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 ....

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), an.......

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 scre.......

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