Transcript Slide 1

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
mRNA
mRNA cleavage and
degradation
AAA
AAA
endogene
QDE2
Nucleus
RISC
siRNA
transgenes
DNA\DNA
interaction
QDE3
epigenetic
modifications
DCR1
DCR2
dicer
QDE3
dsRNA
QDE1
RdRP
aberrant
ssRNA
activity
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RNAi vector for T. reesei
The inverted repeat is placed under the control of a
quinic acid inducible promoter
XmaI
5’end
945nt
350nt
intron
XmaI
qa-2p XmaI trpC T
pIR
dsRNA
3’end
benomyl
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Isolation of multicopy transformants
Southern Blot T. ressei transformed with N.crassa
albino gene (al-1) RNAi vector
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65
3 12 14 24 25 49 51 57 60 M B M B
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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 noninduced 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 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 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
UUAGGUCAU
hairpin
ds-mRNA
AUGACCUAA
PCR
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Small interfering RNA's are present
only in laccase silenced strains
siRNA Northern 24 bp lccB biotin labeled specific probe
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1.
2.
3.
4.
5.
6.
7.
8.
9.
2
3
4
5
6
7
8
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anti-probe 24 bp DNA Oligo (positive control)
P37 expressing laccase, base strain (negative control)
P37 expressing laccase, base strain (negative control)
P37; parent strain (negative control)
RNAi strain, lccB1-8 (laccase silenced)
RNAi strain, lccB1-21 (laccase silenced)
RNAi strain, lccB1-26 (laccase silenced)
RNAi strain, lccB2-5 (laccase silenced)
RNAi strain, lccB2-7 (laccase silenced)
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Use of RNAi to manipulate
fungal morphology
The mutations in the cot1 gene can results in compact
morphologies
Normal growth
+RNAi-cot1
vector
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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
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Use RNAi to characterize regulatory
function in protein secretion
Slide by R Prade OSU
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mRNA degradation in cre1-RNAi
hairpin strains
1
2 3 4
5 6 7
8
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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)
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mRNA degradation in cre1RNAi 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
function in protein secretion
Slide by R Prade OSU
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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
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Transformants with RNAi version
of creC
Evidence of DICER
activity
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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
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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
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mRNA cleavage and degradation
mRNA
mRNA cleavage and
degradation
AAA
AAA
endogene
QDE2
Nucleus
RISC
siRNA
transgenes
DNA\DNA
interaction
QDE3
epigenetic
modifications
DCR1
DCR2
dicer
QDE3
dsRNA
QDE1
RdRP
aberrant
ssRNA
activity
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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
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Summary RNAi Pathway
S-PTGS
sense transgene
qde3
aRNA
qde1
dsRNA
dicer
IR-PTGS
Inverted repeat transgene
siRNA
qde2/RISC
mRNA degradation
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N. crassa results
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
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pIR induces higher silencing frequency than a plasmid
(pX16) containing a single copy
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The presence of a single full-length pIR copy
is sufficient to induce silencing
UNSILENCED
INDUCIBLE SILENCED
CONSTITUTIVELY SILENCED
0
5
10
Relative copy number of full-length pIR
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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
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B. Bower & C Lin
Genencor International
E Forrest, G Marcino & C Cogoni
University of Rome
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