用 III 型分泌系统改造农杆菌以表达 III 型效应物

Vidhyavathi  Raman; Kirankumar S. Mysore

doi:10.21769/BioProtoc.4726

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Peer-reviewed

Engineering Agrobacterium tumefaciens with a Type III Secretion System to Express Type III Effectors

用 III 型分泌系统改造农杆菌以表达 III 型效应物

VR Vidhyavathi Raman

KM Kirankumar S. Mysore email

发布: 2023年08月05日第13卷第15期 DOI: 10.21769/BioProtoc.4726 浏览次数: 915

评审: Wenrong HeYao XiaoYe XuAnonymous reviewer(s)

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Abstract

Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens–mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering A. tumefaciens to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer A. tumefaciens to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered A. tumefaciens.

Keywords: Type III secretion system (III型分泌系统)

Type III effectors (III型效应物)

Agrobacterium (农杆菌)

Triparental mating (三亲交配)

Transformation (转化)

Background

The Type III secretion system (T3SS) is a specialized complex structure that translocates Type III effectors (T3Es) into the plant cell, where they interact with the host immune system. T3SS is present in many pathogenic Gram-negative bacteria (Cornelis, 2006). Heterologous expression of the genes encoding the type III secretion apparatus is functional in Pseudomonas fluorescens (Huang et al., 1988) and Escherichia coli (Ham et al., 1998). Gene cluster coding structural and regulatory components of T3SS from P. syringae have been cloned and are available in a plasmid called pLN18 (Jamir et al., 2004). We have transferred this plasmid into Agrobacterium tumefaciens to express T3SS and deliver T3Es to suppress plant defense and enhance plant genetic transformation (Raman et al., 2022). Because of the large size, we used triparental mating to mobilize pLN18 to A. tumefaciens in the presence of helper plasmid (pRK2013; containing tra genes) containing E. coli, using the protocol by Wise et al. (2006) with some modifications. In addition, we optimized culture growth and fractionation of the liquid culture into pellet and supernatant fractions to avoid cell lysis that leads to false positive results.

Materials and reagents

1.5 mL microcentrifuge tubes (USA Scientific)
50 mL Falcon tubes (Corning, catalog number: 352070)
0.45 μm Durapore PVDF membrane Millipore filter (Steriflip-HV Sterile Centrifuge Tube Top Filter Unit) (Millipore Sigma, catalog number: SE1M003M00)
Amicon Ultra-15 centrifugal filters (Millipore Sigma, catalog number: UFC901024)
Amicon Ultra-0.5 centrifugal filters (Millipore Sigma, catalog number: UFC501024)
A. tumefaciens GV2260 (Rifampicin resistance)
E. coli HB101 (pRK2013) (Kanamycin resistance)
Donor E. coli containing pLN18 (Kanamycin and Tetracycline resistance)
Luria Bertani (LB) medium (MP Biomedicals, catalog number: 3002011)
Luria Bertani (LB) medium with agar (MP Biomedicals, catalog number: 3002201)
PhiLOV-specific antibody (gift from Dr. John M. Christie, University of Glasgow)
Bacto peptone (BD, catalog number: 211677)
Yeast extract (BD, catalog number: 212750)
Bacto agar (BD, catalog number: 214010)
NaCl (J.T. Baker, catalog number: 3624-19)
K₂HPO₄(J.T. Baker, catalog number: 4012-01)
NaH₂PO₄ (Fisher Scientific, catalog number: S369-500)
NH₄Cl (Fisher Scientific, catalog number: A661)
KCl (Fisher Scientific, catalog number: P217-500)
MgSO₄·7H₂O (Sigma-Aldrich, catalog number: M1880)
CaCl₂ (J.T. Baker, catalog number: 1313-01)
FeSO₄·7H₂O (Sigma-Aldrich, catalog number: F8263)
Sucrose (Invitrogen, catalog number: 15503-022)
Rifampicin (Sigma-Aldrich, catalog number: R3501-1G)
Kanamycin sulfate (Sigma-Aldrich, catalog number: 70560-51-9)
Tetracycline (Sigma-Aldrich, catalog number: T3258)
Yeast extract peptone (YEP) medium (1 L) (see Recipes)
20× AB buffer (1 L) (see Recipes)
20× AB salts (1 L) (see Recipes)
Water agar (see Recipes)
25% sucrose (see Recipes)
Agrobacterium minimal (AB)-sucrose agar medium or AB medium (see Recipes)
0.8% NaCl solution medium (see Recipes)
Hrp-derepressing (HDM) medium (see Recipes)

Equipment

28 °C incubator with shaker (New Brunswick Scientific)
37 °C incubator with shaker (New Brunswick Scientific)
Spectrophotometer (Bio-Rad, catalog number: 170-2525)
Eppendorf tabletop centrifuge (Eppendorf, catalog number: 5424)
Eppendorf tabletop refrigerated centrifuge (Eppendorf, catalog number: 5424R)
Eppendorf centrifuge with swing-bucket rotor (Eppendorf, catalog number: 5810R)

Procedure

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Raman, V. and Mysore, K. S. (2023). Engineering Agrobacterium tumefaciens with a Type III Secretion System to Express Type III Effectors. Bio-protocol 13(15): e4726. DOI: 10.21769/BioProtoc.4726.