biology

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

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 ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

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

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mRNA Interactome Capture from Plant Protoplasts

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

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

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

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

MISSION esiRNA for RNAi Screening in Mammalian Cells

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus