Post on 26-Apr-2018
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
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
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
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
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