Systems, methods and composition of using rnase iii mutants to produce srna to control host pathogen infection

Abstract

The current invention includes systems, methods and compositions for the generation of sRNA molecules using select RNase III mutants. In one preferred embodiment, invention includes systems, methods and compositions for the generation of sRNA molecules using RNase III mutants to control a host pathogen through the production and diffusion of sRNA molecules that may initiate an RNAi pathway response directed to a host pathogen. Additional embodiments of the current invention include systems, methods and compositions for the DICER-independent generation of sRNA molecules using select RNase III mutants.

1 . Genetically modified bacteria engineered to co-express a heterologous bacterial RNase III mutant and a heterologous target dsRNA molecule, wherein said target dsRNA molecule is directed to an essential gene of a target pathogen, pest, or herbivore, wherein said RNase III mutant upon expression cleaves said target dsRNA molecule into sRNA molecules of a discrete size capable of inducing RNA silencing in said target pathogen, pest, or herbivore. 
     
     
         2 . The genetically modified bacteria of  claim 1 , wherein said genetically modified bacteria is configured for introduction to a target host that is susceptible to said pathogen, pest, or herbivore. 
     
     
         3 . The genetically modified bacteria of  claim 2 , wherein said genetically modified bacteria is a symbiotic and/or endosymbiotic bacteria with said target host. 
     
     
         4 . The genetically modified bacteria of  claim 2 , wherein said target host is a plant host or an animal host. 
     
     
         5 . The genetically modified bacteria of  claim 2 , wherein said sRNA molecules of a discrete size are generated by said genetically modified bacteria and delivered to said target host or directly to said target pathogen, pest, or herbivore to induce an RNAi pathway response in said target pathogen, pest, or herbivore and protect the target host from said target pathogen, pest, or herbivore. 
     
     
         6 . The genetically modified bacteria of  claim 1 , wherein said genetically modified bacteria comprises a heterologous polynucleotide encoding said heterologous bacterial RNase III mutant and a heterologous polynucleotide encoding said heterologous target dsRNA molecule. 
     
     
         7 . The genetically modified bacteria of  claim 1 , wherein said sRNA molecules of a discrete size consist of 22 to 23 nucleotides (nt). 
     
     
         8 . The genetically modified bacteria of  claim 1 , heterologous bacterial RNase III mutant is derived from a wild type RNase III from a bacteria selected from the group consisting of:  E. coli, Enterobacter  sp.,  Bacillus  sp.,  S. enterica, P. aeruginosa, C. burnetii, R. capsulatus, S. coelicolor, C. jejuni, H. pylori, S. aureus , and  L. lactis.    
     
     
         9 . The genetically modified bacteria of  claim 1 , wherein said essential gene is an essential viral pathogen gene. 
     
     
         10 . A composition for initiating RNA interference (RNAi) in a target pathogen, pest, or herbivore, said composition comprising a plurality of genetically modified bacteria according to  claim 1  dispersed in a carrier. 
     
     
         11 . The composition of  claim 10 , wherein said composition is a wettable powder, spray, emulsion, colloid, aqueous or organic solution, dust, pellet, or colloidal concentrate. 
     
     
         12 . The composition of  claim 10 , wherein said composition is a liquid solution, foam, suspension, or emulsifiable concentrate. 
     
     
         13 . The composition of  claim 10 , wherein composition is an animal feed. 
     
     
         14 . The composition of  claim 10 , further comprising powdered corncobs, rice hulls, and/or walnut shells. 
     
     
         15 . A method of initiating RNA interference (RNAi) in a target pathogen, pest, or herbivore, the method comprising:
 administering or applying to a target host an effective amount of a genetically modified bacteria according to  claim 1 , wherein said target host is infected with or susceptible to said target pathogen, pest, or herbivore;   wherein said genetically modified bacteria expresses said RNase III mutant that cleaves said co-expressed target dsRNA molecule into sRNA molecules of a discrete size, wherein said sRNA molecules of discrete size are generated by said genetically modified bacteria and delivered to said target host or directly to said target pathogen, pest, or herbivore to induce an RNAi pathway response in said target pathogen, pest, or herbivore and protect said target host from said target pathogen, pest, or herbivore.   
     
     
         16 . The method of  claim 15 , wherein said sRNA molecules downregulate said essential gene of said target pathogen, pest, or herbivore pathogen through said a DICER-independent RNAi response pathway. 
     
     
         17 . The method of  claim 15 , wherein said administering or applying comprises feeding said effective amount of said genetically modified bacteria to said target host. 
     
     
         18 . The method of  claim 15 , wherein said administering or applying comprises applying said effective amount of said genetically modified bacteria to a target host plant topically. 
     
     
         19 . The method of  claim 15 , wherein said genetically modified bacteria is administered or applied prophylactically. 
     
     
         20 . The method of  claim 15 , wherein said sRNA molecules of discrete size consist of 22 to 23 nucleotides.