RNA干扰载体的构建

C hapter 40 C onstruction of Hairpin RNA-Expressing Vectors
for RNA-Mediated Gene Silencing in Fungi
Shaobin Zhong,Yueqiang Leng,and Melvin D.Bolton
Abstract
R NA-mediated gene silencing is one of the major tools for functional genomics in fungi and can be achieved by transformation with constructs that express hairpin (hp) RNA with sequences homologous to the target gene(s). To make an hpRNA expression construct, a portion of the target gene can be amplifi ed by PCR and cloned into a vector as an inverted repeat. The generic gene-silencing vectors such as the pSilent1 and pSGate1 have been developed and are available for RNA-mediated gene-silencing studies. In this protocol, we describe construction of hpRNA-expressing constructs using both pSilent1 and pSGate1. With pSilent1, the PCR products of the target gene are inserted into the vector by conventional cloning (i.e., restriction enzyme digestion and ligation). For pSGate1, the PCR products of the target gene are inserted into the vector through the Gateway-directed recombination system. In this chapter, we describe the construction of RNAi vectors for RNA-mediated gene silencing using both pSilent1 and pSGate1.
K ey words:R NAi ,G ene silencing ,G ateway cloning system ,H airpin RNA ,p Silent1 ,p SGate1
1.Introduction
R NA-mediated gene silencing or RNA interference (1)has been
found to be a common gene-silencing phenomenon in eukaryotic
organisms. In this process, double-stranded RNA (dsRNA),
which can be induced by viral RNA, hairpin RNA (hpRNA), or
virus-induced gene-silencing encoded RNA (2), is degraded into
small interfering RNAs (siRNAs). These siRNAs are incorporated
into the RNA-induced silencing complex to target and degrade
mRNA of genes with complementary sequence (2–5). RNAi has
been demonstrated to silence genes in a number of fungi (for
review see ref. (6)). Examples include C ryptococcus neoformans(5),
M agnaporthe oryzae(7–9),V enturia inaequalis(10),H istoplasma Melvin D. Bolton and Bart P.H.J. Thomma (eds.), Plant Fungal Pathogens: Methods and Protocols,
Methods in Molecular Biology,vol. 835, DOI 10.1007/978-1-61779-501-5_40, © Springer Science+Business Media, LLC 2012
623
624S. Zhong et al.
capsulatum ( 11 ) , S chizophyllum commune ( 12 ) , C oprinopsis
cinerea ( 13,
14 ) , M ortierella alpina ( 15 ) , D ictyostelium discoi-deum (
16 ) , A spergillus and F usarium species ( 4 ) , B ipolaris oryzae ( 17 ) , C ladosporium fulvum ( 18– 20 ) , and O phiostoma novo-ulmi
( 21 ) . Most of the studies mentioned above used RNAi vectors containing inverted repeats of the target gene or its partial
sequence separated by a spacer for transformation. Construction
of these RNAi vectors often involves several cloning steps and
thus is laborious and time-consuming. Nakayashiki et al. (
9 ) devel-oped the pHANNIBAL-like silencing vector, pSilent-1 (see Fig.
1 ), for RNA silencing studies in fi lamentous fungi. To make an
RNAi construct expressing the hpRNA of the target gene with
a ) A schematic map of the vector pSilent1 . Unique restriction sites are indicated.
A mpR ampicillin-resistant gene; H phR hygromycin-resistant gene; I ntron intron 2 of the
cutinase (CUT) gene from M agnaporthe oryzae; PtrpC Aspergilus nidulans trpC promoter;
T trpC A. nidulans trpC terminator. ( b ) Restriction sites are u nderlined .A sterisks indicate
unique sites in the silencing vector. I talic nucleotides indicate sequence from the CUT
gene and b old letters represent 5 and 3 splice sites ( 9 ).
625
40 Construction of Hairpin RNA-Expressing Vectors …pSilent-1, a DNA fragment from a target gene is amplifi ed by
PCR with primers containing appropriate restriction sites and
cloned sequentially into each side of the intron spacer in the vec-
tor (see Fig.
2 ). The applicability of pSilent-1 was demonstrated in several phytopathogenic ascomycete fungi, including M . oryzae ,
C olletotrichum lagenarium , and B . oryzae (
9, 17 ) . However, pSi-lent-1 is a restriction enzyme-based cloning vector and therefore
may not be suitable for the construction of large numbers of
RNAi vectors for high-throughput gene-silencing studies.
