Multiple suppressors encoded by tomato leaf curl Java virus,

a monopartite begomovirus associated with betasatellite

M. Ikegami* , T. Kon* *, P. Sharma* * andK. Natsuaki *

* NODAI Research Institute, Tokyo University of Agriculture, Tokyo , Japan**Department of Life Science, Graduate School of Agricultural Science,

Tohoku University, Sendai, Japan

ACPP APPS 2011

Tomato leaf curl disease in Java, Indonesia.

Genome DNA

V2 (MP)

V1 (CP)

C1 (Rep)

C2 (TrAP)

C3 (REn)

C4 (MP)

2752 / 1IR

C1

1354 / 1

A-rich region

Satellite DNA (DNA02)

Genome organizations of ToLCJV and DNA02

Tomato leaf curl Java virus (ToLCJV)

Geminiviridae : Begomovirus : ssDNA

WE REPORT

1. The βC1 protein encoded by DNA β produces leaf curl symptoms in N.benthamiana , functions as a suppressor of PTGS. The βC1 protein localizes into cytoplasm.

2. C2 protein of ToLCJV is a weak suppressor and localizes into nucleus.

3. ToLCJV V2 protein is a pathogenicity determinant that elicits an HR-like response and suppressor of PTGS.

V2

C3

C4

V1

C2

C1 C1

Genome DNA DNA

ToLCJV

ToLCJV + DNA02

LB RB

Genome T-DNA

Binary vector

Agrobacterium tumefaciens

Transformation

Culture Host plants

dsDNA

ssDNA

dsDNAssDNA

DNA02 probe

ToLCJV probe

Mock

ToLCJV

ToLCJVDNA 02

Southern blot analysis

166K 25K 8K CP

12KC1

(357nt, 118aa, 13.7K)

PVX:C1

Expression of C1 from PVX vector (βC1 gene induces severe leaf curing andvein swelling symptoms in N.benthamiana plant)

Cla

ISa

lI

Mock PVX PVX +C1

14 dpi

We examined which genes of ToLCJV DNA with DNAβ

encode suppressor proteins.

GFPPVX :GFP

166K 25K 8K CP

12K

C1PVX:C1

C2PVX:C2

C4PVX:C4

PVX constructs for PTGS suppression assay

PVX/GFP/mC1

Mock PVX：GFPPVX

PVX：GFP＋PVX：C1

Suppression of PTGS by βC1 and C2 proteins

21dpi

PVX:GFP+PVX:C4PVX:GFP+PVX:C2

Analysis of GFP mRNA and GFP siRNA in virally infectedplants using northern blot hybridization. C1 protein issuppressor of PTGS and C2 protein is a week suppressor ofPTGS .

GFP mRNA

GFP siRNA

rRNA

Subcellular localization of GFP alone and GFPC1 fusion protein during transient expression in N.benthamiana leaves and onion epidermis cells. GFPC1 fusion protein localized into cell periphery.

GFP alone GFPβC1 GFPβC1

N. benyhamiana leaves

onion cells

Subcellular localization of GFP C2 fusion protein during in transient expression in N.benthamiana leaves . GFP:C2

fusion protein localized into nucleus.

GFP:C2 GFP:C2

We tested whether V2 protein of ToLCJV DNA is a

suppressor or not.

Potato virus X based expression cassettes of V2 of ToLCJV.PVX-mV2 has a nonsense mutation in the N-terminus region.PVX-V2△N has 58 aa deletion at N-terminus. PVX-V2△Ccontains a deletion of 58 aa at C-terminus.

Suppression of post-transcriptional gene silencing (PTGS) by V2 and itsderivatives using a PVX assay. V2 protein is a suppressor and deletion of 58 aafrom C-terminus abolished V2 silencing activity.

PVX-GFP

PVX-GFP/V2△CPVX-GFP/V2

PVX-GFP/V2△N

PVXmock

1.PVXalone2.PVX-GFP

3.PVX-GFP/V2△C4.PVX-GFP/V25. PVX-GFP/V2△N

Symptoms exhibited by plants following inoculation or co-infiltrated with PVX constructs. V2 protein elicited an HR-like response and deletion of 58 aa from C-terminus abolished the HR-like response.

PVX-V2 PVX-mV2mock

pBIN35S pBIN35S-V2

PVX PVX-V2 △C PVX-V2 △N

N.benthamiana

N. glutinosa

N.benthamiana

Tomato leaf curl Java virus alone does not induce or induces very mild symptoms. When inoculated with DNA , this virus induces severe symptoms,

The C1 gene of ToLCJV DNA functions as --- determinant ofpathogenicity, induces leaf curl symptoms in N. benthamiana inabsence or presence of ToLCJV infection,------ suppressor ofPTGS and localizes into cell periphery.

ToLCJV C2 protein is a week suppressor of PTGS and localizesinto the nucleus.

V2 protein encoded by ToLCJV is a pathogenicity determinantthat elicits an HR-like response and suppressor of PTGS.

Deletion of 58 aa from C-terminus abolished both the HRresponse and V2 silencing activity.

Summary