rnai-based strategies against insect vectors of … · rnai-based strategies against insect vectors...

RNAi-based strategies against insect vectors of

plant pathogens

Bryce W. Falk, Shahideh Nouri, Jared C. Nigg,

Tera L. Pitman, Yen-Wen Kuo, Diogo Manzano

Galdeano, Emilyn Matsumura, Hada

Wuriyanghan, Cristina Rosa, Nida Salem

The understanding of RNA interference

(RNAi) is based in part on 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, and

immune to super-infection.

There is an active RNA-based response

or defense by the plant against the virus

infection.

IT IS RNA INTERFERENCE

Field trial of transgenic 'UH

Rainbow' and 'UH SunUp' was

established in Puna in October

1995. Slides show the progress of

the disease caused by PRSV in

rows of non-transgenic papaya

(left in picture) as compared to the

resistance in rows of 'UH

Rainbow' (right in picture).

RNAi is used in commercial

agriculture.

Aerial view of transgenic field trial

in Puna that was started in October

1995. The solid block of green

papaya trees are 'UH-Rainbow'

while the surrounding papaya trees

that are nearly dead are non-

transgenic papaya trees severely

infected by PRSV.

Papaya ringspot virus in Hawaii

This appears now to be the means for anti-viral transgenic resistance, the

transgenic plant somehow recognizes the transgenic delivered virus RNA

sequence and by targeting it confers specific anti-viral resistance to the

transgenic plant.

John Lindbo and William

Dougherty in 1993, suggested a

mechanism for this to happen.

This is the general basis for RNA

silencing/interference-based

resistance in plants against

viruses.

But RNAi is not limited to plants, or only for plant anti-

viral defense.

It is a normal gene regulation and antiviral defense found in

many different types of organisms including plants, animals,

fungi, invertebrates, etc.

It can be induced by double-stranded (ds) or small (s) RNAs

of known sequence and yield degradation or translational

interference of target (m)RNAs.

RNAi for insect-proof plants. Nat. Biotechnol. 2007 25: 1231

– 1232.

RNA interference – now one of the most widely

studied areas in biology.

When Andrew Fire and Craig Mello won a

Nobel Prize in 2006 for a revolutionary

technique to silence genes, there were high

hopes that the discovery would lead to

new treatments for disease. RNA

interference (RNAi) might help tackle a

wide variety of ailments, such as virus

infections, cancer, and cardiovascular

disease, the Nobel committee noted.

Seven years on, the technology is almost

ready to be applied—but rather than

healing humans, it will kill insects.

Science 16 August 2013:

Vol. 341 no. 6147 pp. 732-733

DOI:

10.1126/science.341.6147.732

•News

A Lethal Dose of RNA

1.Kai Kupferschmidt

A new generation of genetically

modified crops will kill insects

by silencing their genes.

http://www.sciencemag.org/search?author1=Kai+Kupferschmidt&sortspec=date&submit=Submit

http://www.sciencemag.org/search?author1=Kai+Kupferschmidt&sortspec=date&submit=Submit

We have been taking fundamental steps to examine RNAi activity

and strategies against hemipteran vectors of plant pathogens and

we are attempting to use viruses to induce RNAi effects.

Our specific insects of study are:

The glassy-winged sharpshooter, Homalodisca

vitripennis, a xylem feeder and vector of X. fastidiosa

Three phloem-feeders, Bactericera cockerelli and Diaphorina citri, vectors of important

plant pathogenic bacteria, and Planococcus citri, an important pest and vector of some

of the grapevine leafroll-associated viruses.

http://www.acgov.org/cda/awm/agpr

ograms/pestexclusion/sharpshooter.

htm

http://www.ipm.ucdavis.edu/PMG/P/I-HO-PCOC-AD.003.html

Pathways for RNAi

activity in eukaryotes

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.

Homalodisca vitripennis, the glassy-winged

sharpshooter

The Glassy-winged sharpshooter appeared in

southern California (Temecula) in 1997. It feeds on

leaf and stem tissues. It moves through grape

vineyards and PD incidence can be very

widespread, no edge effect.

http://www.cdfa.ca.gov/pdcp/PD_Photos.html

http://www.acgov.org/cda/awm/agprog

rams/pestexclusion/sharpshooter.htm

•Is GWSS susceptible to RNAi activity?

