DOI: 10.1073/pnas.95.17.10206. A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture

A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture

10.1073/pnas.95.17.10206

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp + bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi , a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5α carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to E. coli (pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with E. coli (pHIR11), which encodes a functional P. syringae Hrp system. E. coli (pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.

Bibliography

Ham, J. H., Bauer, D. W., Fouts, D. E., & Collmer, A. (1998). A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture. Proceedings of the National Academy of Sciences, 95(17), 10206â10211.

Authors 4

Jong Hyun Ham (first)
David W. Bauer (additional)
Derrick E. Fouts (additional)
Alan Collmer (additional)

References 48 Referenced 91

10.1128/jb.179.18.5655-5662.1997
10.1146/annurev.ge.24.120190.002311
10.1146/annurev.phyto.34.1.153
10.2307/3870222
S Gopalan, D W Bauer, J R Alfano, A O Loniello, S Y He, A Collmer Plant Cell 8, 1095–1105 (1996). / Plant Cell by Gopalan S (1996)
10.1094/MPMI-9-0252
10.1073/pnas.93.26.15497
10.1126/science.274.5295.2060
10.1126/science.274.5295.2063
10.1046/j.1365-2958.1997.2731623.x
10.1128/jb.173.22.7142-7150.1991
10.1094/MPMI-6-376
10.1094/MPMI-7-0799
10.1094/MPMI.1997.10.2.247
10.1094/MPMI-7-0573
10.1016/S0022-2836(83)80284-8
J Sambrook, E F Fritsch, T Maniatis Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, 2nd Ed., Plainview, NY, 1989). / Molecular Cloning: A Laboratory Manual by Sambrook J (1989)
10.1136/jcp.17.6.627
10.1094/MPMI-8-0484
10.1094/MPMI.1998.11.6.563
10.1128/jb.178.6.1720-1730.1996
10.1046/j.1365-2958.1996.5731077.x
10.1046/j.1365-2958.1996.415946.x
10.1094/MPMI.1997.10.5.580
10.1128/jb.179.12.3866-3874.1997
10.1128/jb.169.12.5789-5794.1987
10.1128/jb.170.10.4846-4854.1988
10.1128/jb.174.5.1604-1611.1992
10.1016/0092-8674(93)90354-S
10.1046/j.1365-2958.1997.6442015.x
10.1073/pnas.95.3.1325
10.1094/MPMI-7-0508
10.1094/MPMI-8-0049
10.1128/JB.180.8.2244-2247.1998
10.1007/978-94-011-0177-6_8
10.1073/pnas.88.3.1079
10.1111/j.1365-2958.1996.tb02499.x
10.1128/JB.180.6.1431-1437.1998
10.1126/science.278.5340.1140
10.1111/j.1365-2958.1996.tb02614.x
10.1128/jb.170.6.2575-2583.1988
10.1016/0022-2836(76)90119-4
10.1128/jb.172.11.6568-6572.1990
10.1016/0378-1119(90)90203-4
10.1128/jb.170.10.4748-4756.1988
10.1126/science.1621099
10.1128/jb.175.24.7958-7967.1993
10.1128/jb.179.5.1690-1697.1997

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:39 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 5:10 p.m.)
Indexed	1 month, 4 weeks ago (July 2, 2025, 11:15 a.m.)
Issued	27 years ago (Aug. 18, 1998)
Published	27 years ago (Aug. 18, 1998)
Published Online	27 years ago (Aug. 18, 1998)
Published Print	27 years ago (Aug. 18, 1998)

