presentación mosaico, maceio 2011

8/6/2019 Presentacin Mosaico, Maceio 2011

1/15

Maximiliano Gmez


2/15

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


3/15

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


4/15

Large Scale Virus Sampling

567 symptomatic leafs

Commercial and

experimental plots

103 locations

104 sugarcane

genotypes


5/15

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


6/15

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


7/15

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


8/15

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.


9/15

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


10/15

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


11/15

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


12/15

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


15/15

Thanks for your attention

[email protected]

presentación mosaico, maceio 2011

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