Methods for eliminating or reducing the expression of genes in filamentous fungal strains by transitive RNA interference

Abstract

The present invention relates to methods of reducing or eliminating expression of a target gene in a filamentous fungal strain by transitive RNA interference.

1 - 8 . (canceled) 
     
     
         9 . A filamentous fungal strain comprising a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation relative to each other and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, is induced by cultivating the filamentous fungal strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the biological substance, and wherein the filamentous fungal strain is an  Aspergillus niger  strain,  Aspergillus oryzae  strain, or a  Trichoderma reesei  strain. 
     
     
         10 . The filamentous fungal strain of  claim 9 , wherein the first homologous region comprises at least 19 nucleotides of the target gene. 
     
     
         11 . The filamentous fungal strain of  claim 9 , wherein the second transcribable region with no effective homology to the target gene comprises at least 19 nucleotides. 
     
     
         12 . The filamentous fungal strain of  claim 9 , wherein the first and second polynucleotides are separated by an intervening sequence. 
     
     
         13 . The filamentous fungal strain of  claim 12 , wherein the two segments complementary to each other in reverse orientation are separated by a linking sequence. 
     
     
         14 . The filamentous fungal strain of  claim 12 , wherein expression of the target gene is reduced by at least 20%. 
     
     
         15 . The filamentous fungal strain of  claim 12 , wherein the interfering RNA interacts with RNA transcripts of one or more homologues of the target gene to reduce or eliminate expression of the one or more homologues of the target gene. 
     
     
         16 . The filamentous fungal strain of  claim 15 , wherein expression of the one or more homologues of the target gene is reduced by at least 20%. 
     
     
         17 . A method for producing a biological substance, comprising:
 (a) cultivating a filamentous fungal strain under conditions conducive for production of the biological substance of interest, wherein the filamentous fungal strain comprises a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to a target gene encoding an undesirable biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation relative to each other and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein RNA transcripts of the double-stranded transcribable nucleic acid construct are produced by cultivating the filamentous fungal strain under conditions to produce the RNA transcripts that are then converted to short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the undesirable biological substance; and wherein the filamentous fungal strain comprises a third polynucleotide encoding the biological substance of interest, and wherein the filamentous fungal strain is an  Aspergillus niger  strain,  Aspergillus oryzae  strain, or a  Trichoderma reesei  strain; and   (b) recovering the biological substance of interest from the cultivation medium.   
     
     
         18 . The method of  claim 17 , wherein the first transcribable region with homology to the target gene comprises at least 19 nucleotides of the target gene. 
     
     
         19 . The method of  claim 17 , wherein the second transcribable region with no effective homology to the target gene comprises at least 19 nucleotides. 
     
     
         20 . The method of  claim 17 , wherein the first and second polynucleotides are separated by an intervening sequence. 
     
     
         21 . The method of  claim 17 , wherein the two segments complementary to each other in reverse orientation are separated by a linking sequence. 
     
     
         22 . The method of  claim 17 , wherein expression of the target gene is reduced by at least 20%. 
     
     
         23 . The method of  claim 17 , wherein the interfering RNA interacts with RNA transcripts of one or more homologues of the target gene to reduce or eliminate expression of the one or more homologues of the target gene. 
     
     
         24 . The method of  claim 23 , wherein expression of the one or more homologues of the target gene is reduced by at least 20%.