The use of RNAi to suppress gene function in industrial fungi
This presentation is the property of its rightful owner.
Sponsored Links
1 / 26

The use of RNAi to suppress gene function in industrial fungi Nigel S. Dunn-Coleman BMS Meeting, Manchester September , 2005 PowerPoint PPT Presentation


  • 162 Views
  • Uploaded on
  • Presentation posted in: General

The use of RNAi to suppress gene function in industrial fungi Nigel S. Dunn-Coleman BMS Meeting, Manchester September , 2005. RNAi pathway in N. crassa. mRNA cleavage and degradation. AAA. QDE2. mRNA. RISC. AAA. endogene. Nucleus. siRNA. transgenes. DNA\DNA interaction. QDE3. DCR1.

Download Presentation

The use of RNAi to suppress gene function in industrial fungi Nigel S. Dunn-Coleman BMS Meeting, Manchester September , 2005

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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


Rnai pathway in n crassa

RNAi pathway in N. crassa


Mrna cleavage and degradation

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


Rnai vector for t reesei

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


Isolation of multicopy transformants

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


Evidence for the rnai pathway activity dicer in t reesei

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.


Rnai reporter system for fungi

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)


Rnai hairpin construct targeting t reesei expressed stacchybotyri s laccase b gene

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


Small interfering rna s are present only in laccase silenced strains

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)  


Use of rnai to manipulate fungal morphology

Use of RNAi to manipulate fungal morphology

The mutations in the cot1 gene can results in compact morphologies

Normal growth

+RNAi-cot1

vector


Use rnai to characterize regulatory function in protein secretion

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


Use rnai to characterize regulatory function in protein secretion1

Use RNAi to characterize regulatory function in protein secretion

Slide by R Prade OSU


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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)


Mrna degradation in cre1 rnai hairpin strains

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

Use RNAi to characterize regulatory function in protein secretion

Slide by R Prade OSU


Creb and crec

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.


Transformants with rnai version of crec

Transformants with RNAi version of creC

Evidence of DICER

activity


Sds gel from supernatants

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

S-PTGS

sense transgene

qde3

aRNA

qde1

dsRNA

dicer

siRNA

IR-PTGS

qde2/RISC

Inverted repeat transgene

mRNA degradation

Summary RNAi Pathway


N crassa results

N. crassa results

pIR induces higher silencing frequency than a plasmid (pX16) containing a single copy


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

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


The use of rnai to suppress gene function in industrial fungi nigel s dunn coleman bms meeting manchester september

B. Bower & C Lin

Genencor International

E Forrest, G Marcino & C Cogoni

University of Rome


  • Login