•Can we discover dsRNAs that can induce RNAi-

mediated negative phenotypes in GWSS?

•Can they be delivered via the plant xylem?

We were able to induce specific

(and non-specific?) RNAi effects

in cells after treatment with

either dsRNAs or siRNAs.

Actin and sar1 mRNA levels in

GWSS cells after RNAi induction,

measured by real time RT-PCR.

RNAi effects can be induced in insects by

injecting dsRNAs

Injected dsRNAs cause reduction of

corresponding target mRNAs.

RNAi effects do not necessarily

equal a predicted phenotype.

Actin dsRNA effects on GWSS nymphs

But, the correct dsRNA,

delivered at the correct stage,

in the correct amount can

induce a desired negative

phenotype.

Actin dsRNA effects on GWSS nymphs

But, the correct dsRNA,

delivered at the correct stage,

in the correct amount can

induce a desired negative

phenotype.

Injection is artificial and delivers

very large amounts of dsRNAs,

oral acquisition is more realistic.

1. The adult psyllid is a small insect (about 3.2mm)

2. The adults have white or yellowish markings on the thorax, clear wings,

and lines on the abdomen that separate segments.

3. Newly emerged adults remain green for a day or so before turning

darker.

Asian Citrus Psyllid and Tomato Psyllid

1. Eggs are small, 0.8mm long.

2. Orange-yellow and supported by small stalks.

3. Frequently deposited along leaf margins but may occur on either leaf

surface.

4. Hatch in 6 to 10 days.

1. Nymph have scalelike flattened, oval, yellowish green to

orangish bodies with red eyes and three pairs of short legs.

2. Older nymphs are greenish and fringed with hairs and have

wing buds.

3. While feeding, psyllid nymphs excrete small, waxy beads of

"psyllid sugar," which resembles granulated sugar. This

material may cover leaves during heavy psyllid infestations.

4. The nymph stage usually lasts from 14 to 22 days.

Both psyllids are vectors of important,

phloem-limited plant pathogenic bacteria.

Fed B

C 1

BC

+ 0

.004ngG

FP

DN

A

0.0

04ng G

FP

DN

A

Fed B

C 2

0.0

004ng G

FP

DN

A

BC

Membrane Feeding on Artificial Diets Containing dsRNAs

Crushed in 50ul

GES or GGB buffer

Psyllids fed on artificial food

with GFP PCR product

Purification

PCR detection

3. Cy3-labelled

dsRNA

2. Food dye

* BC: B. cockerelli

1. PCR Detection

Time, dose

and sequence

are all

important.

But even GFP

dsRNA gives

some RNAi

effects.

Gu

t

RO

Whole

inse

ct

Ab

do

men

Hea

d

Th

ora

x

ATPase

Actin

Head Thorax Abdomen

Gut

Reproductive

organ

Where are we inducing RNAi effects?

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

GFP Actin GFP Actin

Re

lati

ve

Ac

tin

/rR

NA

Whole insect Gut

0

0.2

0.4

0.6

0.8

1

1.2

1.4

GF

P

AT

Pa

se

Rela

tive A

TP

ase/r

RN

A

Whole insect

**

**

Gu

t

RO

Whole

inse

ct

Ab

do

men

Hea

d

Th

ora

x

ATPase

Actin

Head Thorax Abdomen

Gut

Reproductive

organ

Where are we inducing RNAi effects?

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

GFP Actin GFP Actin

Re

lati

ve

Ac

tin

/rR

NA

Whole insect Gut

0

0.2

0.4

0.6

0.8

1

1.2

1.4

GF

P

AT

Pa

se

Rela

tive A

TP

ase/r

RN

A

Whole insect

**

**

RNAi effects are induced in the gut, where the inducer is acquired.

Non-Cell Autonomous, systemic RNAi occurs in plants and the nematode C.

elegans. But insects and most vertebrates appear to lack conventional RdRPs and

thus may not be able to amplify siRNAs, thus no systemic response.

RNAi effects can be induced in feeding hemipteran vectors of plant

pathogens.

If RNAi inducers are acquired by feeding, effects appear to be

prominent in the gut, but the right inducer still can induce desirable

phenotypes.

Several important questions are:

• Which RNA targets are the best?

• Must the RNA targets be those in the gut?

• What form of RNAi inducer is best for specificity and efficacy?

• What is the best way to deliver sufficient quality and quantity of

interfering RNAs.











agriculture.








infected by PRSV.