Funders 0

None

BibTeX

@article{Ham_1998, title={A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture}, volume={95}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.95.17.10206}, DOI={10.1073/pnas.95.17.10206}, number={17}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Ham, Jong Hyun and Bauer, David W. and Fouts, Derrick E. and Collmer, Alan}, year={1998}, month=aug, pages={10206–10211} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        7,
        2
      ]
    ],
    "date-time": "2025-07-02T15:15:00Z",
    "timestamp": 1751469300414
  },
  "reference-count": 48,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "17",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        1998,
        8,
        18
      ]
    ]
  },
  "abstract": "<jats:p>\n            The Hrp (type III protein secretion) system is essential for the plant parasitic ability of\n            <jats:italic>Pseudomonas syringae</jats:italic>\n            and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two\n            <jats:italic>P. syringae</jats:italic>\n            proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp\n            <jats:sup>+</jats:sup>\n            bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in\n            <jats:italic>Escherichia coli</jats:italic>\n            to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The\n            <jats:italic>hrp</jats:italic>\n            gene cluster, carried on cosmid pCPP2156, was cloned from\n            <jats:italic>Erwinia chrysanthemi</jats:italic>\n            , a pathogen that differs from\n            <jats:italic>P. syringae</jats:italic>\n            in being host promiscuous.\n            <jats:italic>E. coli</jats:italic>\n            DH5\u03b1 carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in\n            <jats:italic>Nicotiana clevelandii</jats:italic>\n            only when also expressing\n            <jats:italic>avrB</jats:italic>\n            in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to\n            <jats:italic>E. coli</jats:italic>\n            (pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with\n            <jats:italic>E. coli</jats:italic>\n            (pHIR11), which encodes a functional\n            <jats:italic>P. syringae</jats:italic>\n            Hrp system.\n            <jats:italic>E. coli</jats:italic>\n            (pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.\n          </jats:p>",
  "DOI": "10.1073/pnas.95.17.10206",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2002,
        7,
        26
      ]
    ],
    "date-time": "2002-07-26T14:39:15Z",
    "timestamp": 1027694355000
  },
  "page": "10206-10211",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 91,
  "title": "A cloned\n            <i>Erwinia chrysanthemi</i>\n            Hrp (type III protein secretion) system functions in\n            <i>Escherichia coli</i>\n            to deliver\n            <i>Pseudomonas syringae</i>\n            Avr signals to plant cells and to secrete Avr proteins in culture",
  "prefix": "10.1073",
  "volume": "95",
  "author": [
    {
      "given": "Jong Hyun",
      "family": "Ham",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203"
        }
      ]
    },
    {
      "given": "David W.",
      "family": "Bauer",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203"
        }
      ]
    },
    {
      "given": "Derrick E.",
      "family": "Fouts",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203"
        }
      ]
    },
    {
      "given": "Alan",
      "family": "Collmer",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        1998,
        8,
        18
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_3_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.179.18.5655-5662.1997"
    },
    {
      "key": "e_1_3_3_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.ge.24.120190.002311"
    },
    {
      "key": "e_1_3_3_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.phyto.34.1.153"
    },
    {
      "key": "e_1_3_3_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.2307/3870222"
    },
    {
      "key": "e_1_3_3_5_2",
      "first-page": "1095",
      "volume": "8",
      "author": "Gopalan S",
      "year": "1996",
      "unstructured": "S Gopalan, D W Bauer, J R Alfano, A O Loniello, S Y He, A Collmer Plant Cell 8, 1095\u20131105 (1996).",
      "journal-title": "Plant Cell"
    },
    {
      "key": "e_1_3_3_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-9-0252"
    },
    {
      "key": "e_1_3_3_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.93.26.15497"
    },
    {
      "key": "e_1_3_3_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.274.5295.2060"
    },
    {
      "key": "e_1_3_3_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.274.5295.2063"
    },
    {
      "key": "e_1_3_3_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1365-2958.1997.2731623.x"
    },
    {
      "key": "e_1_3_3_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.173.22.7142-7150.1991"
    },
    {
      "key": "e_1_3_3_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-6-376"
    },
    {
      "key": "e_1_3_3_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-7-0799"