Recently, a pHELLSGATE-like RNAi vector (pTroya) based on
Invitrogen’s Gateway technology has been developed and shown
to be useful in C . gloeosporioides (
22 ) . A similar type of RNAi vec-tor (pSGate1) (see Fig.
3 ) based on the Gateway system has also been developed and used for gene silencing in the spot blotch
fungal pathogen of barley and wheat C ochliobolus sativus (
23 ) . To make an RNAi construct with pSGate1, a portion of the target
gene is amplifi ed by PCR with primers containing the a tt B 1 and
a tt B 2 sites, and cloned into an entry plasmid (pDONR221) to
create an entry clone by BP reaction (see Fig.
4 ). The insert in the entry clone is then recombined into both sides of the intron
spacer in pSGate1 in opposite orientation with the LR reaction,
resulting in an RNAi construct expressing the hpRNA of the tar-
get gene (see Fig.
4 ). In this protocol, we describe the construc-tion of RNAi vectors for RNA-mediated gene silencing using
both pSilent1 and pSGate1.
F ig. 2. A fl owchart showing the construction of a hairpin (hp) RNA-expressing RNAi vector using pSilent1. A DNA fragment from a target gene was amplifi ed by PCR with primers containing appropriate restriction sites. The PCR product was cloned into each side of the intron in pSilent1 sequentially, resulting in an RNAi construct expressing the hpRNA of the target gene.
626S. Zhong et al.
1. B ench top microcentrifuge.
2. E nvironmental incubator shaker.
3. p H meter.
2.Materials
2.1.Equipment
and Consumables F ig. 4. F lowchart showing the construction of a hairpin (hp) RNA-expressing RNAi vector using pSGate1. A DNA fragment from a target gene is amplifi ed by PCR with primers containing the a tt
B1and att B 2 sites. The PCR product is cloned into the plasmid pDONR221 to create an entry clone by BP reaction. The insert in the entry clone is then replaced by the c cdB genes of pSGate1 by LR reaction, resulting in an RNAi construct expressing the hpRNA of the target gene.
Hind III F ig. 3. A schematic map of the Gateway cloning system-based RNAi vector, pSGate1 .The ccdB cassettes were derived from pHellsgate-12 ( 24 ) and cloned into the silencing vector pSilent1 ( 9 ) . Unique restriction sites are indicated. A mpR ampicillin-resistant gene; H phR hygromycin-resistant gene; I ntron intron 2 of the cutinase (CUT) gene from M agnaporthe oryzae; PtrpC Aspergilus nidulans trpC promoter; T trpC A. nidulans trpC terminator.
62740 Construction of Hairpin RNA-Expressing Vectors … 4. T hermal cycler. 5. E lectrophoresis equipment. 6. W ater bath. 7. P etri plates. 8. C ell culture tubes. 9. M icrocentrifuge tubes. 10. P CR tubes. 1. L B medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 20 g of agar (for plates only) to 900 mL deionized H 2 O . Adjust pH to 7.0 with NaOH solution. Add deionized H 2
O to a fi nal
volume of 1 L. Autoclave. Cool to 55–60°C and add 1 mL of ampicillin (100 mg/mL; fi lter-sterilized with 0.2- m m mem-brane fi lter) or kanamycin (50 mg/mL; fi lter-sterilized with 0.2- m m membrane fi lter) as necessary.
2. S OC medium: 2% (w/v) tryptone, 0.5% (w/v) yeast extract,
10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4
, 20 mM glucose. Autoclave. 3. E lectrophoresis-grade agarose.
4. X ho I and H ind I II restriction enzymes, including 10× reaction
buffers as provided by supplier.
5. T 4 ligase, including 10× ligation buffer as provided by supplier.
6. P CR-grade Taq polymerase, including 10× PCR amplifi cation
buffer as provided by supplier.
7. P CR-grade dNTPs.
8. C hemically competent E scherichia coli cells.
9. P roteinase K: supplied with Gateway ® BP or LR kits.
10. T E (10 mM Tris–HCl pH 8.0, 1 mM EDTA).
11. 30%PEG/MgCl 2 : supplied with Gateway ®
BP or LR kits.
12. D NA purifi cation Kit.
13. P lasmid miniprep Kit.
14. G ene-specifi c primers with appropriate restriction sites at the
5 ¢ end (for construction of RNAi vectors with pSilent1) (see Note 2).
