RNA interference Activity in Glassy-winged Sharpshooter Cells and Whole Insects - Falk - Pierce's Disease Conference 2008
<ul><li>RNA interference Activity in Glassy-winged Sharpshooter Cells and Whole Insects </li></ul>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. <ul><li>( a ) Map of the expression cassette. </li></ul><ul><li>( b ) Mean root damage ratings for eight F1 populations, the parental inbred line (negative control) and the corn rootworm–protected Cry3Bb event MON863; NIS, nodal injury score (Iowa State ranking system). </li></ul><ul><li>( c ) The plant on left is a non-transgenic control with average root damage, whereas the plant on the right shows the average root protection seen when the transgene is expressed. </li></ul>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 <ul><li>To develop RNAi-inducers capable of reducing the survival of Homalodisca vitripennis. </li></ul><ul><li>To generate transgenic plants expressing Homalodisca vitripennis RNAi molecules within their xylem . </li></ul><ul><li>To evaluate transgenic plants for inducing RNAi vs. Homalodisca vitripennis . </li></ul>
Our available tools <ul><li>GWSS cell line Z15. </li></ul><ul><li>(G. Kamita and B. Hammock, UCD) </li></ul><ul><li>GWSS insects. </li></ul><ul><li>(R. Almeida, UCB) </li></ul>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.
First genes chosen for their cellular activities to trigger gene silencing. <ul><li>Actin , a contractile protein found in muscle cells. Together with myosin, actin provides the mechanism for muscle contraction. </li></ul><ul><li>Sar1 is involved in transport from the endoplasmic reticulum to the Golgi apparatus. It belongs to the small GTPase superfamily, SAR1 family. </li></ul>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 <ul><li>siRNA </li></ul><ul><li>dsRNA </li></ul><ul><li>Hairpin loop </li></ul><ul><li>generating </li></ul><ul><li>plasmids. </li></ul>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 <ul><li>The most effective RNAi was obtained 72 hpt </li></ul>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
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 <ul><li>Injection </li></ul><ul><li>Feeding </li></ul>
The dsRNA constructs identified in the GWSS cell transfection experiments were also used to test RNAi effects in whole insects. <ul><li>20 GWSS larvae were injected with 1 µg of actin dsRNA, or sar1 dsRNA (the dsRNA was delivered in 1µl volume) or with 1µl of injection buffer. </li></ul><ul><li>Surviving insects were collected 72 hours post injection in groups of 5, and from each group total RNA was extracted and tested by semi-quantitative RT-PCR and real time PCR. </li></ul><ul><li>Experiments were repeated in triplicate. </li></ul>
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
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.
<ul><li>Total nucleic acids were extracted from the top of the infused stem and leaves and electrophoresis was performed on 2% agarose gel. </li></ul>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 <ul><li>The xylem specific promoter CAD2 from Eucalyptus gunii was cloned upstream the β-glucuronidase (GUS) reporter gene (Govindarajulu Manjula, C.G. Tylor lab Donald Danforth Plant Science Center, St. Louis). </li></ul><ul><li>The CAD 2-GUS cassette was inserted into the binary vector pCB301. </li></ul><ul><li>This construct will be evaluated in basil plants. </li></ul><ul><li>The GUS sequence will be replaced by an RNAi inducer and transgenic plants will be regenerated. </li></ul>
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 <ul><li>RNAi can be induced in GWSS cells and insects after delivery of dsRNA. </li></ul><ul><li>GWSS cells can be used to screen candidate gene silencing targets. </li></ul><ul><li>This study provides new evidence that RNAi can be induced in leafhopper species. </li></ul>
Future work includes <ul><li>the choice of suitable RNAi targets. </li></ul><ul><li>the production of transgenic plants expressing dsRNAs in their xylem and </li></ul><ul><li>the study of the fate of ds/siRNA delivery in insects after feeding. </li></ul>