Methods for modifying plants with a recombinant RNA molecule
Synthetic endornaviral satellite RNA molecules and satellite particles containing the same are disclosed. The synthetic endornaviral satellite RNA molecules can include coding and/or non-coding cargo sequences, and are heritable through generations of plants. Also disclosed are methods of using the endornaviral satellite RNA molecules and satellite particles containing the same to change plant phenotypes, improve plant stress resistance, and improve plant pest and pathogen resistance.
1 . A method of increasing a plant's resistance and/or tolerance to an insect, comprising introducing into a plant a recombinant RNA molecule comprising, in 5′ to 3′ order:
(a) a bell pepper endornavirus (BPEV) 5′ replicase recognition sequence recognized by a BPEV RNA-dependent RNA polymerase (RDRP);
(b) a cargo RNA sequence comprising:
(i) an RNA sequence that forms a double-stranded RNA which suppresses expression of at least one target gene of the insect, or
(ii) RNA encoding an insecticidal protein, or
(iii) both (i) and (ii); and
(c) a BPEV 3′ replicase recognition sequence recognized by the BPEV RDRP; wherein the cargo RNA sequence is heterologous to the BPEV 5′ and/or 3′ replicase recognition sequences;
wherein the plant comprises the BPEV RDRP protein that recognizes the BPEV 5′ replicase recognition sequence and BPEV 3′ replicase recognition sequence, whereby the BPEV RDRP protein catalyzes synthesis of a synthetic endornaviral satellite RNA from the recombinant RNA molecule; and
wherein the synthesis of the synthetic endornaviral satellite RNA comprising the cargo RNA sequence results in an increase in the plant's resistance and/or tolerance to the insect relative to the resistance and/or tolerance to the insect in a control plant lacking the recombinant RNA molecule.
2 . The method of claim 1 , wherein the insect is a Leptinotarsa sp.
3 . The method of claim 1 , further comprising the step of selfing the plant that comprises the recombinant RNA molecule or sexually crossing it, resulting in a population of F 1 seed.
4 . The method of claim 3 , further comprising the steps of growing the F 1 seed to produce a population of F 1 plants and selecting an F 1 progeny plant that comprises the recombinant RNA molecule from the population of F 1 plants.
5 . The method of claim 3 , further comprising screening or selecting for the presence of the recombinant RNA molecule in the plant, in floral tissue of the plant, in male reproductive tissue of the plant, or in female reproductive tissue of the plant.
6 . The method of claim 3 , wherein the F 1 seed lacks DNA that encodes the recombinant RNA molecule.
7 . The method of claim 3 , wherein the BPEV RDRP is provided to the F 1 seed by a commensal non-pathogenic bell pepper endornavirus endemic in at least one parent plant of the F 1 seed.
8 . The method of claim 1 , wherein the plant is a grafted plant and the recombinant RNA molecule is introduced into the grafted plant by contacting the scion, the rootstock, or both the scion and the rootstock with a composition comprising the recombinant RNA molecule prior to grafting the scion onto the rootstock to produce the grafted plant.
9 . The method of claim 1 , wherein the recombinant RNA molecule is introduced into the plant by contacting the plant with a formulation including the recombinant RNA molecule.
10 . The method of claim 1 , wherein the recombinant RNA molecule further comprises an operably linked encapsidation recognition sequence and is encapsidated by a viral coat protein.
11 . The method of claim 10 , wherein the viral coat protein is a tobamoviral coat protein.
12 . The method of claim 1 , wherein the RNA sequence that forms the double-stranded RNA which suppresses expression of at least one target insect gene comprises a hairpin RNA (hpRNA), an RNA that forms multiple stem-loops, an siRNA precursor, or a miRNA precursor.
13 . The method of claim 12 , wherein the insect is a Leptinotarsa sp.
14 . The method of claim 13 , wherein the target insect gene is an actin gene.
15 . The method of claim 1 , wherein the BPEV 5′ replicase recognition sequence comprises an RNA molecule having at least 90% sequence identity to the BPEV 5′ replicase recognition sequence of SEQ ID NO: 580 and the BPEV 3′ replicase recognition sequence comprises an RNA molecule having at least 90% sequence identity to the BPEV 3′ replicase recognition sequence of SEQ ID NO: 581.
16 . The method of claim 1 , wherein the BPEV RDRP is encoded by an RNA molecule with at least 90% sequence identity to the RNA equivalent of the BPEV RDRP coding sequence of SEQ ID NO: 494.
17 . The method of claim 1 , wherein the BPEV 5′ replicase recognition sequence comprises at least 200 nucleotides of SEQ ID NO: 580.
18 . The method of claim 1 , wherein the BPEV 3′ replicase recognition sequence comprises at least 100 nucleotides of SEQ ID NO: 581.
19 . The method of claim 1 , wherein the plant that comprises the BPEV RDRP and the introduced recombinant RNA molecule lacks DNA that encodes the recombinant RNA molecule.
20 . The method of claim 1 , wherein the insecticidal protein is an entomopathogenic bacterial insecticidal protein.
21 . The method of claim 20 , wherein the entomopathogenic bacterial insecticidal protein is a Bacillus thuringiensis insecticidal protein.
22 . The method of claim 21 , wherein the Bacillus thuringiensis insecticidal protein is a Cry protein.
23 . The method of claim 1 , wherein the cargo RNA sequence comprises both (i) an RNA sequence that forms a double-stranded RNA which suppresses expression of at least one target gene of the fungal insect, and (ii) RNA encoding an insecticidal protein; and wherein the insecticidal protein is an entomopathogenic bacterial insecticidal protein.
24 . The method of claim 23 , wherein the target insect gene is actin.
25 . The method of claim 23 , wherein the entomopathogenic bacterial insecticidal protein is a Cry protein.
26 . The method of claim 23 , wherein the insect is a Leptinotarsa sp.
27 . The method of claim 23 , wherein the insect is Colorado potato beetle, Leptinotarsa decemlineata and the entomopathogenic bacterial insecticidal protein is a Cry protein.
28 . The method of claim 27 , wherein the Cry protein is Cry3a.