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

Summary

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

Abstract

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.

Introduction

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 silencing^3,4,5. 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 silencing^7,8. 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 culture^{9,10,11,12,13}.

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 3−9 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 viruses^7,8.

Recently, several effector proteins that can inhibit the cellular RNA silencing activity have been characterized from bacterial, oomycete, and fungal plant pathogens^14,15,16. 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.

Protocol

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

1. 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.
2. Determine the signal peptide cleavage site of each effector using a software tool such as SignalP¹⁸.
3. 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.
4. Transform 3−5 µL of LR reaction mixture into 100 µL of competent E. coli cells (e.g., TOP10, DH5α), spread the transformed bacterial cells on Luria-Bertani (LB) agar medium supplemented with 50 µg/mL kanamycin, and incubate at 37 °C for 16−20 h.
   NOTE: Positive transformants can be identified by colony PCR²⁰ and further analyzed by plasmid miniprep and sequencing.
5. Introduce 50−100 ng of recombinant plasmids carrying effector protein into 100 µL 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 °C water bath for 5 min.
6. Add 500 µL of LB broth and incubate at 30 °C for 4−6 h with gentle shaking, and then transfer and spread all the agrobacteria cells on LB agar medium supplemented with 50 µg/mL kanamycin and 50 µg/mL rifampicin at 30 °C for 2 days.
   NOTE: Positive clones can be verified by colony PCR.
7. 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.

2. Preparation of N. benthamiana 16c plants

1. Prepare potting soil mixes consisting of (by volume) 50% peat moss, 30% perlite, and 20% vermiculite, and autoclave at 120 °C for 20 min.
2. 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).
3. 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.
4. Place the tray under light and temperature-controlled growth chambers with a temperature of 23−25 °C, 50−60% relative humidity, and a long-day photoperiod (14 h light/10 h dark, with illumination at 130−150 µE·m^-2s^-1).
5. Take the plastic dome off after the seeds germinate (3−4 days) and allow the seedlings to grow under the same conditions used for the germination step.
6. Add an appropriate amount of water, keeping soil moist but not soaking, every 2−3 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).
7. Use 3−4 week-old leaves of N. benthamiana 16c for local RNA silencing assays, and young N. benthamiana 16c plants (10−14 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.

3. Preparation of Agrobacterium culture for infiltration

1. Pick a positive colony from the LB plate and inoculate the cells into glass tube containing 5 mL of LB medium supplemented with 50 µg/mL kanamycin and 50 µg/mL rifampicin. Grow the cells at 30 °C with shaking at 200 rpm for 24−48 h.
2. Transfer 100 µL 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 µM acetosyringone (AS). Grow bacteria at 30 °C with shaking at 200 rpm for 16−20 h.
3. 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.
4. Repeat step 3.3 to ensure the complete removal of antibiotics.
5. Determine the density of the Agrobacterium culture by measuring the optical density at 600 nm (OD₆₀₀). Adjust to an OD₆₀₀ of 1.5−2.0 with 10 mM MgCl₂ buffer.
6. Add 10 mM MES (pH 5.6) and 150 µM 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.

4. Co-infiltration N. benthamiana leaves

1. 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).
2. Carefully and slowly infiltrate the mixed agrobacterium suspensions on the abaxial sides of N. benthamiana 16c leaves using a 1 mL needleless syringe.
3. Remove the remaining bacterial suspension from the leaves with soft tissue wipers and circle the margins of the infiltrated patches with a maker pen.
4. 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.

5. GFP imaging analysis

1. Visually detect GFP fluorescence in newly grown leaves of whole plants 2 weeks post-infiltration (for systemic RNA silencing) or infiltrated patches of leaves 3−4 dpi using a long-wave ultraviolet (UV) lamp without leaves collection.
2. 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.

6. Northern blot analysis of GFP mRNA levels in infiltrated leaves

1. Isolation of total RNA from leaf tissue using the RNA isolation reagent (Table of Materials)
   1. Collect leaf tissues from infiltrated N. benthamiana 16c patches at 4−7 dpi, pulverize in liquid nitrogen to a fine powder, and transfer the powder to a sterile 2 mL tube.
   2. 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.
   3. Add chloroform (200 µL/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 °C.
   4. 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.
   5. Precipitate the RNA pellet by centrifugation at 12,000 x g for 15 min at 4 °C.
   6. 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 °C water bath for 10−20 min.
2. Northern blot analysis of GFP mRNA level in the infiltrated leaves
   1. Prepare a 1.2% formaldehyde denaturing agarose gel in 1x MOPS running buffer.
   2. Mix RNA 1:1 with RNA loading dye and denature by incubation at 65 °C for 10 min, immediately chill the denatured samples on ice for 1 min.
   3. Load the samples on the gel and electrophorese at 100 V for 50 min until the RNA is well separated.
   4. 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.
   5. Add the appropriate amount of hybridization buffer (10 mL per 100 cm² membrane; Table of Materials) in a hybridization tube and agitate at 60 °C in a hybridization oven.
   6. Put the membrane into the hybridization tube and incubate for 60 min at 60 °C. Dilute a 5'DIG-labeled DNA probe (final concentration, 50 ng/mL) into the hybridization solution containing prewarmed hybridization buffer.
   7. Discard the prehybridization buffer and immediately replace with prewarmed hybridization solution containing the DIG-labeled probe. Incubate the blot with probe at 60 °C overnight, with gentle agitation.
   8. After hybridization, wash the membrane twice in buffers of increasing stringency at 60 °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).
   9. Detect hybridized signals by chemiluminescent reaction combined with the imaging system.

Representative Results

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 5−6 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 2−3 dpi. Green fluorescence intensity remained strong in patches co-infiltrated with 35S-GFP plus CMV2b during a 6−9-day period of observation²¹. Co-infiltration of GFP with PSR1 also resulted in stronger GFP fluorescence during a 3.5−4.5 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 4−5 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 3−4 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).

Figure 1: Phytophthora sojae RxLR effector PSR1 suppresses local RNA silencing in Nicotiana benthamiana (N. benthamiana) 16c plants. Fully developed leaves of 3−4-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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

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 pathway^22,23. 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 0.75−1.0 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 accumulation^15,16,17. 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.

Materials

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