mistérios moleculares da vassoura de bruxa

www.lge.ibi.unicamp.br

Mistérios Moleculares da

Vassoura de Bruxa

Gonçalo Guimarães PereiraUNICAMP

Salvador - Bahia

23/04/04


Basis

Organism Gene

BiochemistryDiscover the function of the Gene A

in the Organism X

GenomicsDiscover that the Organism Y

has a gene similar to the Gene A found in the Organism X

A(Y) ~ A(X)


Genomics Tools

Organism

Raw Sequence

Putative Genes

Cellular Program

Sequencing

Bioinformatics

Expression Analysis


Genomics Mission

Genomics X

Trial and Error

Hypothesis for Targets Definition


Genome Xylella fastidiosa

3 Mb

50 kb

1 a 2 kb

50 kb

Sequencing

ATGGGCCTTTGCTTTGCCCCCAAA

TGCCCCCAAAAGGGGGAAAGGGGGAAAAG

ATGGGCCTTTGCCCCCAAAAGGGGGAAAAGCCTGGGGGATGGGCCTTTGC AGGGGGAAAAGTTTGC

Identification of genes

ATG CT

Bioinformatics


BLAST

Virtual Metabolism


A.J.G. Simpson1, F.C. Reinach2, P. Arruda3, F.A. Abreu4, M. Acencio5, R. Alvarenga2,

L.M.C. Alves6, J.E. Araya7, G.S. Baia2, C.S. Baptista8, M.H.Barros8, E.D. Bonaccorsi2, S.

Bordin9, J.M. Bové10, M.R.S. Briones7, M.R.P. Bueno11, A.A. Camargo1, L.E.A.

Camargo12, D.M. Carraro12, H. Carrer12, N.B. Colauto13, C. Colombo14, F.F. Costa9, M.C.R.

Costa15, C.M. Costa-Neto16, L.L. Coutinho12, M. Cristofani17, E. Dias-Neto1, C. Docena2,

H. El-Dorry2, A.P. Facincani6, A.J.S. Ferreira2, V.C.A. Ferreira18, J.A. Ferro6, J.S. Fraga4,

S.C. França19, M.C. Franco20, M. Frohme21, L.R. Furlan22, M. Garnier10, G.H. Goldman23,

M.H.S. Goldman24, S.L. Gomes2, A. Gruber4, P.L. Ho25, J. D. Hoheisel21, M.L. Junqueira26,

E.L. Kemper3, J.P. Kitajima27, J.E. Krieger26, E.E. Kuramae28, F. Laigret10, M.R.

Lambais12, L.C.C. Leite25, E.G.M. Lemos6, M.V.F. Lemos29, S.A. Lopes19, C.R. Lopes13,

J.A. Machado30, 36 , M.A. Machado17, A.M.B.N. Madeira4, H.M.F. Madeira12, 37, C.L.

Marino13, M.V. Marques8, E.A.L. Martins25, E.M.F. Martins18, A.Y. Matsukuma2, C.F.M.

Menck8, E.C. Miracca5, C.Y. Miyaki11, C.B. Monteiro-Vitorello12, D.H. Moon20, M.A.

Nagai5, A.L.T.O. Nascimento25, L.E.S. Netto11, A. Nhani Jr.6, F.G. Nobrega8, 38, L.R.

Nunes31, M.A. Oliveira32, M.C. de Oliveira33, R.C. de Oliveira31, D.A. Palmieri13, A.

Paris13, B.R. Peixoto2, G.A.G. Pereira32, H.A. Pereira Jr.6, J.B. Pesquero16, R.B. Quaggio2,

P.G. Roberto19, V. Rodrigues34, A.J. de M. Rosa34, V.E. de Rosa Jr.28, R.G. de Sá34, R.V.

Santelli2, H.E. Sawasaki14, A.C.R. da Silva2, A.M. da Silva2, F.R. da Silva3,27, W.A. Silva

Jr.15, J.F. da Silveira7, M.L.Z. Silvestri2, W.J. Siqueira14, A.A. de Souza17, A.P. de Souza3,

M.F. Terenzi23, D. Truffi12, S.M. Tsai20, M.H. Tsuhako18, H. Vallada35, M.A. Van Sluys33,

S. Verjovski-Almeida2, A.L. Vettore3, M.A. Zago15, M. Zatz11, J. Meidanis*27 & J.C.

Setubal*27.

