RNA interference Activity in Glassy-RNA interference Activity in Glassy-winged Sharpshooter Cells and winged Sharpshooter Cells and

Whole InsectsWhole Insects

Co-PI C. Rosa, PI B.W. Falk, Department of Plant Pathology, University of Department of Plant Pathology, University of

California, Davis, CACalifornia, 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 Control of coleopteran insect pests through RNA insect pests through RNA

interferenceinterference

Nature Biotechnology 25, 1322 - 1326 (2007)Nature Biotechnology 25, 1322 - 1326 (2007)

James A BaumJames A Baum11, Thierry Bogaert, Thierry Bogaert22, William Clinton, William Clinton11, Gregory R Heck, Gregory R Heck11, Pascale , Pascale FeldmannFeldmann22, Oliver Ilagan, Oliver Ilagan11, Scott Johnson, Scott Johnson11, Geert Plaetinck, Geert Plaetinck22, Tichafa , Tichafa

Munyikwa1, Michael Pleau1, Ty Vaughn1 & James Roberts1 Munyikwa1, Michael Pleau1, Ty Vaughn1 & James Roberts1

1 Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, Missouri 1 Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017-1732, USA.63017-1732, USA.

2 Devgen N.V., Technologiepark 30, B-9052 Ghent–Zwijnaarde, Belgium.2 Devgen N.V., Technologiepark 30, B-9052 Ghent–Zwijnaarde, Belgium.

F1 plants expressing a F1 plants expressing a V-V-ATPaseATPase A A dsRNAdsRNA are protected from are protected from Diabrotica virgifera Diabrotica virgifera virgiferavirgifera western corn western corn

rootworm (WCR)rootworm (WCR) feeding damage.feeding damage.

• ((aa) Map of the expression ) Map of the expression cassette. cassette.

• ((bb) Mean root damage ratings ) Mean root damage ratings for eight F1 populations, the for eight F1 populations, the parental inbred line (negative parental inbred line (negative control) and the corn rootworm–control) and the corn rootworm–protected Cry3Bb event protected Cry3Bb event MON863; NIS, nodal injury score MON863; NIS, nodal injury score (Iowa State ranking system). (Iowa State ranking system).

• ((cc) The plant on left is a non-) The plant on left is a non-transgenic control with average transgenic control with average root damage, whereas the plant root damage, whereas the plant on the right shows the average on the right shows the average root protection seen when the root protection seen when the transgene is expressed. transgene is expressed. Nature Biotechnology 25, 1322 - Nature Biotechnology 25, 1322 -

1326 (2007)1326 (2007)

Homalodisca vitripennisHomalodisca vitripennis overviewoverview

ClassificationClassification:: 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 It is one of the vectors of vectors of X. X. fastidiosafastidiosa..Its host range Its host range includes many native, includes many native, ornamental and crop ornamental and crop plants. plants. One of the preferred One of the preferred hosts in Southern hosts in Southern California and other California and other areas is citrus.areas is citrus.

http://www.acgov.org/cda/awm/agprograms/pestexclusion/sharpshooter.htm

Specific Specific objectivesobjectives

• To develop RNAi-inducersTo develop RNAi-inducers capable of capable of

reducing the survival of reducing the survival of Homalodisca Homalodisca vitripennis.vitripennis.

• To generate To generate transgenic plantstransgenic plants expressing expressing Homalodisca vitripennisHomalodisca vitripennis RNAi molecules RNAi molecules within their xylemwithin their xylem..

• To To evaluate transgenic plantsevaluate transgenic plants for inducing for inducing RNAi vs.RNAi vs. Homalodisca vitripennisHomalodisca vitripennis..

Our available toolsOur available tools

• GWSS cell line Z15. GWSS cell line Z15. (G. Kamita and B. Hammock, UCD)(G. Kamita and B. Hammock, UCD)

• GWSS insects.GWSS insects. (R. Almeida, UCB)(R. Almeida, UCB)

http

://w

ww

.acg

ov.o

rg/c

da/a

wm

/ag

prog

ram

s/pe

stex

clus

ion/

shar

psho

oter

.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 experimentsGWSS cell experiments

First genes chosen for their First genes chosen for their cellular activities to trigger cellular activities to trigger gene silencing.gene silencing.

• ActinActin, a contractile protein , a contractile protein found in muscle cells. Together found in muscle cells. Together with myosin, actin provides the with myosin, actin provides the mechanism for muscle mechanism for muscle contraction. contraction.

• Sar1Sar1 is involved in transport is involved in transport from the endoplasmic reticulum from the endoplasmic reticulum to the Golgi apparatus. It to the Golgi apparatus. It belongs to the small GTPase belongs to the small GTPase superfamily, SAR1 family.superfamily, SAR1 family.

Actin filaments stained using Actin filaments stained using Alexa-Fluor phalloidin (Molecular Alexa-Fluor phalloidin (Molecular

probe, Invitrogen)probe, Invitrogen)

Genes screened in GWSS cells as Genes screened in GWSS cells as candidate gene silencing targets.candidate gene silencing targets.

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

The same genes were also cloned from GSS

RNAi inducers tested in the RNAi inducers tested in the GWSS Z15 cell lineGWSS Z15 cell line

1.1. siRNAsiRNA2.2. dsRNAdsRNA3.3. Hairpin loop Hairpin loop generating generating plasmids.plasmids.

pGEMTeasy

T7

SP6

actin

Nco

I (38)

PstI (1423)SacI (1444)

SacII (50)

MluI (1449)

NotI (44)

NotI (1412)

6X his tag

actin

Tribolium

actin rc

polyadenilation region

methallothionein promoter

pMT 48

1

3

2

siRNA

Efficacy of RNAi inducers: Efficacy of RNAi inducers: siRNA vs. dsRNA vs. hairpin siRNA vs. dsRNA vs. hairpin loop RNA.loop RNA.