MP CP Insert

pJL36 flanking primer

Insert gene primer

In planta delivery of effector RNAs using plant

viruses.

There is an active

RNA-based

response or

defense by the

plant against the

virus infection.

IT IS RNA

INTERFERENCE

The virus infection

yields both

dsRNAs

and siRNAs, both

powerful inducers

of RNAi.

Figure 1. Cartoon of psyllid feeding and acquiring siRNAs.

Theoretical virus-based delivery of siRNAs to phloem

Courtesy of Dr. W. O. Dawson

RT-PCR detection of GFP mRNA from psyllids which fed on

pJL24-infiltrated tomato plants. Total RNA was isolated from

tomato plants or psyllids. GFP specific primers were used for

RT-PCR. The psyllids showed products for TMV GFP.

Lanes: 1Kb, 1Kb ladder; 1, pJL24 plasmid control; 2, Tomato;

3, pJL24-infiltrated tomato; 4, psyllid; 5, Psyllid fed on pJL24-

infiltrated tomato; 6, H2O control.

1Kb 1 2 3 4 5 6

Psyllids Fed on Recombinant TMV-infected Tomato and Tomatillo Plants

DEVELOPING

NYMPHS ARE

BETTER TARGETS,

AND ON PLANT

ASSAYS ARE MORE

REALISTIC.

Fig.5: P. citri feeding on N. benthamiana plants inoculated

with different treatments

Mealy bug crawlers feeding on: A) N. benthamiana plants

infected with wildtype TMV (pJL36) show healthy crawlers

emerging while B and C and D) N. benthamiana plants

infected with recombinant pJL36+V-ATPase (B), actin (C), or

CHS (D) show high mortality in crawlers but less in adults.

Relative expression levels of chs1 (E)

and V-ATPase (F) in Planococcus citri

after feeding on N. benthamiana

plants infected with the corresponding

recombinant Tobacco mosaic virus

constructs.

E

F A B

C D

Planococcus citri, the citrus

mealybug

We know that we can induce RNAi effects in insects,

including phloem-feeding hemipterans.

We can use recombinant plant viruses, or probably

transgenic plants engineered to generate the interfering RNA

(typically as a dsRNA).

But RNAi activity in plants gives a population of siRNAs.

• Will the RNAi effects be specific only to the target?

• What approaches might we take to increase specificity?

“exposure to

insecticidal small

RNAs will

probably occur at a

previously

unrealized scale”

Hit: M. persicae mRNA for nicotinic

acetylcholine receptor alpha 2 subunit

No hits

No hits

No hits

No hits

GFP from

transgenic

plants

“exposure to

insecticidal small

RNAs will

probably occur at a

previously

unrealized scale”

We are taking two approaches in

attempts to maximize RNAi efficacy

and minimize potential off-target

effects:

1) Artificial micro-RNAs to specific

RNA targets;

2) insect virus delivery.

Pathways for RNAi

activity in eukaryotes

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.

These should all be

the same!

These can be slightly

different!

Engineering to deliver primary microRNAs

• Our ability to deliver the small RNAs into the insects by using plant

viruses (psyllids and mealybugs) made us think in the direction of

delivery of microRNAs in insects.

Experimental steps

TMV = Tobacco mosaic virus (ssRNA)

(cytoplasmic replication)

TAV = Tomato mottle begomovirus

(ssDNA) (nuclear replication)

35S = Binary plasmid with 35S

promoter.

AmiRNA accumulation

Identify the best way to express amiRNAs in plants.

Mapping of small RNAs by deep sequencing: Although easy to use, the TMV-

based vector produced lower levels and non-specific sequences of small RNAs in

plants.

Mapping of small RNA deep sequencing: The results show that the modified

TAV DNA virus vector produced high levels and excellent quality of specific

amiRNAs in plants.

Identify the best way to express amiRNAs in plants.

Comparative expression of amiRNAs in plants.

Virus type Viral

replication

Replication

location

amiRNA

accumulation

amiRNA peak

reads

TMV RNA Yes Cytoplasm + <190,000

TAV DNA Yes Nucleus +++ >2,400,000

pGWB2 -- -- -- ++ ~1,500,000

Total reads for each sample: ~60,000,000

TMV = Tobacco mosaic virus; TAV = chromosome A of the Begomovirus, Tomato

mottle virus; pGWB2 is a binary plasmid containing the 35S promoter (such as would

be used in conventional transgenic plants).

