# US 2020/0087676 A1 · Biological control of cucumber green mottle mosaic virus

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- Number: US 2020/0087676 A1
- Document ID: US20200087676A1
- Status: Published application
- Kind: A1
- Application number: 16/558,069
- Filed: 2019-08-31
- Published: 2020-03-19
- Earliest claimed: 2018-09-14
- Assignees: A&L Canada Laboratories Inc.
- Applicants: A&L Canada Laboratories, Inc.
- Inventors: Keri WANG; George LAZAROVITS; Yibin LIU; Magda KONOPKA; Greg PATTERSON
- Web page: https://patentgrep.com/patent/US20200087676A1

## CPC

- A01H 6/346: {Cucumis sativus[cucumber]}
- C12N 15/8283: {for virus resistance}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2770/00021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2770/00062: by genetic engineering
- C12N 2770/14021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

An attenuated strain of cucumber green mottle mosaic virus (CGMMV) is useful to protect cucumber plants from infection with the wild-type infectious CGMMV strain. The genome of the attenuated virus contains at least one mutation or group of mutations selected from c.4969G>A, c.3334C>T, and a group of at least six of the mutations c.315G>A; c.1498A>G; c.1660C>T; c.3430C>T; c.3528A>G; c.4144C>T; c.4248C>T; and c.6228C>T. These mutated genomes encode one or more mutations selected from R1637H in the 186 kDa readthrough replication protein, A1092V in the 129 kDa replication protein and/or the 186 kDa readthrough replication protein, and at least six mutations selected from G86S, E480G, S534F, A1124V, N1157D, P1362L, P1397S in the 129 kDa replication protein and/or the 186 kDa readthrough replication protein, and the A156V mutation in the coat protein.

## Claims

### Claim 1

An attenuated strain of cucumber green mottle mosaic virus (CGMMV) comprising a genome, wherein the genome is a polyribonucleotide having a sequence functionally equivalent to a variant of SEQ ID NO:18, the sequence comprising at least one residue or group of residues selected from:
a) A at a position corresponding to position 4969 of SEQ ID NO:18;
b) U at a position corresponding to position 3334 of SEQ ID NO: 18; and
c) a group of at least six residues selected from:
A at a position corresponding to position 315 of SEQ ID NO: 18;
G at a position corresponding to position 1498 of SEQ ID NO:18;
U at a position corresponding to position 1660 of SEQ ID NO: 18;
U at a position corresponding to position 3430 of SEQ ID NO: 18;
G at a position corresponding to position 3528 of SEQ ID NO:18;
U at a position corresponding to position 4144 of SEQ ID NO:18;
U at a position corresponding to position 4248 of SEQ ID NO:18; and
U at a position corresponding to position 6228 of SEQ ID NO:18.

### Claim 2 (depends on 1)

The attenuated strain according to claim 1 wherein the sequence of the polyribonucleotide comprises one or more of A at the position corresponding to position 4969 of SEQ ID NO: 18 and U at the position corresponding to position 3334 of SEQ ID NO: 18.

### Claim 3 (depends on 1)

The attenuated strain according to claim 1 wherein the sequence of the polyribonucleotide comprises:
A at the position corresponding to position 315 of SEQ ID NO: 18;
G at the position corresponding to position 1498 of SEQ ID NO: 18;
U at the position corresponding to position 1660 of SEQ ID NO:18;
U at the position corresponding to position 3430 of SEQ ID NO: 18;
G at the position corresponding to position 3528 of SEQ ID NO: 18;
U at the position corresponding to position 4144 of SEQ ID NO:18;
U at the position corresponding to position 4248 of SEQ ID NO: 18; and
U at the position corresponding to position 6228 of SEQ ID NO: 18.

### Claim 4 (depends on 3)

The attenuated strain according to claim 3 wherein the sequence of the polyribonucleotide further comprises one or more of A at the position corresponding to position 4969 of SEQ ID NO:18 and U at the position corresponding to position 3334 of SEQ ID NO:18.

### Claim 5 (depends on 1)

A polydeoxyribonucleotide having a sequence functionally equivalent to the sequence of the polyribonucleotide according to claim 1, wherein the sequence of the polydeoxyribonucleotide is a variant of SEQ ID NO: 18 comprising at least one residue or group of residues selected from:
a) A at the position corresponding to position 4969 of SEQ ID NO: 18;
b) T at the position corresponding to position 3334 of SEQ ID NO:18; and
c) a group of at least six residues selected from;
A at the position corresponding to position 315 of SEQ ID NO:18;
G at the position corresponding to position 1498 of SEQ ID NO:18;
T at the position corresponding to position 1660 of SEQ ID NO:18;
T at the position corresponding to position 3430 of SEQ ID NO:18;
G at the position corresponding to position 3528 of SEQ ID NO:18;
T at the position corresponding to position 4144 of SEQ ID NO:18;
T at the position corresponding to position 4248 of SEQ ID NO:18; and
T at the position corresponding to position 6228 of SEQ ID NO: 18.

### Claim 6 (depends on 5)

The polydeoxyribonucleotide according to claim 5 wherein the sequence of the polydeoxyribonucleotide comprises one or more of A at the position corresponding to position 4969 of SEQ ID NO:18 and T at the position corresponding to position 3334 of SEQ ID NO:18.

### Claim 7 (depends on 5)

The polydeoxyribonucleotide according to claim 5 wherein the sequence of the polydeoxyribonucleotide comprises:
A at the position corresponding to position 315 of SEQ ID NO:18;
G at the position corresponding to position 1498 of SEQ ID NO: 18;
T at the position corresponding to position 1660 of SEQ ID NO:18;
T at the position corresponding to position 3430 of SEQ ID NO:18;
G at the position corresponding to position 3528 of SEQ ID NO:18;
T at the position corresponding to position 4144 of SEQ ID NO:18;
T at the position corresponding to position 4248 of SEQ ID NO: 18; and
T at the position corresponding to position 6228 of SEQ ID NO:18.