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

Transfection efficiency was Transfection efficiency was measured by labeled siRNA, measured by labeled siRNA,

and was almost 50%.and was almost 50%.

GWSS cells GWSS cells

dsRNA was the most efficient RNAi inducer

Time course transfection Time course transfection experiments in cellsexperiments in cells

• The most effective RNAi was The most effective RNAi was obtained 72 hptobtained 72 hpt

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 mRNA 72 hpt

0

0.2

0.4

0.6

0.8

1

1.2

GFP dsRNA

actindsRNA

argkin dsRNA

transfectionreagentcontrol

treatments

sar1 mRNA 72 hpt

0.00

0.50

1.00

1.50

2.00

2.50

GFP dsRNA

sar1 dsRNA

argkindsRNA

transfectionreagent

control

treatments

lin

ear

ct

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.

C= transfectionreagent transfectedcontrol cells

A=actin dsRNAtransfectedcells

S= sar1 dsRNAtransfectedcells

G=GFP dsRNA transfectedcells

C C A A S G

mRNA actin

Input dsRNA

1.5% agarosegel

Analysis of large RNAs from Analysis of large RNAs from actin dsRNA transfected cellsactin dsRNA transfected cells

C C A A S G

C C A A S G

AnalysisAnalysis ofof small RNAs from actin small RNAs from actin dsRNA transfected cellsdsRNA 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= transfectionreagent transfectedcontrol cells

A=actin dsRNAtransfectedcells

S= sar1 dsRNAtransfectedcells

G=GFP dsRNA transfectedcells

GWSS whole insect GWSS whole insect experimentsexperiments

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 dsRNA delivery systems for GWSS insectsGWSS insects

• InjectionInjection

• FeedingFeeding

The dsRNA constructs identified in the GWSS The dsRNA constructs identified in the GWSS cell transfection experiments were also used cell transfection experiments were also used to test RNAi effects in whole insects.to test RNAi effects in whole insects.

• 20 GWSS larvae were injected with 120 GWSS larvae were injected with 1µg of actin µg of actin dsRNA, or sar1 dsRNA (the dsRNA was dsRNA, or sar1 dsRNA (the dsRNA was delivered in 1µl volume) or with 1µl of injection delivered in 1µl volume) or with 1µl of injection buffer.buffer.

• Surviving insects were collected 72 hours post Surviving insects were collected 72 hours post injection in groups of 5, and from each group injection in groups of 5, and from each group total RNA was extracted and tested by semi-total RNA was extracted and tested by semi-quantitative RT-PCR and real time PCR. quantitative RT-PCR and real time PCR.

• Experiments were repeated in triplicate.Experiments were repeated in triplicate.

15 cycles 18 cycles 21 cycles

Day 3Day 3Day 3 Day 1Day 1Day 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 1Day 3 Day 3 Day 3Day 1Day 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

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

actin mRNA 72 hpi

0

0.2

0.4

0.6

0.8

1

1.2

dsRNA actin control dsRNA GFP

Series1

Actin and sar1

silencing: real time RT-PCR

results

sar1 mRNA72 hpi

0

0.2

0.4

0.6

0.8

1

1.2

1.4

control dsRNA GFP dsRNA sar1

Series1

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.

In progressIn progress

dsDNA plant infusion.dsDNA plant infusion.

Nucleic acids

Cotton leaves, petioles and stems

infused with dsDNA GW

SS

kep

t on

co

ntro

l bas

il

GW

SS

kep

t on

bas

il in

fuse

d w

ith d

sDN

A

Negative control

Negative control

Cotton or basil

young plants

GWSS nymphs

Infusion experiments

32P-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.

Total nucleic acids were extracted from the top of the infused stem and leaves and electrophoresis was performed on 2% agarose gel.

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

A discrete band confirmed the presence of the infused 700bp dsRNA in the analyzed tissues.

Total nucleic acids were electrophoresed on a 7M urea 15% polyacrilamide gel

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

The predominance of dsRNA in plant tissues was not degraded, as evidenced by the absence of specific siRNA.

Cloning of a xylem specific Cloning of a xylem specific promoterpromoter• 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).

• The CAD 2-GUS cassette was inserted into the binary vector pCB301.

• This construct will be evaluated in basil plants.

• The GUS sequence will be replaced by an RNAi inducer and transgenic plants will be regenerated.

AK K 1431 C A D8 6 4 8 bp

AM P(R )

SU In t ron

N LS-GU S

M 13 Forward

M 13 R ev erse

EX

EY

AIA

AIH

AB J

C AD pro m oter

OR I - C o lE1

tN OS

CAD 2-GUSRestriction site 1,

R1Restriction site 2,

R2

Basil Plant with Xylem specific promoter, CAD 2, + gene

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.

ConclusionsConclusions

•RNAi can be induced in GWSS cells and insects after delivery of dsRNA.

•GWSS cells can be used to screen candidate gene silencing targets.

•This study provides new evidence that RNAi can be induced in leafhopper species.

Future work includes

• the choice of suitable RNAi targets.

• the production of transgenic plants expressing dsRNAs in their xylem and

• the study of the fate of ds/siRNA delivery in insects after feeding.