Insect salivary enzyme triggers systemic resistance

• US 6,303,326 B1
• Filed: 12/03/1998
• Issued: 10/16/2001
• Est. Priority Date: 12/04/1997
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of The University of Arkansas NA
• Original Assignee: University of Arkansas (University of Arkansas System)
• Inventors: Murphy, John B., Felton, Gary W., Mathews, Mary C., Bi, Jianlong
• Primary Examiner(s): Low, Christopher S. F.
• Assistant Examiner(s): Mohamed, Abdel A.
• Application Number:
  US09/205,630
• Time in Patent Office: 1,048 Days
• Field of Search: 435/14, 435/28, 435/39, 435/68.1, 435/183, 435/190, 435/805, 435/808, 435/814, 435/815, 436/95, 436/166, 436/169, 426/8, 426/9, 426/10, 426/12, 426/56, 426/57, 426/58, 514/12
• US Class Current: 435/14
• CPC Class Codes: A01N 37/46 N-acyl derivatives
• CPC Class Codes: A01N 63/14 Insects
• CPC Class Codes: A01N 63/50 Isolated enzymes; Isolated proteins peptides A01N37/46
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification
• CPC Class Codes: Y10S 435/815 by sorption
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

The present invention includes the characterization of the major salivary protein or enzyme of the corn earworm Helicoverpa zea for triggering resistance to bacterial blight and frogeye leafspot in soybeans and for triggering resistance to insects in tomatoes. The invention includes an enzyme or a novel protein secreted from the salivary glands of certain insects including the saliva of species belonging to the order Hymenoptera and Lepidoptera. The regurgitant of Helocoverpa zea obtained from the functional salivary glands contains a protein that possesses glucose oxidase activity. The amino acid sequence of the protein is unique and when the protein is applied to plants, it triggers disease and insect resistance systematically. The physical and kinetic attributes of the enzyme are a pH of 7.0, a pI of 4.4 and a molecular weight of 88 kd. The k_m and V_max of the enzyme for glucose is 26.9 mmol and 26.7 μmol min⁻¹ mg⁻¹, respectively. The enzyme may be expressed in crops for plant resistance and/or applied to crops for inhibiting foliar pathogens and/or other pests, in food applications for improving the shelf life or quality of the food products as well as the production of low alcohol products. Biomedical uses include using the enzyme for glucose monitoring of blood, urine, etc., as well as using the enzyme in the development of anti-cancer and/or anti-tumor agents and the production of antimicrobial products and the like. Various biochemical applications include the use of the enzyme in immunochemistry as well as for the enzymatic iodination of proteins and enzymatically amplified sensors for amperometry and voltammetry. Another use of the gene from the enzyme is an expression in a Baculovirus for pesticide usage.

Claims

1. A glucose oxidase salivary enzyme isolated and purified from Helicoverpa having the following physiochemical properties:

action;

catalyzing the following reaction;

glucose+O₂→

gluconic acid+hydrogen peroxide

optimum pH;

approximately 7.0

isoelectric point;

approximately 4.4

molecular weight;

approximately 88 kDa.

Michealis-Menton constant;

approximately 27 millimoles

maximum reaction velocity;

approximately 27 micromoles per minute per milligram

Enzymatic activity;

greater than 20,000 nanomoles per minute per milligram.

2. The enzyme of claim 1 having the N terminal sequence of SEQ ID NO:

1.

3. A glucose oxidase insect salivary enzyme purified and isolated from an insect from the order of Lepidoptera, the super family of Noctuiodea, having the following physicochemical properties:

action;

catalyzing the following reaction;

glucose+O₂→

gluconic acid+hydrogen peroxide

molecular weight;

approximately 88 kDa.

optimum pH;

approximately 7.0

isoelectric point;

approximately 4.4

Michealis-Menton constant;

approximately 27 millimoles,

maximum reaction velocity;

approximately 27 micromoles per minute per milligram

enzyrrmtic activity;

greater than 20,000 nanomoles per minute per milligram.

4. The glucose oxidase salivary enzyme of claim 3 having the N-terminal sequence of SEQ ID NO:

1.

5. A glucose oxidase salivary enzyme purified and isolated from an insect of the order of hymenpotra, from the family of Apis mellifera, having the following physicochemical properties:

action;

catalyzing the following reaction;

Glucose+O₂→

gluconic acid+hydrogen peroxide

molecular weight;

approximately 88 kDa.

optimum pH;

approximately 7.0

isoelectric point;

approximately 4.4

Michealis-Menton constant;

approximately 27 millimoles

maximum reaction velocity;

approximately 27 micromoles per minute per milligram

enzymatic activity greater than 20,000 nanomoles per minute per milligram.

6. The glucose oxidase salivary enzyme of claim 5, having the N-terminal sequence of SEQ ID NO 1.

7. A method of increasing plant resistance to pathogens comprising the steps of;

lacerating at least one leaf of said plant;

applying at least 10 micrograms of an enzynme to the lacerated portion of said leaf;

wherein said lacerating step is accomplished using a method selected from the group consisting of cutting, puncturing or excising of a small portion of leaf tissue;

wherein said enzyme comprises a glucose oxidase salivary enzyme isolated and purified from Helicoverpa having the following physiochemical properties;

action;

catalyzing the following reaction;

glucose+O₂→

gluconic acid+hydrogen peroxide

optimum pH;

approximately 7.0

isoelectric point;

approximately 4.4

molecular weight;

approximately 88 kDa.

Michealis-Menton constant;

approximately 27 millimoles

maximum reaction velocity;

approximately 27 micromoles per minute per milligram

Enzymatic activity;

greater than 20,000 nanomoles per minute per milligram; and

,

for a time and under conditions that increase a plant'"'"'s resistance to pathogens.

8. The method of claim 7 wherein said lacerating step consists of excising at least one 0.5 centimeter disk from a leaf of said plant.