Methods and compositions for microbial delivery of double stranded RNA
Methods and materials for improved delivery of dsRNA are presented. In particular, methods for controlling an insect pest by stably delivering large quantities of dsRNA to the insect pest are provided. Compositions comprising a bacterium containing a large quantity of dsRNA targeting an insect gene in which the dsRNA exhibits increased stability and activity are also provided.
1 . A method for controlling an insect pest, the method comprising: contacting the insect pest in a body of water or insect saliva with a composition comprising a microorganism comprising:
(i) a promoter operably linked to a DNA sequence encoding a dsRNA molecule that specifically inhibits expression of a target gene in an insect pest, wherein the target gene is selected from extracellular signal-regulated kinase 1 (erkA), tubulin, vATPase, acetyl choline esterase, chitin synthase gene A, beta-actin, and genes coding for inhibitors of apoptosis; and
(ii) a promoter operably linked to a DNA sequence comprising a coat protein gene encoding a capsid protein or N-terminus of the capsid protein coding sequence (CDS); and wherein the dsRNA in the composition comprising the microorganism exhibits at least 60% undegraded dsRNA for at least 7 days in the body of water or insect saliva relative to the dsRNA when isolated.
2 . The method according to claim 1 , wherein the capsid protein is encoded by a leviviridae coat protein gene.
3 . The method according to claim 1 , wherein the capsid protein is encoded by the coat protein gene of bacteriophage MS2 or bacteriophage Qβ.
4 . The method according to claim 1 , wherein the DNA sequence encoding the dsRNA and the DNA sequence of the coat protein gene encoding the capsid protein are present on one plasmid or extrachromosomal element within the microorganism.
5 . The method according to claim 4 , wherein the plasmid or extrachromosomal element is pAPSE10180 encoded by SEQ ID NO: 1, pAPSE10269 encoded by SEQ ID NO: 8, pAPSE10216 encoded by SEQ ID NO: 10, pAPSE10219 encoded by SEQ ID NO: 12, pAPSE10279 encoded by SEQ ID NO: 14, pAPSE10270 encoded by SEQ ID NO: 16, pAPSE10271 encoded by SEQ ID NO: 17, pAPSE10272 encoded by SEQ ID NO: 18, pAPSE10292 encoded by SEQ ID NO: 19, pAPSE10291 encoded by SEQ ID NO: 20, pAPSE10359 encoded by SEQ ID NO: 25, pAPSE10372 encoded by SEQ ID NO: 27, pAPSE10429 encoded by SEQ ID NO: 29, pAPSE10430 encoded by SEQ ID NO: 31, pAPSE10431 encoded by SEQ ID NO: 32, pAPSE10432 encoded by SEQ ID NO: 33, pAPSE10434 encoded by SEQ ID NO: 35, pAPSE10436 encoded by SEQ ID NO: 37, pAPSE10440 encoded by SEQ ID NO: 40, pAPSE10442 encoded by SEQ ID NO: 42, pAPSE10444 encoded by SEQ ID NO: 44, or pAPSE10445 encoded by SEQ ID NO: 45.
6 . The method according to claim 1 , wherein the microorganism is an Escherichia coli strain.
7 . The method according to claim 1 , wherein the microorganism is a Corynebacterium glutamicum strain.
8 . The method according to claim 1 , wherein the target gene is beta-actin or the genes coding for inhibitors of apoptosis.
9 . The method according to claim 1 wherein the microorganism is alive or is killed.
10 . The method according to claim 1 , wherein the insect pest is a member of the order Lepidoptera, the order Coleoptera, the order Hymenoptera, or the order Diptera.
11 . The method according to claim 1 , wherein the insect pest is a diamond back moth or a Colorado potato beetle.
12 . The method of claim 1 , wherein the body of water is selected from puddle water and pond water.