The cytoplasmic replicating TMV gave lower amounts and less specific small

RNAs.

The nuclear replicating TAV have high yields of specific amiRNAs.

Plant viruses can be used to generate anti-insect siRNAs and amiRNAs in

plants.

But we still have problems with:

• inducing systemic RNAi effects in recipient insects.

• in having the means to spread the RNAi inducer recombinant virus in

the target area.

Plant viruses can be used to generate anti-insect siRNAs and amiRNAs in

plants.

But we still have problems with:

• inducing systemic RNAi effects in recipient insects.

• in having the means to spread the RNAi inducer recombinant virus in

the target area.

Can we use insect infecting viruses to induce specific, desirable RNAi effects

in target insects? If so this offers the means to increase specificity (avoiding

off-target potential) and to induce systemic RNAi effects in insects as the virus

spreads in the insect body.

Pathogen

Pagliai et al. (2014) PLoS Pathog 10(4) :

e1004101. doi:10.1371/journal.ppat.1004101

https://cisr.ucr.edu/citrus_greening.html

http://www.cdfa.ca.gov/plant/acp/gallery/p

hotos/acp70.jpg

Insect vector

Host

• Viruses are the most abundant microbes on the planet and many viruses are

not pathogens and thus remain to be discovered.

• If viruses can be identified, recovered and their genomes cloned as cDNAs to

generate infectious viruses, then they can assessed for biological effects.

small RNA deep sequencing and transcriptome profiles analysis in

world populations of Diaphorina citri

Collected D. citri: US (FL, TX, HI, CA)

and many foreign locations (Taiwan,

China, Brazil and Pakistan).

Generated small RNA and

transcriptome libraries

Sequencing Bioinformatics analysis

Confirm virus presence

by (RT)PCR Small RNA

(HiSeq)

Deep sequencing of small RNAs and

transcriptomes for identifying viruses

associated with Diaphorina citri

RNA-seq

(HiSeq)

Category Family Genus Species E-

value

Population

dsRNA virus Reoviridae Fijivirus Nilaparvata lugens reovirus 7e-70 CH, TW, FL, TX,

HW

ssRNA virus Flaviviridae Unclassified Gentian Kobu-sho associated virus

7e-56 CH, FL, HW

ssRNA virus Iflaviridae Iflavirus Deformed wing virus 8e-112 BR, CH, TW

ssDNA virus Parvoviridae Ambidensovirus Mythimna loreyi densovirus 1e-13 CH, TW, PK

ssRNA virus Bunyaviridae Phasmavirus Wuchang cockraoch virus 1 5e-40 CH, TW, FL

ssRNA virus Unclassified Unclassified Chronic bee paralysis virus 9e-04 CH, CA, TX, FL

dsRNA virus

Unclassified

phages

Unclassified WO prophage

0.0 BR, CH, HW, FL,

CA. TX

CH: China; TW: Taiwan; BR: Brazil; PK: Pakistan; FL: Florida; CA: California; TX: Texas; HW: Hawaii

Significant BLASTx hits to the viral database using contigs created from

small RNA/RNA-seq libraries as query sequences

Virus/Virus-related sequences Encoded Protein % Query coverage % Identity Closely related species/genus

Diaphorina citri reovirus (DcRV)

Seg.1: RdRP 97 36

Nilaparvata lugens reovirus

Seg.2: 136.6KD 33 28

Seg.3: major core

capsid protein 87 30

Seg.4: 130KD 99 26

Seg.7: 73.5KD 96 23

Seg.8: major outer

capsid protein 87 26

Seg.10: polypeptide 82 24

Diaphorina citri Picorna-like virus

(DcPLV)

Polyprotein 70 33 Deformed wing virus

Diaphorina citri densovirus

(DcDNV)

NS1 37 35 Uncharacterized protein in D. citri

NS2 96 31 Cherax quadricarinatus

densovirus

VP1 37 30 Densovirus SC1065

VP2 43 42 Periplaneta fuliginosa densovirus

Diaphorina citri bunyavirus (DcBV)

RdRp 85 31

Wuchang Cockroach Virus 1 Glycoprotein

Precursor 80 32

Nucleocapsid 90 36

Diaphorina citri associated C virus

(DcACV)