    },
    {
      "key": "e_1_3_3_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI.1997.10.2.247"
    },
    {
      "key": "e_1_3_3_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-7-0573"
    },
    {
      "key": "e_1_3_3_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0022-2836(83)80284-8"
    },
    {
      "key": "e_1_3_3_17_2",
      "volume-title": "Molecular Cloning: A Laboratory Manual",
      "author": "Sambrook J",
      "year": "1989",
      "unstructured": "J Sambrook, E F Fritsch, T Maniatis Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, 2nd Ed., Plainview, NY, 1989).",
      "edition": "2"
    },
    {
      "key": "e_1_3_3_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1136/jcp.17.6.627"
    },
    {
      "key": "e_1_3_3_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-8-0484"
    },
    {
      "key": "e_1_3_3_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI.1998.11.6.563"
    },
    {
      "key": "e_1_3_3_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.178.6.1720-1730.1996"
    },
    {
      "key": "e_1_3_3_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1365-2958.1996.5731077.x"
    },
    {
      "key": "e_1_3_3_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1365-2958.1996.415946.x"
    },
    {
      "key": "e_1_3_3_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI.1997.10.5.580"
    },
    {
      "key": "e_1_3_3_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.179.12.3866-3874.1997"
    },
    {
      "key": "e_1_3_3_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.169.12.5789-5794.1987"
    },
    {
      "key": "e_1_3_3_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.170.10.4846-4854.1988"
    },
    {
      "key": "e_1_3_3_28_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.174.5.1604-1611.1992"
    },
    {
      "key": "e_1_3_3_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(93)90354-S"
    },
    {
      "key": "e_1_3_3_30_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1365-2958.1997.6442015.x"
    },
    {
      "key": "e_1_3_3_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.95.3.1325"
    },
    {
      "key": "e_1_3_3_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-7-0508"
    },
    {
      "key": "e_1_3_3_33_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1094/MPMI-8-0049"
    },
    {
      "key": "e_1_3_3_34_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JB.180.8.2244-2247.1998"
    },
    {
      "key": "e_1_3_3_35_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/978-94-011-0177-6_8"
    },
    {
      "key": "e_1_3_3_36_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.88.3.1079"
    },
    {
      "key": "e_1_3_3_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1365-2958.1996.tb02499.x"
    },
    {
      "key": "e_1_3_3_38_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JB.180.6.1431-1437.1998"
    },
    {
      "key": "e_1_3_3_39_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.278.5340.1140"
    },
    {
      "key": "e_1_3_3_40_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1365-2958.1996.tb02614.x"
    },
    {
      "key": "e_1_3_3_41_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.170.6.2575-2583.1988"
    },
    {
      "key": "e_1_3_3_42_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(76)90119-4"
    },
    {
      "key": "e_1_3_3_43_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.172.11.6568-6572.1990"
    },
    {
      "key": "e_1_3_3_44_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0378-1119(90)90203-4"
    },
    {
      "key": "e_1_3_3_45_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.170.10.4748-4756.1988"
    },
    {
      "key": "e_1_3_3_46_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1621099"
    },
    {
      "key": "e_1_3_3_47_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.175.24.7958-7967.1993"
    },
    {
      "key": "e_1_3_3_48_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/jb.179.5.1690-1697.1997"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.95.17.10206",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        4,
        13
      ]
    ],
    "date-time": "2022-04-13T21:10:52Z",
    "timestamp": 1649884252000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.95.17.10206"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1998,
        8,
        18
      ]
    ]
  },
  "references-count": 48,
  "journal-issue": {
    "issue": "17",
    "published-print": {
      "date-parts": [
        [
          1998,
          8,
          18
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.95.17.10206"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.95.17.10206",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        1998,
        8,
        18
      ]
    ]
  },
  "assertion": [
    {
      "value": "1998-02-23",
      "order": 0,
      "name": "received",
      "label": "Received",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1998-05-26",
      "order": 1,
      "name": "accepted",
      "label": "Accepted",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1998-08-18",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}