15. p Silent1 (for construction of RNAi vectors with pSilent1; see
Note 3).
16. G ene-specifi c primers with a tt B 1 and a tt B 2 sequences at the
5 ¢ end (for construction of RNAi vectors with pSGate1; see Note 4).
17. p SGate1 (for construction of RNAi vectors with pSGate1; see
Note 5).
2.2.Media
and Reagents
(see N ote 1 )
628S. Zhong et al.
18. p DONR221 (Invitrogen, Carlsbad, CA).
19. G ateway ® BP Clonase ® II enzyme mix (Invitrogen).
20.
G ateway ® LR Clonase ® II enzyme mix (Invitrogen). 1. D esign the fi rst set of primers containing appropriate restric-tion sites added to the 5 ¢ end of each primer that amplify the sense sequence of the target gene (see Note 2). For example
purposes, we have chosen the forward primer to contain the X ho I restriction site and the reverse primer to contain H ind I II restriction site.
2. A mplify the targeted gene using the primer pair designed above in a standard PCR reaction following Taq polymerase suppli-er’s recommendations. The thermal cycling conditions are as follows: initial denaturation (95°C, 2 min), followed by 35 cycles of denaturation (94°C, 30 s), annealing (58°C, 30 s; see Note 6), and extension (72°C, 1 min), and then one fi nal cycle of extension (72°C, 10 min).
3. D igest the PCR product with X ho I and H ind I II following restriction enzyme supplier’s digestion recommendations and buffers. Digestion is performed in a 50 m L reaction which con-tains 40 m L of PCR product, 5 m L of 10× restriction enzyme buffer, 0.5 m L 100× bovine serum albumin (BSA), 5 units of each enzyme, and water up to 50 m L . The reaction is incubated at 37°C overnight (see Note 7).
4. P urify the PCR product using a commercial PCR purifi cation kit following the manufacturers’ recommendations.
5. L inearize pSilent-1 by H ind I II and X ho I double-digestion fol-lowing restriction enzyme supplier’s digestion recommenda-tions and buffers.
6. P urify the linearized pSilent-1 vector using a commercial puri-fi cation kit following supplier’s recommendations.
7. L igate the X ho I / H ind I II-digested PCR product in the X ho I / H ind I II-digested pSilent1. Ligation is performed in a 10 m L reaction which contains 50 ng of purifi ed PCR product, 100 ng of pSilent-1, 1 m L of 10× ligation buffer, 1 m L of T4 Ligase, and water up to 10 m L . Incubate the reaction at room temperature for 1–2 h (see Note 8).
8. A dd 2–5 m L of the ligation reaction to 50 m L of competent E . coli cells and incubate on ice for 30 min. Heat-shock cells by incubating at 42°C for 60 s without shaking. Remove the vial from 42°C water bath and place on ice for 2 min. 3.Methods
3.1.Construction of RNAi Vectors with pSilent1
62940 Construction of Hairpin RNA-Expressing Vectors … 9. A dd 250 m L of SOC medium and incubate at 37°C for 1 h with shaking at 150 rpm. 10. S pread an aliquot of the transformation reaction onto LB agar plates with 100 m g /mL ampicillin (see Note 9). Incubate plates at 37°C for 24 h. 11. P ick single colonies and inoculate culture tubes containing 2 mL of LB containing 100 m g /mL ampicillin and incubate at 37°C with shaking at 250 rpm overnight (around 14–16 h). 12. I solate the plasmid DNA using a commercial plasmid purifi ca-tion kit following the manufacturer’s recommendations. 13. V erify the integration of the PCR product into pSilent1 by double digestion of the plasmid with H ind I II and X ho I follow-ing restriction enzyme supplier’s digestion recommendations and buffers. Alternatively, use PCR to confi rm integration. 14. D esign the second set of primers containing appropriate restric-tion sites to amplify the sense sequence of the target gene (see Note 2). For example purposes, we have chosen the forward primer to contain the K pn I restriction site and the reverse primer to contain B gl I I restriction site. 15. R epeat steps 2–13 above but utilize K pn I and B gl I I for the design, integration, and confi rmation of the anti-sense copy of the gene to be silenced into pSilent1. 16. V erify the hpRNA expression construct by double digestion ( X ho I / H ind I II or B gl I I/ K pn I ) and visualize pattern in stan-dard gel electrophoresis (see Note 10). 17. T he verifi ed silencing construct is linearized by S pe I and can be used for RNAi via PEG-mediated transformation (see Note 11). 1. D esign a primer pair with the appropriate attB sequences added at the 5 ¢ end of each primer (see Note 4). 2. A mplify the targeted gene using the primer pair designed above
in a standard PCR reaction following Taq polymerase suppli-er’s recommendations. The thermal cycling conditions are as follows: initial denaturation (95°C, 2 min), followed by 35 cycles of denaturation (94°C, 30 s), annealing (58°C, 30 s; see Note 6), and extension (72°C, 1 min), and then one fi nal cycle of extension (72°C, 10 min).
