The use of RNAi to suppress gene function in industrial fungi Nigel S. Dunn-Coleman BMS Meeting, Manchester September ,

1. The use of RNAi to suppress gene function in industrial fungiNigel S. Dunn-ColemanBMS Meeting, Manchester September , 2005

2. RNAi pathway in N. crassa

3. mRNA cleavage and degradation AAA QDE2 mRNA RISC AAA endogene Nucleus siRNA transgenes DNA\DNA interaction QDE3 DCR1 DCR2 dicer QDE3 epigenetic modifications dsRNA QDE1 RdRP activity aberrant ssRNA mRNA cleavage and degradation

4. RNAi vector for T. reesei The inverted repeat is placed under the control of a quinic acid inducible promoter XmaI intron XmaI 5’end XmaI qa-2p 350nt trpC T 945nt pIR dsRNA benomyl 3’end

5. Isolation of multicopy transformants Southern Blot T. ressei transformed with N.crassa albino gene (al-1) RNAi vector 13 65 3 12 14 24 25 49 51 57 60 M B M B

6. Evidence for the RNAi pathway activity DICER in T. reesei Small interfering RNAs corresponding to the al-1 dsRNA.The transformants 1, 24 and 42 show a clear accumulation of siRNA. The RNA was extracted from cultures either in quinic induced (i) or non-induced conditions (ni). The 6xw is a Neurospora silenced strain with multiple copies of transgene, used as positive control. The strains B1 and B7 are also positive controls.

7. RNAi reporter system for fungi Genencor in collaboration with academic researchers has developed laccase as a reporter system for gene activity for A. niger and T. reesei (submitted) laccase gene over expressed in T. reesei strain P37(ABTS indicator plates)

8. AUGACCUAA UUAGGUCAU RNAi hairpin construct targeting T.reesei expressed Stacchybotyris laccase B gene repeat, 500 bp lccB anti-sense strand 500 bp lccB sense strand unpaired ATGACCTAA TTAGGTCAT transcription lccB Effective suppression of laccase activity PCR hairpin ds-mRNA

9. Small interfering RNA's are present only in laccase silenced strains siRNA Northern 24 bp lccB biotin labeled specific probe 1 2 3 4 5 6 7 8 9 1. anti-probe 24 bp DNA Oligo (positive control) 2.  P37 expressing laccase, base strain (negative control) 3.  P37 expressing laccase, base strain (negative control) 4.  P37; parent strain (negative control) 5.  RNAi strain, lccB1-8 (laccase silenced) 6.  RNAi strain, lccB1-21 (laccase silenced) 7.  RNAi strain, lccB1-26 (laccase silenced) 8. RNAi strain, lccB2-5 (laccase silenced) 9. RNAi strain, lccB2-7 (laccase silenced)  

10. Use of RNAi to manipulate fungal morphology The mutations in the cot1 gene can results in compact morphologies Normal growth +RNAi-cot1 vector

11. Use RNAi to characterize regulatory function in protein secretion areA is a positively acting regulatory gene which has been shown to be essential for activating genes encoding enzymes, permeases, needed to acquire nitrogen for the environment areA has recently been shown in Aspergillus to play a positive role in cellulase expression creB and creC play a role in conjunction with cre1 in the regulation of cellulases. Make RNAi versions of these genes to determine impact on cellulase expression. The genes for all three of these regulators are found in the JGI T. reesei genome sequence No mutants for areA, creB or creC exist in T. reesei

12. Use RNAi to characterize regulatory function in protein secretion Slide by R Prade OSU

13. mRNA degradation in cre1-RNAi hairpin strains 1 2 3 4 5 6 7 8 9 cre1 mRNA Probable creA mRNA degradation product Lanes 1-7: P-37 independent cre1-RNAi transformants Lane 8. P-37 transformed with IRal-1 (control) Lane 9: P-37 untransformed (control)

14. mRNA degradation in cre1-RNAi hairpin strains cre1 phenotype Second demonstration that RNAi can be used to regulate morphology in T. reesei These transformants are also carbon catabolite de-repressed

15. Use RNAi to characterize regulatory function in protein secretion Slide by R Prade OSU

16. creB and creC Mutations in creA, creB and creC lead to significant carbon catabolite de-repression of cellulase in A. nidulans The role of the CREB/CREC complex is to remove ubiquitin from specific substrates Mutants examined to-date appear to be loss of function mutations (K Kelly et al) Two T. reesei homologs in JGI T. reesei genome The small, 76-residue, protein is found both as free monomer in eukaryotic cells, and co-valently attached to itself and other proteins. The C-terminus of ubiquitin forms an isopeptide bond with the amino group of a lysine side chain in a target protein. In this way proteins can be covalently modified by the addition of ubiquitin (cf. phosphorylation) which may alter the target protein's function. If a chain of multiple copies of ubiquitin is atached to a target proteins this appears to target the protein for degradation by the large intacellular protease known as the 26S proteasome. However, recent evidence suggests that ubiquitination (or ubiquitinylation - whatever you prefer!) can target proteins for other fates besides degradation by the proteasome. Ubiquitinylation has been compared to phosphorylation (hence the change in the word) , and indeed the emeging scope and universality of this protein modification suggests this comparison is not fanciful. A great deal of interest is focusing on the multiple roles of ubiquitinylation, not just from the basic science viewpoint, but also because of its importance in disease.

17. Transformants with RNAi version of creC Evidence of DICER activity

18. SDS Gel from supernatants 1 2 3 4 5 6 8 9 10 11 12 Line 1: Standard Line 2: control P3-37 Line 3: Sample A2 Line 4: Sample A8 Line 5: Sample A9 Line 6: Sample A34 Line 8: control P-37 Line 9: Sample CB 9 Line 10: Sample CB 21 Line 11: Sample CB 4 Line 12: Sample CB 5

19. SDS Gel from supernatants 1 3 4 5 6 7 8 Line 1: Standard Line 3: control P-37 Line 4: Sample CC1 Line 5: Sample CC5 Line 6: Sample CC53 Line 7: Sample CC19 Line 8: Sample CC 48

20. mRNA cleavage and degradation AAA QDE2 mRNA RISC AAA endogene Nucleus siRNA transgenes DNA\DNA interaction QDE3 DCR1 DCR2 dicer QDE3 epigenetic modifications dsRNA QDE1 RdRP activity aberrant ssRNA mRNA cleavage and degradation

21. Conclusion for T. reesei • The expression of dsRNA by a transgenic inverted repeat is expected to by-pass both qde3 and qde1 but NOT dicer and qde2 These are similar results to those obtained earlier in N. crassa

22. S-PTGS sense transgene qde3 aRNA qde1 dsRNA dicer siRNA IR-PTGS qde2/RISC Inverted repeat transgene mRNA degradation Summary RNAi Pathway

23. N. crassa results pIR induces higher silencing frequency than a plasmid (pX16) containing a single copy

24. The presence of a single full-lengthpIR copy is sufficient to induce silencing UNSILENCED INDUCIBLE SILENCED CONSTITUTIVELY SILENCED 5 10 0 Relative copy number of full-length pIR

25. Considerations on the induction of gene silencing The presence of a single full-length copy of pIR is sufficient to induce silencing of al-1 gene. However, very few (less than 10%) of the transformants strains show an “inducible” silencing IT IS IMPORTANT TO USE A VERY TIGHTLY REGULATED PROMOTER

26. B. Bower & C Lin Genencor International E Forrest, G Marcino & C Cogoni University of Rome