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[object Object],Co-PI C. Rosa,   PI B.W. Falk,   Department of Plant Pathology, University of California, Davis, CA
Two pathways are shown, the miRNA (nuclear) and siRNA (cytoplasmic) pathways. The miRNA pathway is a natural means of gene regulation while the siRNA pathway is believed to have evolved primarily as a defense against cytoplasmic RNA viruses. For both, double stranded RNAs are initial targets, and the resulting small RNAs serve as “guide RNAs” to direct the silencing complex to the target. The enzyme  dicer   trims double stranded RNA, to form  small interfering RNA  or  microRNA .  These processed RNAs are incorporated into the  RNA-induced silencing complex  (RISC), which targets  messenger RNA  to prevent  translation . [wikipedia ]
RNAi-based virus resistance is derived from a discovery reported in 1928, that virus-infected plants could “recover” from a virus infection. A gradual decline of virus symptoms can be seen by examining leaves from the bottom up.  The top leaves are asymptomatic. There is an active response or defense of the plant against the virus infection. RNAi is already used in agriculture to control specific plant diseases.
RNAi for insect-proof plants. Nat Biotechnol. 2007 Nov;25(11):1231-2. Is RNAi activity found in insects?
Control of coleopteran insect pests through RNA interference Nature Biotechnology 25, 1322 - 1326 (2007)   James A Baum 1 , Thierry Bogaert 2 , William Clinton 1 , Gregory R Heck 1 , Pascale Feldmann 2 , Oliver Ilagan 1 , Scott Johnson 1 , Geert Plaetinck 2 , Tichafa Munyikwa 1 , Michael Pleau 1 , Ty Vaughn 1  & James Roberts 1   1 Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017-1732, USA. 2 Devgen N.V., Technologiepark 30, B-9052 Ghent–Zwijnaarde, Belgium.
F1 plants expressing a V- ATPase  A  dsRNA  are protected from  Diabrotica virgifera virgifera  western corn rootworm (WCR)   feeding damage. ,[object Object],[object Object],[object Object],Nature Biotechnology 25, 1322 - 1326 (2007)
Homalodisca vitripennis  overview Classification :   Order: Hemiptera (True Bugs, Cicadas, Hoppers, Aphids and Allies)  Family: Cicadellidae (Leafhoppers)  Subfamily: Cicadellinae  Genus:  Homalodisca   Species:  vitripennis  ( Glassy-winged sharpshooter )   It is one of the vectors of  X. fastidiosa . Its host range includes many native, ornamental and crop plants.  One of the preferred hosts in Southern California and other areas is citrus. http://www.acgov.org/cda/awm/agprograms/pestexclusion/sharpshooter.htm
Specific objectives ,[object Object],[object Object],[object Object]
Our available tools ,[object Object],[object Object],[object Object],[object Object],http://www.acgov.org/cda/awm/agprograms/pestexclusion/sharpshooter.htm
UC Davis Biosafety Level 3P, Contained Research Facility GWSS is a quarantined pest in California and is regulated by CDFA. At the present, we are only able to rear and maintain prolific colonies of GWSS in the UC Davis CRF.
GWSS cell experiments
First genes chosen for their cellular activities to trigger gene silencing. ,[object Object],[object Object],Actin filaments stained using Alexa-Fluor phalloidin (Molecular probe, Invitrogen)
Genes screened in GWSS cells as candidate gene silencing targets. The same genes were also cloned from GSS RT-PCR products for selected GWSS mRNAs. Lanes 3 and 15 show products for Sar1 and actin, respectively.   Lanes 1, 2, 4, 5, 6 and 8 – 14 show RT-PCR products for vitellogenin, histone 3, RAB1 1, kinase C receptor, ubiquitin conjugating enzyme, tropomyosin, mitochondrial porin, Delta 9 saturase, Fructose 1,6 biphosphate aldolase, rhodopsin, ferritin, and arginine kinase, respectively. 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15
