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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

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 th.......

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 pathway22,23. Several screening assays have been developed to identify RSSs10.

Here, we describe an improved protocol for scre.......

Acknowledgements

This work was supported by grants from the "Shuguang Program" of Shanghai Education Development Foundation and Shanghai Municipal Education Commission, the National Key Research and Development Program of China National Key R&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).

....

Materials

NameCompanyCatalog NumberComments
2-Morpholinoethanesulfonic Acid (MES)Biofroxx1086GR500Buffer
2xTaq Master MixVazyme BiotechP112-AAPCR
3-(N-morpholino) propanesulfonic acid (MOPS)Amresco0264C507-1KGMOPS Buffer
Acetosyringone (AS)Sigma-AldrichD134406-5GInduction of Agrobacterium
AgarSigma-AldrichA1296-1KGLB medium
AgaroseBiofroxx1110GR100Electrophoresis
Amersham Hybond -N+GE HealthcareRPN303 BNothern blot
Amersham ImagerGE HealthcareAmersham Imager 600Image
Bacto TryptoneBD Biosciences211705LB medium
Bacto Yeast ExtractionBD Biosciences212750LB medium
CameraNikonD5100Photography
ChemiDoc MP Imaging SystemBio-Rad
Chemiluminescent detection module component of dafa kitsThermo Fisher Scientific89880Luminescence detection
ChloramphenicolAmresco0230-100GAntibiotics
ClonExpress II One Step Cloning KitVazyme BiotechC112-01Ligation
DIG Easy HybSigma-Aldrich11603558001Hybridization buffer
Easypure Plasmid Miniprep kitTransGen BiotechEM101-02Plasmid Extraction
EasyPure Quick Gel Extraction KitTransGen BiotechEG101-02Gel Extraction
EDTA disodium salt dihydrateAmresco/VWR0105-1KGMOPS Buffer
Electrophoresis Power SupplyLiuYiDYY6DNucleic acid electrophoresis.
FastDigest EcoRVThermo Fisher ScientificFD0304Vector digestion
Gel Image SystemTanonTanon3500Image
GentamycinAmresco0304-5GAntibiotics
Kanamycin SulfateSigma-AldrichK1914Antibiotics
LR Clonase II enzymeInvitrogen11791020LR reaction
Nitrocellulose Blotting membrane 0.45umGE Healthcare10600002Northern
NORTH2south biotin random prime dna labeling kitThermo Fisher Scientific17075Biotin labeling
PCR Thermal CyclersBio-RadT100PCR
Peat mossPINDSTRUPDark Gold + clayPlants
Peters Water-Soluble FertilizerICEPeter Professional 20-20-20Fertilizer
Phanta Max Super-Fidelity DNA PolymeraseVazyme BiotechP505-d1Enzyme
RifampicinMP Biomedicals219549005Antibiotics
RNA Gel Loading Dye (2X)Thermo Fisher ScientificR0641RNA Gel Loading Dye
Sodium Acetate HydrateAmresco/VWR0530-1KGMOPS Buffer
Sodium ChlorideAmresco0241-10KGLB medium
Tri-Sodium citrateAmresco0101-1KGSSC Buffer
Trizol ReagentInvitrogen15596018RNA isolation reagent
UV lampAnalytik JenaUVP B-100APObservation
UVP Hybrilinker OvenAnalytik JenaOV2000Northern

References

  1. Waterfield, N. R., et al. Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts. Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15967-15972 (2008).
  2. Rovenich, H., Boshoven, J. C., Thomma, B. P.

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