3. A dd 75 m L of TE and 50 m L of 30% PEG/MgCl 2
solution to
25 m L of the PCR reaction to purify the PCR product. Mix well and centrifuge for 15 min at 14,000 rpm in a microcentrifuge. 4. R emove the supernatant carefully and re-suspend the pellet in
10 m L TE.
5. Q uantify the PCR product. The concentration of the purifi ed
PCR product should be more than 10 ng/ m L (see Note 12).
3.2.Construction
of RNAi Vectors
with pSGate1
630S. Zhong et al.
6. M ix 1–7 m L (15–150 ng) of the purifi ed PCR products with
1 m L of pDONR221, and add TE to the fi nal volume of 8 m L.
Keep the reaction on ice and add 2 m L BP Clonase II enzyme
mix (see Note 13). Mix well and incubate at 25°C for 2 h.
7. A dd 1 m L Proteinase K solution to the sample and incubate at
37°C for 10 min to terminate the reaction.
8. A dd 3–5 m L of BP reaction to 50 m L of competent E. coli cells
and incubate on ice for 30 min. Heat-shock cells by incubating
at 42°C for 60 s without shaking. Remove the vial from 42°C
water bath and place on ice for 2 min.
9. A dd 250 m L of SOC medium and incubate at 37°C for 1 h
with shaking at 150 rpm.
10. S pread an aliquot of the transformation reaction onto LB agar
plates containing 50 m g/mL kanamycin. Incubate plates at
37°C for 24 h (see Note 9).
11. P ick single colonies and inoculate culture tubes containing
2 mL of LB with 50 m g/mL kanamycin and incubate at 37°C
with shaking at 250 rpm overnight (around 14–16 h).
12. I solate the plasmid DNA using a commercial plasmid purifi ca-
tion kit following the manufacturer’s recommendations.
13. C arry out PCR using the isolated plasmid DNA as template
and the primers designed in step 1 to verify whether the plas-
mid contains the DNA insert from the target gene. The veri-
fi ed plasmid is then used as entry clone for the LR reaction.
14. P erform LR reaction by mixing 1 m L entry clone (150 ng/ m L),
1 m L destination clone (pSGate1, 100 ng/ m L), and 6 m L TE.
Keep the reaction on ice and add 2 m L LR clonase enzyme
mix (see Note 14). Mix the reaction well and incubate at
25°C for 2 h.
15. A dd 1 m L Proteinase K solution to the sample and incubate at
37°C for 10 min to terminate the reaction.
16. A dd 3–5 m L of the BP reaction to 50 m L of competent E. coli
cells and incubate on ice for 30 min. Heat-shock cells by incu-
bating at 42°C for 60 s without shaking. Remove the vial from
42°C water bath and place on ice for 2 min.
17. A dd 250 m L of SOC medium and incubate at 30°C (see Note
15) for 1 h with shaking at 150 rpm. Spread 100 m L of trans-
formation reaction onto LB agar plates containing 50 m g/mL
ampicillin. Incubate plates at 30°C (see Note 15) for 24 h.
18. P ick 6–10 single colonies and inoculate them individually to
culture tubes containing LB medium with 50 m g/mL ampicil-
lin. Incubate the cultures at 30°C (see Note 15) with shaking
at 250 rpm overnight (around 14–16 h).
631
40 Construction of Hairpin RNA-Expressing Vectors…
19. I solate the plasmid DNA using a commercial plasmid purifi ca-
tion kit following the manufacturer’s recommendations.
20. V erify the hpRNA-expressing construct by single restriction
enzyme ( X ho I) and double restriction enzyme ( B gl I I and K pn I) digestions (see Note 16).