RNAi inducers tested in the GWSS Z15 cell line ,[object Object],[object Object],[object Object],[object Object],[object Object],1 3 2 siRNA pGEMTeasy  T7 SP6 actin Nco I (38) Pst I (1423) Sac I (1444) Sac II (50) Mlu I (1449) Not I (44) Not I (1412) 6X his tag actin Tribolium actin rc polyadenilation region methallothionein promoter pMT 48
Efficacy of RNAi inducers: siRNA vs. dsRNA vs. hairpin loop RNA. Transfection efficiency was measured by labeled siRNA, and was almost 50%. GWSS cells  dsRNA was the most efficient RNAi inducer Actin mRNA 72 hpt 0 0.2 0.4 0.6 0.8 1 1.2 control actin dsRNA pMT48 actin hairpin loop actin siRNA
Time course transfection experiments in cells ,[object Object],actin   mRNA 0 0.5 1 1.5 2 2.5 control actin dsRNA  pMT48 actin hairpin loop control actin dsRNA  pMT48 actin hairpin loop control actin dsRNA  pMT48 actin hairpin loop Series1 24 hpt 48 hpt 72 hpt
actin dsRNA vs sar1 dsRNA: real time RT-PCR results in cells Actin dsRNA induced better RNAi effects than sar1 dsRNA, when compared to transfection reagent transfected cells.  actin mRNA 72 hpt 0 0.2 0.4 0.6 0.8 1 1.2 GFP  dsRNA actin dsRNA argkin dsRNA transfection reagent control treatments sar1 mRNA 72 hpt 0.00 0.50 1.00 1.50 2.00 2.50 GFP  dsRNA sar1  dsRNA argkin dsRNA transfection reagen t control treatments linear ct
Analysis of large RNAs from actin dsRNA transfected cells C=  transfection reagent  transfected control cells  A= actin dsRNA transfected cells S= sar1  dsRNA transfected cells G=GFP  dsRNA transfected cells C  C  A  A  S  G   mRNA actin Input dsRNA 1.5%  agarose gel C  C  A  A  S  G   C  C  A  A  S  G
Analysis   of   small RNAs from actin dsRNA transfected cells 7M urea 15% polyacrylamide denaturing gel   C  C  A  A  S  G  M 26  nt 24nt 22  nt C  C  A  A  S  G  M C  C  A  A  S  G  M M  C  C  A  A  S  G  C=  transfection reagent  transfected control cells  A= actin dsRNA transfected cells S= sar1  dsRNA transfected cells G=GFP  dsRNA transfected cells
GWSS whole insect experiments
We have very good colonies of GWSS in the UC Davis CRF.  We use a mix of basil, cowpea and cotton plants, 24C and 70% RH. Thanks to Rodrigo Almeida and Elaine Backus for advice.
dsRNA delivery systems for GWSS insects ,[object Object],[object Object]
The dsRNA constructs identified in the GWSS cell transfection experiments were also used to test RNAi effects in whole insects. ,[object Object],[object Object],[object Object]
Semi-quantitative real time RT-PCR results Injection of dsRNA of corresponding endogenous genes in insects induced a reduction of the transcribed mRNA, 3 dpi 15 cycles 18 cycles 21 cycles Day 3 Day 3 Day 3 Day 1 Day 1 Day 1 A  C  G  S  A  C  G  S  H  A  C  G  S  A  C  G  S  H  A  C  G  S  A  C  G  S  H sar1 PRIMERS 15 cycles 18 cycles 21 cycles Day 1 Day 3 Day 3 Day 3 Day 1 Day 1 ACTIN PRIMERS A  C  G  S  A  C  G  S  H  A  C  G  S  A  C  G  S  H  A  C  G  S  A  C  G  S  H H = negative PCR control S = sar1 dsRNA treated insects G = GFP dsRNA treated insects C = control buffer treated insects A = actin dsRNA treated insects
Actin and sar1 silencing: real time RT-PCR results Real time RT-PCR confirmed that injection of dsRNA of corresponding endogenous genes, sar1 and actin in insects induced a reduction of the transcribed mRNA, indicating RNAi activity in GWSS whole insects. actin mRNA  72 hpi 0 0.2 0.4 0.6 0.8 1 1.2 dsRNA actin   control dsRNA GFP   Series1 sar1 mRNA 72 hpi 0 0.2 0.4 0.6 0.8 1 1.2 1.4 control dsRNA GFP   dsRNA sar1 Series1
In progress
dsDNA plant infusion. Nucleic acids Cotton leaves, petioles and stems infused with dsDNA   GWSS kept on control basil  GWSS kept on basil   infused with dsDNA  Negative control Negative control Cotton or basil young plants GWSS nymphs
Infusion experiments 32 P-Radio-labeled dsRNA infused in basil 32 -P-labeled actin dsRNA was infused into basil cuttings. Top leaves were subjected to autoradiography, showing dsRNA uptake through xylem elements.
