Post on 28-Mar-2015
transcript
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
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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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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