21. T he verifi ed silencing construct is linearized by S pe I and can be
used for RNAi via PEG-mediated transformation (see Note 11).
1. A ll media used for culturing bacteria should be sterilized by
autoclaving at 121°C for 20 min.
2. T wo sets of primers must be designed to amplify the target
gene. The fi rst primer pair amplifi es a fragment that is cloned into the H ind I II -Sna B I -Xho I polylinker of pSilent1 and the second primer pair amplifi es the same fragment, which is cloned into the B gl I I -Sph I-Stu I-Kpn I-Apa I polylinker of pSilent1 (see Fig. 1).The region of the target gene to be amplifi ed should not contain the same restriction enzyme sites as those used in primers. Gene-specifi c primers should be 18–25 bp with melt-
ing temperature ( T
M ) ranging from 55 to 60°C. The size of
fragment to be amplified from the target gene should range
from 300 to 600 bp to achieve effective gene silencing.
3. p Silent-1 is available from the Fungal Genetics Stock Center
(FGSC#634).
4. T he forward primer is 5 ¢-GGGGACAAGTTTGTACAAAAAA
GCAGGCT- g ene-s pecifi c primer-3 ¢and the reverse primer is
5 ¢-GGGGACCACTTTGTACAAGAAAGCTGGGT- g ene-
s pecifi c primer-3 ¢. Gene-specifi c sequences should be 18–25 bp
with annealing temperature ranging from 55 to 60°C. The size
of fragment to be amplifi ed from the target gene should range
from 300 to 600 bp to achieve effective gene silencing.
5. p SGate1 is available from the Fungal Genetics Stock Center.
6. A nnealing temperature depends on T
M
of the primers used.
7. D igestion of PCR productions may take as little as 2 h.
However, if a large amount of DNA is digested, an overnight
digestion is recommended.
8. F or more effi cient ligation, the ligation reaction can be incu-
bated at 16°C overnight. The following formula shows how to
calculate the amount of insert and vector used in the ligation
reaction: amount of insert/100 ng vector = 100 × size of insert
(Kb) × 3/size of vector (Kb).
4.Notes
632S. Zhong et al.
9. W hen plating E. coli cells on LB agar plates, plate with two dif-
ferent volumes (50 and 100 m L) to ensure that at least one
plate has well-separated colonies.
10. D ouble-digestion by H ind I II and X ho I or B gl I I and K pn I
should generate a fragment with the same size as the PCR
product amplifi ed from the target gene. Sequencing could be
used to further confi rm the insert in the vector construct.
11. T he hpRNA-expressing construct cassette can also be released
with appropriate restriction enzymes and cloned into an appropri-
ate T-DNA vector for A grobacterium-mediated transformation.
12. A fter purifi cation, 10 m L of TE is usually added to the purifi ed
PCR products and 3 m L of the DNA solution is used for BP
reaction. If the concentration of the purifi ed PCR product is
too low, a larger volume (50 m L or more) of the PCR reaction
can be purifi ed to achieve a higher concentration.
13. T haw the BP clonase enzyme mix on ice before use and store
at −20 or −80°C immediately after use.
14. T haw the LR clonase enzyme mix on ice before use and store
at −20 or −80°C immediately after use.
15. I ncubation and culture of bacteria containing the hrRNA-
expressing plasmid construct at 30°C is highly recommended
to optimize bacterial growth and stabilize the construct.
16. D igestion by single restriction enzyme ( X ho I) and double
restriction enzymes ( B gl I I and K pn I) should generate the same
size of fragments. Sequencing could be used to further confi rm
the vector. Approximately 50% of the colonies are likely to
contain the right construct.
Acknowledgments
T he authors thank Dr. H. Nakayashiki (Kobe University, Japan)
for generously providing pSilent-1.
