The use of RNAi to suppress gene The use of RNAi to suppress gene function in industrial fungifunction in industrial fungi

Nigel S. Dunn-ColemanNigel S. Dunn-Coleman

BMS Meeting, Manchester September , 2005

RNAi pathway in N. crassa

3

mRNA cleavage and degradationmRNA cleavage and degradation

transgenesNucleus

endogene

epigeneticmodifications

QDE3

DNA\DNA interaction

QDE3

aberrant ssRNA

mRNAAAA

RdRPactivity

QDE1

DCR2

dsRNA

siRNA

AAA

QDE2

RISC

DCR1 dicer

mRNA cleavage and degradation

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RNAi vector for RNAi vector for T. reeseiT. reesei

trpC T

benomyl

pIR

XmaI

XmaI XmaIintron

The inverted repeat is placed under the control of a quinic acid inducible promoter

dsRNA

945nt350nt

5’end

3’end

qa-2p

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Isolation of multicopy transformantsIsolation of multicopy transformants

Southern Blot T. ressei transformed with N.crassa albino gene (al-1) RNAi vector

3 12 14 24 25 49 51 57 60 M B M B

13 65

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Evidence for the RNAi pathway Evidence for the RNAi pathway activity DICER in activity DICER in T. reeseiT. 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.

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RNAi reporter system for fungiRNAi 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)

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RNAi hairpin construct targeting RNAi hairpin construct targeting T.reesei T.reesei expressed expressed StacchybotyriStacchybotyris laccase B genes laccase B gene

ATGACCTAA unpaired

500 bp lccB sense strandrepeat, 500 bp lccB anti-sense strand

transcription

AU

GA

CC

UA

A

UU

AG

GU

CA

U

hairpinds-mRNA

TTAGGTCAT

PCR

lccB Effective suppression of

laccase activity

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Small interfering RNA's are present Small interfering RNA's are present only in laccase silenced strainsonly in laccase silenced strains

siRNA Northern 24 bp lccB biotin labeled specific probe

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)  

1 2 3 4 5 6 7 8 9

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Use of RNAi to manipulate Use of RNAi to manipulate fungal morphologyfungal morphology

+RNAi-cot1

vector

Normal growth

The mutations in the cot1 gene can results in compact morphologies

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Use RNAi to characterize regulatory Use RNAi to characterize regulatory function in protein secretionfunction 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

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Use RNAi to characterize regulatory Use RNAi to characterize regulatory function in protein secretionfunction in protein secretion

Slide by R Prade OSU

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cre1 mRNA

1 2 3 4 5 6 7 8 9

Lanes 1-7: P-37 independent cre1-RNAi transformants Lane 8. P-37 transformed with IRal-1 (control)Lane 9: P-37 untransformed (control)

Probable creA mRNA degradation product

mRNA degradation in mRNA degradation in cre1cre1-RNAi-RNAi hairpin strainshairpin strains

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mRNA degradation in mRNA degradation in cre1cre1--RNAiRNAi hairpin strains 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

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Use RNAi to characterize regulatory Use RNAi to characterize regulatory function in protein secretionfunction in protein secretion

Slide by R Prade OSU

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creBcreB and and creCcreC

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

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Transformants with RNAi version Transformants with RNAi version of of creCcreC

Evidence of DICER

activity

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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

1 2 3 4 5 6 8 9 10 11 12

SDS Gel from supernatantsSDS Gel from supernatants

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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

1 3 4 5 6 7 8

SDS Gel from supernatants

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mRNA cleavage and degradationmRNA cleavage and degradation

transgenesNucleus

endogene

epigeneticmodifications

QDE3

DNA\DNA interaction

QDE3

aberrant ssRNA

mRNAAAA

RdRPactivity

QDE1

DCR2

dsRNA

siRNA

AAA

QDE2

RISC

DCR1 dicer

mRNA cleavage and degradation

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Conclusion for Conclusion for T. reeseiT. 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

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Summary RNAi PathwaySummary RNAi Pathway

IR-PTGS Inverted repeat transgene

aRNA

dsRNA

siRNA

qde3

qde1

qde2/RISC

S-PTGS

sense transgene

dicer

mRNA degradation

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Strain Silenced/total % I pX16 (al-1 single copy plasmid) %

WT 54/70 77 32

qde-1 87/112 78 3

qde-3 57/83 68 2

qde-2 0/85 0 0

dcr1/dcr2 0/73 0 0

dcr1 130/180 72 30

dcr2 63/81 77 30

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

N. crassa N. crassa results results

24Relative copy number of full-length pIR

0 5 10

UNSILENCED

CONSTITUTIVELY SILENCED INDUCIBLE SILENCED

The presence of aThe presence of a single single full-leng full-lengthth pIRpIR copy copy is sufficient to induce silencingis sufficient to induce silencing

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ConConsiderations on the induction of siderations on the induction of gene silencinggene 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

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B. Bower & C Lin

Genencor International

E Forrest, G Marcino & C Cogoni

University of Rome