Publication


More


Xylella fastidiosa - Pierce's Disease Strain: Grape phytopathogen

Leifsonia xyli subsp. xyli (Sugarcane phytopathogen)

Leptospira

Eucalyptus

Coffee

Caw

and more....


Regional Genomes

Leishmania chagasi

Schistosoma mansoni

Trypanossoma cruzi

Paracoccidioides brasiliensis

Human diseases

Agriculture improvement

Herbaspirillum seropedicae

Gluconacetobacter diazotrophicus

Eucalyptus

Phytopathogen

Crinipellis perniciosa


Samba, football and .....

Genomics

Brazilian science

Fruits of co-operationJul 20th 2000 | SAO PAULOFrom The Economist print editionSAMBA, football and...genomics. The list of things for which Brazil is renownedhas suddenly got longer. Only a few days after publishing, on July 13th, the first-ever sequence of the genome of a plant pathogen, scientists at Sao Paulo’s stateresearch agency, Fapesp, were due to announce, on July 21st, anothersuccess—the composition of 279,000 human expressed-sequence tags, smallpieces of DNA that allow genes to be located along chromosomes. Only inAmerica and Britain have more than that number of human ESTs beenidentified.

Though they are of global significance, both of these advances are also of particularinterest to Brazilians. A number of the ESTs in question are derived from geneslinked to cancer of the head and neck, which for some reason is unusually common inBrazil. And the plant pathogen sequenced, Xylella fastidiosa, is an insect-bornebacterium that has been ravaging Brazil’s orange groves, causing their trees toproduce shrivelled fruit and costing growers an estimated $100m a year. ............


Practical Results

????????????


Cacau Production in Brazil

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Production

Th

ousa

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ons


International Price

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Cacau Income

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lion


Witche’s Broom Loses

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1983

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Loses

U$

Mil

lion

U$ 1.380.000.000,00300.000 jobs

100.000 ha Forest


Goals of the Witch's Broom Genome Project

Pragmatic

2. Create the basis to understand the Witch's Broom disease, indicating possible solutions

1. Build a Genomics Network at the State of Bahia to face biological problems


Symptoms - 1

Fotos - João de Cássia - CEPEC-CEPLAC



Symptoms - 2



Symptoms - 3


Crinipellis perniciosa



Life Cycle

Differentiation

Meiose


Complexity X Strategy

Prokaryotes

Eukaryotes

Fungi - Saccharomyces cerevisiae

Human

ATG CT

ATGCT

ATG CT

ATGCT

~ 3 Mb1G/1Kb

~ 20 Mb1G/2Kb

~3.000 Mb1G/75 Kb

ATG (?) CT


0,0%

20,0%

40,0%

60,0%

80,0%

100,0%

0,0% 100,0% 200,0% 300,0% 400,0% 500,0% 600,0%

Sequenced

Effe

ctiv

e c

ove

rag

e

Normal % singlets % contigs % assembled

Sequencing X Coverage

Singlets

Contigs

www.lge.ibi.unicamp.brG ap x c over age

60539

29778

12161

6032

2962

1201

597

303

129

356110

175474

93509

42908

2091413739

6110

2982

1322

1

10

100

1000

10000

100000

1000000

1% 10% 100% 1000%

C over age ( r eads)

0

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2000

3000

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5000

6000

7000

8000

9000

Average Maximum > 1000 > 1

1X 2X

303

>1000bp

Gaps X Coverage


Sequencing Strategy

20 Kb

40 reads: 500 bp/each

3 reads

Genomic Shotgun


Bahia Genomics Network

UESCCEPLACCENARGENUNICAMP

DNA CoordinationUNICAMP

UFBA-FarmáciaUFBA- IBUEFSUCSAL

Databank

BioinformáticaUNICAMP/UESC

Interface

Cacau Research Community

Libraries Libraries

Sequences Sequences


Data Treatment: “on going” annotation

Shotgun

Blast

Pre-Annotated Read

Local clustering

ORFs

Assembling

BACs

Complete Genome

Representative Read

Chips Molecular Mapping

InactivationStructural Analysis

Proteomics

Metabolic Maps


Bioinformatics Services

Serviços

Labs productivity Labs productivity graphs Labs productivity graphs (cumulative) Quality

