presentación mosaico, maceio 2011
TRANSCRIPT
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Maximiliano Gmez
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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)
Mosaic Disease
SCMV /
SrMVgenome
Viral particlesSymptomatic leaf
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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
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Large Scale Virus Sampling
567 symptomatic leafs
Commercial and
experimental plots
103 locations
104 sugarcane
genotypes
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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)
533 + for SCMV (94%)
16 + for SrMV (2.8%)
3 + + showed coinfection (0.5%)
10 + with degenerate primers forPoeacea potyviruses
5 - for all primers (0.9%), including SCSMV primers
RT-PCR and Sequencing
Results:SCMVSrMV
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Sequence Analysis
475 viral sequences analyzed with Identity Matrixes andPhylogenetic 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 thegroup
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DQ315493
DQ31
5495
DQ315498
AJ421468DQ846832
DQ973170AJ310105
AY195610
DQ6476
60
DQ64765
1
AY629311
DQ
647659
EF419173
AJ31010
2
AJ
31
01
04
SrMV-H
0.05
W group
SCE group
Brazil group
SrMV
MZ group
NCSE
group
Thailand group
-H
Phylogenetic Analysis
4 groups with IDvalues under 0.9
amongst them
need to be addressed
separately for the
design of RNA
silencing construct
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Further Study of SCMV W group
SrMV
SCMV W
SCMV SCE
SCMV W shows an
interesting pattern of
silent mutations in
the core of the Coat
Protein
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 allSCMV and SrMV
samples.
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SCMV W whole genome sequencing
P1
HC-Pro P
3
6K1
CI
6K2
VPg
NIa-Pro
NIb
CP
3'UTR
5UTR
Entropy plot between W and SCMV Brisbane isolate
Silent mutations accumulate in CP and 3 of Nib genes
http://www.ncbi.nlm.nih.gov/nuccore/24415606?from=9342&to=9596&report=gbwithpartshttp://www.ncbi.nlm.nih.gov/nuccore/24415606?from=9342&to=9596&report=gbwithparts -
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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
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RNA Silencing Construct Design
Intron UBI PromTerm Act Prom NptII TermPlasmid
pUBI VIR3 NptIIP3 Sr
P3SrMZMZ
Antibiotic ResistanceVirus Resistance
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
Construct targets all 4 viral groups found in survey
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Sugarcane Transformation
5 sugarcane varieties
susceptible to mosaictransformed with RNA
silencing construct
Particle Inflow Gun
transformation and selection inGeneticin
395 putative events obtained,
3600 plantlets rusticated in the
greenhouse
R ti i I l i
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Rusticacin e Inoculacin 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
healthy mosaicsymptoms
manual inoculation3-5 leaf stage
Greenhouse virus resistance trial
221 events selected for field trial toscreen under natural infection
conditions: Planted January 2011
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Conclusions
Mosaic disease resistance: a relevant project forArgentina
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
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Thanks for your attention