RdRp 70 33 Pea enation mosaic virus 2

Hypothetical protein 8 39 Chronic bee paralysis virus

Diaphorina citri flavi-like virus

(DcFLV)

Polyprotein 21 39 Gentian Kobu-sho-associated virus

D. citri virome

Diaphorina citri picorna-like virus

(DcPLV):+ssRNA

http://viralzone.expasy.org/


Diaphorina citri reovirus (DcRV):

dsRNA



(DcDNV):ssDNA Diaphorina citri associated C virus

(DcACV):+ssRNA

Diaphorina citri bunyavirus (DcBV):

-ssRNA


Nouri, et al. J. Virol, 90: 2434-45

Diaphorina citri flavi-like

virus: +ssRNA

Genome organization and coding regions of DcPLV

VP1 VP3

5`UTR 3`UTR

VP2 Helicase Protease Polymerase (RdRp)

Iflavirus genome

organization

5`NTR

Vpg (A)n

L VP2

VP

4 VP3 VP1 Hel Pro RdRp

Structural Proteins Non-Structural Proteins

3`NTR

Non-Structural Proteins Structural Proteins

5`NTR

Calici coat protein

VP2 VP1

?

Hel RdRp

Vpg

L

?

VP3 3`NTR

(A)n

Diaphorina citri picorna-like virus (DcPLV) genome organization

M) 1 kb plus; 1, 2, 4, 5, 7, 8, 10, 11) China; 3, 6, 9, 12) Brazil

M 1 2 3 4 5 6 7 8 9 10 11

12 1 2 3 4 ~10 kb

F1/R1 F2/R2 F3/R3 F4/R4

Nouri, et al. 2015. J. Virol, 90: 2434-45

5`NTR

Vpg (A)n

L VP2

VP

4

VP3 VP1 Hel Pro RdRp

Structural Proteins Non-Structural Proteins

A

3`NTR

Non-Structural Proteins Structural Proteins

5`NTR

Calici coat protein

VP2 VP1

?

Hel RdRp

Vpg

L

?

VP3

B

3`NTR

(A)n

Iflavirus

DcPLV 10,281 nt

DWV

KV

VdV-1

FeV2

HeIFV

ApIFV

LdIFV

LsHDV1

NlHDV1

BbPLV

GnV-1

SBPV

DcPLV

SBV

ALPV

ABPV

CrPV

GDCV

PYFV

RTSV

HaRNAv

HAV

EMCV

PV

FCV

RHDV

PVY

RGMV100

86

100

93

100

100

100

100

97

100

99

100

89

54

87

79

70

98

32

3853

98

69

39

79

0.5

Screening of D. citri populations for DcPLV & its phylogenetic relationships based on the RdRp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

M) 1 kb plus; 1) China B; 2) China STE; 3) Taiwan 2; 4) Taiwan 4; 5) Brazil

17; 6) Brazil 11; 7) Pakistan; 8) Hawaii F6; 9) Hawaii Morita; 10) Florida 9;

11) Florida 5; 12) Florida fecal; 13) California 1; 14) Texas 2; 15) Texas 4;

16) CRF (negative control)

Iflaviridae

Potyviridae

Caliciviridae

Dicistroviridae

Secoviridae

Marnaviridae

Picornaviridae

D. citri virome


(DcPLV):+ssRNA




dsRNA



(DcDNV):ssDNA Diaphorina citri associated C virus

(DcACV):+ssRNA


-ssRNA




virus: +ssRNA

Diaphorina citri densovirus (DcDNV) Nouri, et al. 2015. J. Virol, 90: 2434-45; Nigg et al. 2016. Genome Announcements: 2016 Jul 28;4(4). pii: e00589-16. doi:

10.1128/genomeA.00589-16

5,071 nt

5`NTR

Non-Structural ORFs

3`NTR

ITR ITR

NS

NS1 (A)n

(A)n

3`NTR

Structural ORFs

5`NTR

ITR ITR

VP (A)n

(A)n VP1

Screening of D. citri populations for DcDNV & its phylogenetic relationships based on the NS1

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

16

M) 1 kb plus; 1) China B; 2) China STE; 3) Taiwan 2; 4) Taiwan 4; 5) Brazil 17; 6) Brazil 11; 7) Pakistan;

8) Hawaii F6; 9) Hawaii Morita; 10) Florida 9; 11) Florida 5; 12) Florida fecal; 13) California 1; 14) Texas