References
1. C oppin E, Debuchy R, Arnaise S, Picard M (1997) Mating types and sexual development in fi lamentous ascomycetes. Microbiol Mol Biol Rev 61:411–428
2. W aterhouse PM, Helliwell CA (2003) Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 4:29–38
3. N akayashiki H,Nguyen QB (2008) RNA inter-ference: roles in fungal biology. Curr Opin Microbiol 11:494–502
4. M cDonald T, Brown D, Keller NP, Hammond TM (2005) RNA silencing of mycotoxin pro-duction in A spergillus and F usarium species. Mol Plant-Microbe Interact 18:539–545
5. L iu H, Cottrell TR, Pierini LM et al (2002) RNA interference in the pathogenic fungus C ryptococcus neoformans. Genetics 160:463–470
6. Kück U, Hoff B (2010) New tools for the genetic manipulation of fi lamentous fungi. Appl Microbiol Biotechnol 86:51–62
633 40 Construction of Hairpin RNA-Expressing Vectors…
7. J eon J, Park SY, Chi MH et al (2007) Genome-
wide functional analysis of pathogenicity genes
in the rice blast fungus. Nat Genet 39: 561–565
8. K adotani N, Nakayashiki H, Tosa Y, Mayama S
(2003) RNA silencing in the phytopathogenic
fungus M agnaporthe oryzae. Mol Plant-Microbe
Interact 16:769–776
9. N akayashiki H, Hanada S, Quoc NB et al
(2005) RNA silencing as a tool for exploring
gene function in ascomycete fungi. Fungal Genet Biol 42:275–283
10. F itzgerald A, van Kan JAL, Plummer KM
(2004) Simultaneous silencing of multiple genes in the apple scab fungus, V enturia
inaequalis, by expression of RNA with chimeric
inverted repeats. Fungal Genet Biol 41: 963–971
11. R appleye CA, Engle JT, Goldman WE (2004)
RNA interference in H istoplasma capsulatum
demonstrates a role for a-(1, 3)-glucan in viru-
lence. Mol Microbiol 53:153–165
12. D e Jong JF, Deelstra HJ, Wosten HAB, Lugones
LG (2006) RNA-mediated gene silencing in
monokaryons and dikaryons of S chizophyllum
commune. Appl Environ Microbiol 72: 1267–1269
13. N amekawa SH, Iwabata K, Sugawara H et al
(2005) Knockdown of L IM15/DMC1in the
mushroom C oprinus cinereus by double-stranded
RNA-mediated gene silencing. Microbiology 151:3669–3678
14. W alti MA, Villalba C, Buser RM et al (2006)
Targeted gene silencing in the model mush-
room C oprinopsis cinerea( C oprinus cinereus)
by expression of homologous hairpin RNAs.
Eukaryot Cell 5:732–744
15. T akeno S, Sakuradani E, Tomi A et al (2005)
Improvement of the fatty acid composition of
an oil-producing filamentous fungus, M ortierella
alpina1S-4, through RNA interference with
D12-desaturase gene expression. Appl Environ
Microbiol 71:5124–5128
16. M artens H, Novotny J, Oberstrass J et al (2002)
RNAi in D ictyostelium: the role of RNA-
directed RNA polymerases and double-stranded
RNase. Mol Biol Cell 13:445–453
17. M oriwaki A, Ueno M, Arase S, Kihara J (2007)
RNA mediated gene silencing in the phyto-
pathogenic fungus B ipolaris oryzae. FEMS Microbiol Lett 269:85–89
18. v an Esse HP, Bolton MD, Stergiopoulos I et al
(2007) The Chitin-Binding C ladosporium ful-
vum effector protein Avr4 is a virulence factor.
Mol Plant-Microbe Interact 20:1092–1101 19. B olton MD, van Esse HP, Vossen JH et al
(2008) The novel C ladosporium fulvum lysin
motif effector Ecp6 is a virulence factor with
orthologues in other fungal species. Mol Microbiol 69:119–136
20. v an Esse HP, van’t Klooster JW, Bolton MD
et al (2008) The C ladosporium fulvum viru-
lence protein Avr2 inhibits host proteases required for basal defense. Plant Cell 20:1948–1963
21. C arneiro JS, de la Bastide PY, Chabot M et al
(2010) Suppression of polygalacturonase gene
expression in the phytopathogenic fungus O phiostoma novo-ulmi by RNA interference.
Fungal Genet Biol 47:399–405
22. S hafran H, Miyara I, Eshed R et al (2008)
Development of new tools for studying gene
function in fungi based on the Gateway system.
Fungal Genet Biol 45:1147–1154
23. L eng Y, Wu C, Liu Z et al (2011) RNA-
mediated gene silencing in the cereal fungal
pathogen C ochliobolus sativus. Mol Plant Pathol
12:289–298
24. W esley SV, Helliwell CA, Smith NA et al (2001)
Construct design for efficient, effective and high throughput gene silencing in plants. Plant J
27:581–590。