Administrative services

Access control Access control (graph)

Bug reports

Sequence submission Nomenclature Nomenclature edition Submission test

Keyword search in Blast files Anotation (test) Annotated seeds Contigs progress Sequence pattern search Blast against C. perniciosa Blast results - All reads Sequence analysis tools (Emboss)

Library Control Strain

Geração Controle Análise

Gene Project


Gene Project*

Data bank: Blasted reads

Selected reads

Assembled ReadsConsensus

Annotated Gene

*Carazzolle, MF, Digiampietri, L, Araújo, MRS, Formighieri, EF, Tsukumo, F & Pereira, GAG

Selection: File, Keyword, Blast, Seq. pattern, all

Assembling: PPC, SaturationBlast of consensus

Analysis: ORF finder, Comparison, Prediction


Chitin Metabolism

UDP-N-AcetilglicosaminaUDP-N-Acetilglicosamina

CHITINCHITIN

N-Acetylglicosamine-1- phosphateN-Acetylglicosamine-1- phosphate

Glucosamine-6- phosphateGlucosamine-6- phosphate

GlutamineGlutamine

GlutamateGlutamate

AcetylCoAAcetylCoA

CoACoA

UTPUTP

N-AcetylglucosamineN-Acetylglucosamine

UDPUDP

2. 7. 7. 232. 7. 7. 23

ChitobioseChitobiose

N-Acetylglucosamine-6-hosphateN-Acetylglucosamine-6-hosphate

2. 3. 1. 42. 3. 1. 4

5. 4. 2. 35. 4. 2. 3

2. 7. 1. 592. 7. 1. 59pyrophosphatepyrophosphate

2. 6. 1. 162. 6. 1. 16

ChitosanChitosan 3. 5. 1. 413. 5. 1. 41

3. 5. 1. 25

2. 4. 1. 16

3. 2. 1. 143. 2. 1. 143. 2. 1. 523. 2. 1. 52

Fructose-6- phosphateFructose-6- phosphateGlucose-Glucose-6-phosphate6-phosphate

5. 3. 1. 95. 3. 1. 9

Glucosaminide

3. 2. 1. 1323. 2. 1. 132

GlucosamineGlucosamine3. 2. 1. -3. 2. 1. - 2. 7. 1. 12. 7. 1. 1

ATPATP

ADPADP

ATPATP ADPADPNc, Sc, Sp, CpNc, Sc, Sp, Cp Nc, Sc, CpNc, Sc, Cp

Only Only NcNc NoneNone

Goes-Neto, Priminho, et. AlUEFS


Control Infected

1 3

2 7

3 14

4 21

DAIPhases

5 35

6 61

7 130

Biochemistry of Infection - 1

Ph.D. Work: Leandra Scarpari; Supervisor: Gonçalo Pereira; Co-supervisors: Paulo Mazzafera e Alan PomellaAcknowledge: Almirante Cacau



Component > Control > Inoculated No Diference

Açucares- Solúveis totais XXX- Sacarose XXX- Amido XXX- Hemicelulose XXX- Açúcares redutores XXX- Celulose XXX- Pectinas XXX

AminoácidosQuantitativo XXXQualitativo *** ASP, GLU ASN

Fenóis totais XXXTaninos XXXNitrato XXXFlavonóides totais XXXClorofilas e carotenóides XXX

HormôniosEtileno XXXAuxinaCitocinina

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Açúcares Solúveis Totais (ug/mg)