### Claim 8 (depends on 7)

The polydeoxyribonucleotide according to claim 7 wherein the sequence of the polydeoxyribonucleotide comprises one or more of A at the position corresponding to position 4969 of SEQ ID NO:18 and T at the position corresponding to position 3334 of SEQ ID NO: 18.

### Claim 9 (depends on 5)

The polydeoxyribonucleotide according to claim 5 wherein the sequence of the polydeoxyribonucleotide is selected from SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO:60, and variants thereof.

### Claim 10 (depends on 5)

The polydeoxyribonucleotide according to claim 5 wherein the sequence of the polydeoxyribonucleotide is selected from SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:60, and variants thereof.

### Claim 11 (depends on 5)

The polydeoxyribonucleotide according to claim 5, wherein the polydeoxyribonucleotide encodes one or more proteins selected from:
a 129 kDa protein having an amino acid sequence selected from SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:61 and variants thereof;
a 186 kDa protein having an amino acid sequence selected from SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:62 and variants thereof; and
a coat protein having an amino acid sequence selected from SEQ ID NO:32 and variants thereof.

### Claim 12 (depends on 5)

A vector comprising the polydeoxyribonucleotide according to claim 5.

### Claim 13 (depends on 5)

A genetically modified cell, wherein the cell expresses the polydeoxyribonucleotide according to claim 5.

### Claim 14 (depends on 13)

The genetically modified cell according to claim 13 wherein the cell is a microorganism.

### Claim 15 (depends on 13)

The genetically modified cell according to claim 13 wherein the cell is a plant cell.

### Claim 16 (depends on 1)

A composition for preventing symptoms associated with infection by wild-type CGMMV in a plant or for increasing resistance of a plant to infection by wild-type CGMMV, wherein the composition comprises an attenuated strain of CGMMV according to claim 1 and an agriculturally acceptable carrier.

### Claim 17 (depends on 13)

A composition for preventing symptoms associated with infection by wild-type CGMMV in a plant or for increasing resistance of a plant to infection by wild-type CGMMV, wherein the composition comprises a genetically modified cell according to claim 13 and an agriculturally acceptable carrier.

### Claim 18 (depends on 1)

A method for preventing symptoms associated with infection by wild-type CGMMV in a plant or for increasing resistance of a plant to infection by wild-type CGMMV, wherein the method comprises inoculating the plant with an attenuated strain of CGMMV according to claim 1.

### Claim 19 (depends on 13)

A method for preventing symptoms associated with infection by wild-type CGMMV in a plant or for increasing resistance of a plant to infection by wild-type CGMMV, wherein the method comprises inoculating the plant with a genetically modified cell according to claim 13.

### Claim 20 (depends on 16)

A method for preventing symptoms associated with infection by wild-type CGMMV in a plant or for increasing resistance of a plant to infection by wild-type CGMMV, wherein the method comprises inoculating the plant with a composition according to claim 16.

### Claim 21 (depends on 17)

A method for preventing symptoms associated with infection by wild-type CGMMV in a plant or for increasing resistance of a plant to infection by wild-type CGMMV, wherein the method comprises inoculating the plant with a composition according to claim 17.

### Claim 22

An attenuated strain of cucumber green mottle mosaic virus (CGMMV) comprising a genome, wherein the genome is a polyribonucleotide encoding a 186 kDa protein having an amino acid sequence comprising at least one residue or group of residues selected from:
histidine at the position corresponding to position 1637 of SEQ ID NO:64;
valine at the position corresponding to position 1092 of SEQ ID NO:64; and
a group of at least five residues selected from:
serine at the position corresponding to position 86 of SEQ ID NO:64;
glycine at the position corresponding to position 480 of SEQ ID NO:64;
phenylalanine at the position corresponding to position 534 of SEQ ID NO:64;
valine at the position corresponding to position 1124 of SEQ ID NO:64;
aspartic acid at the position corresponding to position 1157 of SEQ ID NO:64;
leucine at the position corresponding to position 1362 of SEQ ID NO:64; and
serine at the position corresponding to position 1397 of SEQ ID NO:64.

### Claim 23 (depends on 22)

The attenuated strain according to claim 22, wherein the polyribonucleotide further encodes a coat protein having an amino acid sequence comprising valine at the position corresponding to position 156 of SEQ ID NO:65.

### Claim 24 (depends on 22)

The attenuated strain according to claim 22, wherein the amino acid sequence of the 186 kDa protein comprises at least one residue selected from histidine at the position corresponding to position 1637 of SEQ ID NO:64 and valine at the position corresponding to position 1092 of SEQ ID NO:64.

### Claim 25 (depends on 22)

The attenuated strain according to claim 22, wherein the amino acid sequence of the 186 kDa comprises at least five residues selected from:
serine at the position corresponding to position 86 of SEQ ID NO:64;
glycine at the position corresponding to position 480 of SEQ ID NO:64;
phenylalanine at the position corresponding to position 534 of SEQ ID NO:64;
valine at the position corresponding to position 1124 of SEQ ID NO:64;
aspartic acid at the position corresponding to position 1157 of SEQ ID NO:64;
leucine at the position corresponding to position 1362 of SEQ ID NO:64; and
serine at the position corresponding to position 1397 of SEQ ID NO:64.

### Claim 26 (depends on 25)

The attenuated strain according to claim 25, wherein the amino acid sequence of the 186 kDa protein further comprises at least one residue selected from histidine at the position corresponding to position 1637 of SEQ ID NO:64 and valine at the position corresponding to position 1092 of SEQ ID NO:64.