[object Object],A discrete band confirmed the presence of the infused 700bp dsRNA in the analyzed tissues. T=Total nucleic acid S= solution dsRNA = control  dsRNA 700   bp T  S   Control  dsRNA 1 hour   exposure 48 hours   exposure T  S   Control  dsRNA
Total nucleic acids were electrophoresed on a 7M urea 15% polyacrilamide gel The predominance of dsRNA in plant tissues was not degraded, as evidenced by the absence of specific siRNA. 26nt S  T   dsRNA 6 hour exposure   S  T  dsRNA 1 hour exposure   T=Total nucleic acid S= solution dsRNA = control  dsRNA 1:10 1:1
Cloning of a xylem specific promoter ,[object Object],[object Object],[object Object],[object Object]
CAD 2-GUS Restriction site 1, R1 Restriction site 2, R2 pCB301 R1 R2 KAN The xylem specific promoter CAD2 from  Eucalyptus gunii  (from the U C Davis arboretum)   was cloned in pGEMTeasy (Promega) and subcloned in the AKK1431 vector upstream the  β-glucuronidase  (GUS) reporter gene (Govindarajulu Manjula, C.G. Tylor lab Donald Danforth Plant Science Center, St. Louis). The CAD 2-GUS cassette was then inserted into the binary vector pCB301.   The construct will be evaluated in basil plants and then the GUS sequence will be replaced by an RNAi inducer to obtain transgenic plants. Basil Plant with Xylem specific promoter, CAD 2, + gene
Conclusions ,[object Object],[object Object],[object Object]
Future work includes ,[object Object],[object Object],[object Object]

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RNA interference Activity in Glassy-winged Sharpshooter Cells and Whole Insects - Falk - Pierce's Disease Conference 2008

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  • 2. Two pathways are shown, the miRNA (nuclear) and siRNA (cytoplasmic) pathways. The miRNA pathway is a natural means of gene regulation while the siRNA pathway is believed to have evolved primarily as a defense against cytoplasmic RNA viruses. For both, double stranded RNAs are initial targets, and the resulting small RNAs serve as “guide RNAs” to direct the silencing complex to the target. The enzyme dicer trims double stranded RNA, to form small interfering RNA or microRNA . These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation . [wikipedia ]
  • 3. RNAi-based virus resistance is derived from a discovery reported in 1928, that virus-infected plants could “recover” from a virus infection. A gradual decline of virus symptoms can be seen by examining leaves from the bottom up. The top leaves are asymptomatic. There is an active response or defense of the plant against the virus infection. RNAi is already used in agriculture to control specific plant diseases.
  • 4. RNAi for insect-proof plants. Nat Biotechnol. 2007 Nov;25(11):1231-2. Is RNAi activity found in insects?
  • 5. Control of coleopteran insect pests through RNA interference Nature Biotechnology 25, 1322 - 1326 (2007) James A Baum 1 , Thierry Bogaert 2 , William Clinton 1 , Gregory R Heck 1 , Pascale Feldmann 2 , Oliver Ilagan 1 , Scott Johnson 1 , Geert Plaetinck 2 , Tichafa Munyikwa 1 , Michael Pleau 1 , Ty Vaughn 1 & James Roberts 1 1 Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017-1732, USA. 2 Devgen N.V., Technologiepark 30, B-9052 Ghent–Zwijnaarde, Belgium.
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  • 7. Homalodisca vitripennis overview Classification : Order: Hemiptera (True Bugs, Cicadas, Hoppers, Aphids and Allies) Family: Cicadellidae (Leafhoppers) Subfamily: Cicadellinae Genus: Homalodisca Species: vitripennis ( Glassy-winged sharpshooter ) It is one of the vectors of X. fastidiosa . Its host range includes many native, ornamental and crop plants. One of the preferred hosts in Southern California and other areas is citrus. http://www.acgov.org/cda/awm/agprograms/pestexclusion/sharpshooter.htm
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  • 10. UC Davis Biosafety Level 3P, Contained Research Facility GWSS is a quarantined pest in California and is regulated by CDFA. At the present, we are only able to rear and maintain prolific colonies of GWSS in the UC Davis CRF.