Açucar Solúvel

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Inoculado

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Hemicelulose

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CONTROLE1 CONTROLE2 CONTROLE3 CONTROLE4 CONTROLE5 CONTROLE6

ASP

GLU

ASN

AA - Controle

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INOCULADO1 INOCULADO2 INOCULADO3 INOCULADO4 INOCULADO5 INOCULADO6

ASP

GLU

ASN

AA - Inoculado

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Inoculado

Aminoácidos (nmoles/mg)

AA - Total

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Inoculado

Amido (ug/mg)

Amido

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Clorofila Total



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ASP GLU ASN SER GLN ALA GABA GLY +THR

CONTROLE1

CONTROLE2

CONTROLE3

CONTROLE4

CONTROLE5

CONTROLE6

C

0,0

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ASP GLU ASN SER GLN ALA GABA GLY +THR

INOCULADO1

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INOCULADO3

INOCULADO4

INOCULADO5

INOCULADO6

I

Acido Glutâmico

Asparagina

The infection triggers the plant Programmed Cell Death - PCD

www.lge.ibi.unicamp.brTunnel


1 2 3 4 5 6 7 8 9 101. S. cerevisiae + H. wingei (Bio Rad)

2. CP02 (Biótipo C)

3. CP09 (Biótipo C)

4. FA42 (Biótipo C)

5. Ilhéus (Biótipo C)

6. FA104 (Biótipo S)

7. SCFT (Biótipo L)

8. FA322 (Biótipo L)

9. S. pombe

10. S. cerevisiae

C S L

Molecular Features

Rincones, J, Meinhardt, L, Vidal, B & Pereira, GAG


CP02 (25-06)

3.12.8(2)

4.65.23.5(3)

29 Mb

Genome Size


Genome Variability

Bahia I II IIAmazônicos

Probably only two clones of C. perniciosa have been introduced in Bahia


Telomeric PCR

I II

BahiaA1-R

1018 bp

1636 bp2036 bp3054 bp

517 bp

I II

BahiaA1

Meinhardt, L, Rincones, J. & Pereira, GAG


Amazônicos

Rondônia????


Genetic Solution

Creation of strict sanitary barriers between Amazonia and Bahia


Transposons

S C C L

Class ISuper-family Gypsy/Ty3

Marisa Queiroz - UFV

M L S U 1 2 3 4 5 6 P B

394

ReverseTrancriptase

S C C L

Impala-like

S C C L

Restless-like

www.lge.ibi.unicamp.brTransposon Distribuition


Variability Dynamics

Homobasidiomycete

Sexual Phase

???

Meioses

1. Cromossomal variations can be immediately fixed2. Variability may raise by mutation andtransposons 3. Why we do not see higher variability in Bahia????


Mitochondria


Senescence

Kalilo - like

109 kb


Senescence Waves

Hugo


Alternative Oxidase

C. perniciosa Alternative Oxidase


Recommendations

1. Strobirulin based fungicides are ineffective at the field

2. Inhibition of mitochondria may be effective to control the disease and could be achieved by combining strobirulin and Sham analogues


Acidocalcisome ???

Nuclei

???