2; 15) Texas 4; 16) CRF (negative control)

Iteravirus

JcDNV

PiDNV

DsDNV

GmDNV

MlDNV

HaDNV

CpDNV

sc116

AdDNV

PcDNV

CeDNV

DpDNV

DcDNV

PfDNV

BgDNV

MpDNV

CqDNV

SSaDV

AeDNV

AgDNV

AAV2

MVM

100

100

95

70

40

22

100

99

100

99

100

55

84

86

70

31

70

45

34

0.5

Ambidensovirus

DcDNV Positive

DcDNV Negative

D. citri virome


(DcPLV):+ssRNA




dsRNA



(DcDNV):ssDNA


-ssRNA




virus: +ssRNA


(DcACV):+ssRNA

Diaphorina citri associated C virus (DcACV) Nouri, et al. 2015. J. Virol, 90: 2434-45, Nouri, et al. 2016. Genome Announc. In Press

RNA1:2,376 nt

RNA2:1,817 nt

Tombusviridae

Chronic bee paralysis virus

5`NTR 3`NTR

ORF1

ORF2 (RdRp)

5`NTR 3`NTR

ORF2

ORF1

Screening of D. citri populations for DcACV & its phylogenetic relationships based on the RdRp

M 1 2 3 4 5 6 7 8 9 10

M) 1 kb plus; 1) California 1; 2) Texas 2; 3) China STE; 4) Brazil 17;

5) China 2; 6) Taiwan 2; 7) Florida 10; 8) Hawaii Morita ; 9) Pakistan;

10) CRF (negative control)

CBPV2

CBPV3

CBPV1

CBPV11

CBPV12

CBPV9

CBPV5

CBPV6

CBPV7

CBPV8

CBPV10

CBPV4

AACV

LSV2-1

LSV2-2

DcACV

TBSV

MNeSV

SmVA

FHV

NoV

100

100

100

96

96

98

99

98

53

72

78

100

32

22

50

92

43

0.5

Tombusviridae

D. citri virome


(DcPLV):+ssRNA




dsRNA



(DcDNV):ssDNA


-ssRNA




virus: +ssRNA


(DcACV):+ssRNA

“flavi-like viruses”

http://education.expasy.org/images/Hepacivirus_genome.jpg

9-11 kb Flaviviridae

22 kb 19 kb

Partial sequences

23 kb

16-26 kb

DcFLV

5`NTR Hel 3`NTR

(A)n Methyltransferase

60S ribosomal

Transmembrane

Unknown function protein

27,724 nt

M 1 2 3 4 5

M 6 7

M) 1 kb plus; 1) Seg. A 1; 2) Seg. B; 3) Seg. C; 4) Seg. D; 5) Negative

control; 6) Seg. E; 7) Seg. F

Matsumura et al. 2016. Genome Announc. Accepted

DcFLV& its phylogenetic relationship based on the NS5

DcFLV FL

DcFLV HW

DcFLV CH

GKaV

DmelFlaviLike 4

WHCeV

TCTV8

SbCNV-5

XZSV2

EsCLV

BLTV4

BHBV1

GMV

XSCV

MeV-1

SALV2

SYSV4

Pestivirus Bungowannah

Pestivirus Pronghorn

BVDV-1

Pestivirus reindeer-1 V60-Krefeld

BDV

WNV

WLSV

GBV-A

SYFV2

WHCV

WHAV1

SAIV 7

WHFV

88

100

94

99

54

94

74

100

100

100

94

96

81

38

34

84

74

39

62

26

24

64

39

96

59

36

37

0.1

• Worldwide populations of D. citri and its prokaryotic

endosymbionts contain several new viruses.

• The DcPLV, DcACV and DcDNV appear to be good

candidates for further studies.

• Our efforts now are to assess wildtype and/or recombinant

viruses for potential biological control efforts with D. citri.

• We are attempting to engineer recombinant viruses to

target and knock down desired genes in D. citri via the

insects natural RNAi response by Virus-induced gene

silencing (VIGS).

Virus-induced gene

silencing (VIGS)

VIGS is done by

inserting a host gene

sequence into a virus

genome.

Then when the organism

responds to the virus

infection by RNAi, it

silences the inserted

sequence.

This is commonly done

in plants, our goal is to

take a similar approach

with D. citri.