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  • 13. Genes screened in GWSS cells as candidate gene silencing targets. The same genes were also cloned from GSS RT-PCR products for selected GWSS mRNAs. Lanes 3 and 15 show products for Sar1 and actin, respectively. Lanes 1, 2, 4, 5, 6 and 8 – 14 show RT-PCR products for vitellogenin, histone 3, RAB1 1, kinase C receptor, ubiquitin conjugating enzyme, tropomyosin, mitochondrial porin, Delta 9 saturase, Fructose 1,6 biphosphate aldolase, rhodopsin, ferritin, and arginine kinase, respectively. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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  • 15. Efficacy of RNAi inducers: siRNA vs. dsRNA vs. hairpin loop RNA. Transfection efficiency was measured by labeled siRNA, and was almost 50%. GWSS cells dsRNA was the most efficient RNAi inducer Actin mRNA 72 hpt 0 0.2 0.4 0.6 0.8 1 1.2 control actin dsRNA pMT48 actin hairpin loop actin siRNA
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  • 17. actin dsRNA vs sar1 dsRNA: real time RT-PCR results in cells Actin dsRNA induced better RNAi effects than sar1 dsRNA, when compared to transfection reagent transfected cells. actin mRNA 72 hpt 0 0.2 0.4 0.6 0.8 1 1.2 GFP dsRNA actin dsRNA argkin dsRNA transfection reagent control treatments sar1 mRNA 72 hpt 0.00 0.50 1.00 1.50 2.00 2.50 GFP dsRNA sar1 dsRNA argkin dsRNA transfection reagen t control treatments linear ct
  • 18. Analysis of large RNAs from actin dsRNA transfected cells C= transfection reagent transfected control cells A= actin dsRNA transfected cells S= sar1 dsRNA transfected cells G=GFP dsRNA transfected cells C C A A S G mRNA actin Input dsRNA 1.5% agarose gel C C A A S G C C A A S G
  • 19. Analysis of small RNAs from actin dsRNA transfected cells 7M urea 15% polyacrylamide denaturing gel C C A A S G M 26 nt 24nt 22 nt C C A A S G M C C A A S G M M C C A A S G C= transfection reagent transfected control cells A= actin dsRNA transfected cells S= sar1 dsRNA transfected cells G=GFP dsRNA transfected cells
  • 20. GWSS whole insect experiments
  • 21. We have very good colonies of GWSS in the UC Davis CRF. We use a mix of basil, cowpea and cotton plants, 24C and 70% RH. Thanks to Rodrigo Almeida and Elaine Backus for advice.
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  • 24. Semi-quantitative real time RT-PCR results Injection of dsRNA of corresponding endogenous genes in insects induced a reduction of the transcribed mRNA, 3 dpi 15 cycles 18 cycles 21 cycles Day 3 Day 3 Day 3 Day 1 Day 1 Day 1 A C G S A C G S H A C G S A C G S H A C G S A C G S H sar1 PRIMERS 15 cycles 18 cycles 21 cycles Day 1 Day 3 Day 3 Day 3 Day 1 Day 1 ACTIN PRIMERS A C G S A C G S H A C G S A C G S H A C G S A C G S H H = negative PCR control S = sar1 dsRNA treated insects G = GFP dsRNA treated insects C = control buffer treated insects A = actin dsRNA treated insects
  • 25. Actin and sar1 silencing: real time RT-PCR results Real time RT-PCR confirmed that injection of dsRNA of corresponding endogenous genes, sar1 and actin in insects induced a reduction of the transcribed mRNA, indicating RNAi activity in GWSS whole insects. actin mRNA 72 hpi 0 0.2 0.4 0.6 0.8 1 1.2 dsRNA actin control dsRNA GFP Series1 sar1 mRNA 72 hpi 0 0.2 0.4 0.6 0.8 1 1.2 1.4 control dsRNA GFP dsRNA sar1 Series1
  • 27. dsDNA plant infusion. Nucleic acids Cotton leaves, petioles and stems infused with dsDNA GWSS kept on control basil GWSS kept on basil infused with dsDNA Negative control Negative control Cotton or basil young plants GWSS nymphs
  • 28. Infusion experiments 32 P-Radio-labeled dsRNA infused in basil 32 -P-labeled actin dsRNA was infused into basil cuttings. Top leaves were subjected to autoradiography, showing dsRNA uptake through xylem elements.
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  • 30. Total nucleic acids were electrophoresed on a 7M urea 15% polyacrilamide gel The predominance of dsRNA in plant tissues was not degraded, as evidenced by the absence of specific siRNA. 26nt S T dsRNA 6 hour exposure S T dsRNA 1 hour exposure T=Total nucleic acid S= solution dsRNA = control dsRNA 1:10 1:1
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  • 32. CAD 2-GUS Restriction site 1, R1 Restriction site 2, R2 pCB301 R1 R2 KAN The xylem specific promoter CAD2 from Eucalyptus gunii (from the U C Davis arboretum) was cloned in pGEMTeasy (Promega) and subcloned in the AKK1431 vector upstream the β-glucuronidase (GUS) reporter gene (Govindarajulu Manjula, C.G. Tylor lab Donald Danforth Plant Science Center, St. Louis). The CAD 2-GUS cassette was then inserted into the binary vector pCB301. The construct will be evaluated in basil plants and then the GUS sequence will be replaced by an RNAi inducer to obtain transgenic plants. Basil Plant with Xylem specific promoter, CAD 2, + gene
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