# US 11,441,158 B1 · Transgenic resistance to cotton leaf curl disease using small hairpin RNA to induce RNA interference

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- Number: US 11,441,158 B1
- Document ID: US11441158B1
- Status: Granted
- Kind: B1
- Application number: 16/370,544
- Filed: 2019-03-29
- Granted: 2022-09-13
- Earliest claimed: 2018-03-29
- Assignees: Arizona Board of Regents on Behalf of the University of Arizona
- Applicants: Arizona Board of Regents on Behalf of the University of Arizona
- Inventors: Judith K. Brown; Muhammad Zia-Ur-Rehman
- Web page: https://patentgrep.com/patent/US11441158B1

## CPC

- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8283: {for virus resistance}
- C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/8201: {Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation}
- C12N 2310/122: Hairpin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to the control of pest infestation by inhibiting or reducing the expression of certain genes implicated in cotton leaf curl virus disease (CLCuD), by simultaneously targeting viral RNA involved in viral replication and movement. More specifically, the invention relates to a method for substantially simultaneously targeting the AC1 gene of a begomovirus and (2) the betasatellite and non-coding region. Strategies using small hairpin RNA (shRNA) constructs are described herein to achieve viral-mediated gene silencing, and an exemplary embodiment is disclosed, for targeting the replication-associated protein gene (AC1) and non-coding region found in numerous species and strains of the cotton leaf curl disease begomovirus complex, and a coding and adjacent non-coding region of the associated betasatellites. The constructs of the invention reduce or prevent replication of the disease-associated virus, and reduce or prevent the ability of a virus to suppress the defenses of the host plant. In an embodiment of the invention, a small interfering RNA construct referred to herein as an shRNA is disclosed. Also disclosed are transgenic cotton plants that are resistant to leaf curl virus disease.

## Claims

### Claim 1

A nucleic acid construct encoding an RNA sequence that forms a hairpin when expressed, said RNA sequence comprising a sense sequence complementary to a portion of SEQ ID NO:1 that simultaneously targets an AC1 gene and associate betasatellite BC1 gene of a begomovirus, wherein expression of the construct in a plant infected with the begomovirus causes a reduction in expression of the AC1 and BC1 genes.

### Claim 2 (depends on 1)

The nucleic acid construct of claim 1 further comprising:
an antisense fragment complementary to a portion of SEQ ID. No. 2.

### Claim 3 (depends on 2)

The nucleic acid construct of claim 2, further comprising:
(a) a promoter functional in a cotton plant; and
(b) a terminator functional in the plant.

### Claim 4 (depends on 1)

An Agrobacterium plasmid vector containing the nucleic acid construct of claim 1 for transforming a cotton plant.