Maximiliano Gmez

Mosaic Disease
Most widespread viral disease in sugarcane Caused by 2 viral species:
Sugarcane Mosaic Virus (SCMV) Sorghum Mosaic Virus (SrMV)

Single stranded + RNA genome

Yield losses up to 10-40% depending on strain and sugarcane variety Disease Management:
Genetic resistance (traditional breeding) Transgenics (experimental)
SCMV / SrMV genome Viral particles Symptomatic leaf

Mosaic Project at Chacra

Objectives: 1) Determine the molecular diversity of the virus in Argentina and neighbouring countries 2) Obtain transgenic sugarcane resistant to mosaic disease through RNA Silencing

Large Scale Virus Sampling

567 symptomatic leafs Commercial and experimental plots 103 locations 104 sugarcane genotypes

RT-PCR and Sequencing

Samples analyzed by RT-PCR (coat protein gene) of SCMV and SrMV from a crude leaf extract and direct sequencing (Gomez et al., 2009, J. Virol Methods)

Results:
533 + for SCMV (94%) 16 + for SrMV (2.8%) 3 + + showed coinfection (0.5%)

SrMV

SCMV

10 + with degenerate primers for Poeacea potyviruses 5 - for all primers (0.9%), including SCSMV primers

Sequence Analysis
475 viral sequences analyzed with Identity Matrixes and Phylogenetic trees High variability within SCMV, classified in 3 groups:
SCE: 97% of samples, ID over 0.95 within the group. MZ: 2 samples, ID of ~ 0.88 with SCE W: 9 samples, newly defined group, ID values ~ 0.90 with SCE

Low variability within SrMV: ID values over 0.97 within the group

Phylogenetic Analysis
5 9 3 49 54Q3 15 3 1 D315 498 DQ D Q

SCE group
5 9 3 49 54Q3 15 3 1 D315 498 DQ D Q

Brazil group

AJ4 2 14 68 DQ 9 7 DQ8 468 31 32 AJ 7 0 31 01 05

W group

4 groups with ID values under 0.9 amongst them

10 56 19 AY

60 76 64 5 1 10 56 D Q 476 1 6 31 19 DQ 629 AY AY DQ647 659 3 191 7 EF4 010 2 AJ3 1 AJ310104 60 76 64 5 1 D Q 476 1 6 31 DQ 629 AY DQ647 659 3 191 7 EF4 010 2 AJ3 1 AJ310104

AJ4 2 14 68 DQ 9 7 DQ8 468 31 32 AJ 7 0 31 01 05

MZ group

Thailand group

need to be addressed separately for the design of RNA silencing construct

NCSE group

-H SrMV-H

SrMV

0.05

Further Study of SCMV W group

SCMV SCE

SCMV W shows an interesting pattern of silent mutations in the core of the Coat Protein
SrMV

SCMV W

High aa identity but low nt identity

Relationship between amino acid (aa) and nucleotide (nt) identity in a set of 217,156 pairwise comparisons between the CP sequences all SCMV and SrMV samples.

SCMV W whole genome sequencing

Entropy plot between W and SCMV Brisbane isolate

HC-Pro

Silent mutations accumulate in CP and 3 of Nib genes

NIa-Pro

5UTR

3'UTR

6K2 VPg

NIb

6K1

CP

P1

P3

CI

RNA Silencing Construct Design

RNA Silencing is a natural viral defense mechanism present in plants, that can be directed to a specific virus Search for other conserved regions in viral genome:
Sequenced P3 gene of 15 samples representing all 4 viral groups Found high ID values between SCE and W samples

Decided to clone 3 viral fragments in a RNA silencing construct

RNA Silencing Construct Design

SCMV / SrMV Genome

SCMV P3 gene targeting to SCE and W group

SrMV CP gene (5) targeting SrMV SCMV CP gene (3) targeting MZ group

Term P3 Sr MZ

Intron

Virus Resistance

Construct targets all 4 viral groups found in survey

P3 Sr MZ

Plasmid pUBI VIR3 NptII

UBI Prom

Act Prom

NptII

Term

Antibiotic Resistance

Sugarcane Transformation
5 sugarcane varieties susceptible to mosaic transformed with RNA silencing construct Particle Inflow Gun transformation and selection in Geneticin 395 putative events obtained, 3600 plantlets rusticated in the greenhouse

Rusticacin e Inoculacin Greenhouse virus resistance trial

Plantlets artificially inoculated by rubbing leaf +1 with carborundum and infective juice (SCMV SCE) Infection assessed by Leaf Printing (Serology). Results:
Low infection rate (11%) in non transgenic controls Events with at least one infected plant discarded

221 events selected for field trial to screen under natural infection conditions: Planted January 2011

3-5 leaf stage

manual inoculation

healthy

mosaic symptoms

Conclusions Mosaic disease resistance: a relevant project for Argentina Large scale sampling and sequencing found significant virus diversity Four viral groups need to be addressed separately (SrMV, and SCMV SCE, MZ, W ) One isolate of SCMV W fully sequenced RNA silencing construct targeting all groups Transgenic plants obtained for 5 mosaic susceptible varieties Ongoing greenhouse and field trials to evaluate events

Thanks for your attention

mgomez@chacraexperimental.org