Delivery

Cells lines (Transfection) Insect (Injection/bombardment)

http://www.entu.cas.cz/zurovec/eng/research/cultures.html http://ucanr.edu/blogs/anrnews/index.cfm?start=6&tagname=Mark%20Hoddle

Flock House Virus (FHV) Clones: Gifts from Shou-Wei Ding`s lab, UC-

Riverside

pMT+RNA1 pMT+RNA2

MT Promoter MT Promoter

MT: The Drosophila metallothionein gene promoter

Drosophila S2 Cells

FHV pMT

Subgenomic RNA

RNA1

HDV-

Ribozyme

HDV-

Ribozyme

DcACV

Cells lines (Transfection)

Drosophila S2 Cells

MT Promoter MT Promoter

pMT+RNA1 pMT+RNA2

MT: The Drosophila metallothionein gene promoter

pMT DcACV

RNA1

DcACV

Summary

• Phloem-feeding insects can be targeted by RNAi.

• Viruses, both plant and insect-infecting, offer good opportunities

for use as vehicles for delivering interfering RNAs.

• Plant viruses can be used to generate siRNAs and amiRNAs.

• Insect viruses offer good potential for specificity and for

inducing systemic RNAi effects.

• Our goal now is to use D. citri-infecting viruses to induce

desirable VIGS effects in D. citri.

Acknowledgments

We thank the following colleagues for collecting and

supplying D. citri and/or D. citri RNAs:

W. O. Dawson, H. Wuriyanghan, H.H Yeh, Y. Cen, Diogo Manzano Galdeano,

Tatiane da Silva, David Jenkins, Clesson Higashi, Andrew Chow, David

Morgan, Kris Godfrey, K. Pelz-Stelinski, Arif Muhammad Khan

Funding support

Position 180-280 Position 180-280 Position 75-225

in Diet On Plant

GFP

GFP

Nandety RS and Falk BW, Figure adapted from Nandety et., al 2013

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HoCV genomic RNA

smRNA reads mapped on HoCV -1 virus

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smRNA reads mapped on HoVRV S1 genomic RNA

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a b

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Like plants, insects also respond to RNA virus infection by RNAi activity!

Thus we might be able to use insect viruses, but which ones?

24, 21, 22, 23 nt 22, 21 nt

Read Length

Rea

ds

Read Length

Rea

ds

22 nt

in Diet On Plant

GFP

GFP

Virus Delivery

• DcPLV (10.2 kb), DcACV (2.3 kb and 1.8 kb) and DcDNV (5.1 kb)

• Making infectious clones and validating them in insect cell lines

and D. citri

• Engineering recombinant viruses to target and knock down vital

genes in D. citri via RNAi

21 nt 21 nt

Read Length

Rea

ds

Read Length

Rea

ds

Read Length

Rea

ds

in Diet On Plant

24-28 nt

GFP

GFP

But dsRNAs cannot be expressed from chloroplasts

to target phloem-feeding hemipterans.

And dsRNAs may not be transported in the phloem

anyway.

But small RNAs are phloem mobile.

D. citri virome


(DcPLV):+ssRNA




dsRNA



(DcDNV):ssDNA


-ssRNA




virus: +ssRNA


(DcACV):+ssRNA











agriculture.








infected by PRSV.


This appears now to be the means for anti-viral transgenic resistance, the

transgenic plant somehow recognizes the transgenic delivered virus RNA

sequence and by targeting it confers resistance to the transgenic plant.

John Lindbo and William

Dougherty in 1993, suggested a

mechanism for this to happen.

This is the general basis for RNA

silencing/interference-based

resistance in plants against

viruses.

Screening of D. citri populations for DcBV & its phylogenetic relationships based on the RdRp

M 1 2 3 4 5 6 7 8 9

M) 1 kb plus; 1) China STE; 2) China 2; 3) Taiwan 2; 4) Brazil 17; 5)

Hawaii Morita; 6) Florida 11; 7) California 2; 8) Texas 4; 9) CRF

(negative control)

WuMV1

KIGV

NOMV

WuCV1

JONV

FERV

SWSV2

DcBV

HnTV

TuLV

OYV

BuNV

WSMV

TSWV

DuBV

HZV

RSV

TuV

RVFV

DaMV

100

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95

99

66

100

88

96

72

76

54

59

0.5

Phasmavirus


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