Homeostase

Ca

Ca

Ca


Necrosis Factor

Phytophthora

Crinipellis

C. perniciosa

VV VS G M

RT-PCR do gene indutorde etileno e necrose

~ 4 paralogsNo intronsSecreted

UESC & UNICAMPExpressão, Estrutura e

Regulação

Etileno/Massa

20

60

100

140

180

220

260

300

1 2 3 4 5 6 7

coleta

controle

inoculado


Fátima Alvim - UESC

Necrosis Induction


Dr. Lyndel Mainhardt

Plant X Pathogen

BT

NC


Biotrophyc Hyfas


First Global Expression


Ethylene Formation


Chemical Genomics

ATOM 1 N PHE 13 41.568 48.879 96.268 1.00 23.86 N ATOM 2 CA PHE 13 42.820 48.397 96.955 1.00 23.90 C ATOM 3 C PHE 13 43.984 48.568 95.986 1.00 23.35 C ATOM 4 O PHE 13 43.926 49.391 95.071 1.00 21.30 O ATOM 5 CB PHE 13 42.687 46.912 97.362 1.00 22.95 C ATOM 6 N SER 14 45.042 47.799 96.191 1.00 23.62 N ATOM 7 CA SER 14 46.177 47.890 95.297 1.00 24.80 C ATOM 8 C SER 14 45.721 47.249 94.008 1.00 26.03 C ATOM 9 O SER 14 44.772 46.466 94.005 1.00 27.80 O ATOM 10 CB SER 14 47.368 47.133 95.860 1.00 22.46 C ATOM 11 N ASP 15 46.348 47.619 92.900 1.00 27.00 N ATOM 12 CA ASP 15 46.006 47.011 91.632 1.00 25.59 C ATOM 13 C ASP 15 46.328 45.539 91.796 1.00 24.64 C ATOM 14 O ASP 15 45.507 44.694 91.497 1.00 26.72 O ATOM 15 CB ASP 15 46.816 47.642 90.503 1.00 24.84 C

Target Structure

http://www.rcsb.org/pdb/20098 Structures (18/02/03)

Chemical Libraries

Molecular Docking

Molecular Design


“Omics” Era

Genomics

Metabolomics

TranscriptomicsProteomics Economics


Malaria

Nature 419, 498 - 511 (2002)


The Regress


Price Reaction

Preço interno na Bahia - Diferencial s/Bolsa de NY(em $/t)

-200,00

-100,00

0,00

100,00

200,00

300,00

04/0

1/99

13/0

2/99

25/0

3/99

04/0

5/99

13/0

6/99

23/0

7/99

01/0

9/99

11/1

0/99

20/1

1/99

30/1

2/99

08/0

2/00

19/0

3/00

28/0

4/00

07/0

6/00

17/0

7/00

26/0

8/00

05/1

0/00

14/1

1/00

24/1

2/00

02/0

2/01

14/0

3/01

23/0

4/01

02/0

6/01

12/0

7/01

21/0

8/01

30/0

9/01

09/1

1/01

19/1

2/01

28/0

1/02

09/0

3/02

18/0

4/02

28/0

5/02

500

750

1.000

1.250

1.500

1.750Bolsa NIPrêmio/Desconto

Difer. Bolsa

Fonte: Thomas Hartman

Início

.


www.lge.ibi.unicamp.br/vassoura

Homepage


Team

COORDENAÇÃOGonçalo Amarante Guimarães Pereira -Júlio Cascardo

COORDENADORES DE CENTROS CEPLAC Raúl René Meléndez Valle e Karina GramachoEMBRAPA - Dário Gratapaglia UESC -Júlio Cascardo e Dário Ahnert UNICAMP - Gonçalo Guimarães PereiraUFBA - FC - Marilda GonçalvesUFBA - IB – Luciana Veiga UEFS - Aristóteles Goes Neto e Carlos Priminho PirovaniUCSAL - Luzimar Gonzaga

GERÊNCIA E ASSESSORIA JURÍDICAEliane Laranja Dias e Leonardo Grecco

COORDENADORAS DNA Carolina Cotomacci - Dirce Maria Carraro

CONSULTORES DNA Anderson Ferreira da Cunha e Helaine Carrer

PLANTA X PATÓGENOLeandra Scarpari, Allan Pomella e Lyndel Meinhardt

BIOINFORMÁTICA Marcos Renato Araujo, Luciano Digiampetri,Marcelo F. Carazzolle e Eduardo Formigheri

SEQÜENCIAMENTOCarolina Cotomacci, Raquel, Anrdéa de Assis, Camilla RamosVitor Almeida, Robson, Monica, Luana, ReimarMaíza Alves Lopes e Bruno Motta Oliveira

MAPEAMENTO MOLECULARJohana Rincones, Lyndel Maynhardt

CICLO SEXUAL Carolina Cotomacci

ECOLOGIA MOLECULAR / MITOCÔNDRIA Naiara Sardinha Pinto, Eduardo Formigheri

EXPRESSÃO GLOBALMaricene Sabha e Leandra

TRANSFORMAÇÃO DO FUNGO Marisa Queiroz


GENÉTICA VEGETAL Acássia Benjamim Leal Pires Alana Maria Cerqueira de OliveiraFábio Gelape Faleiro Fátima Cerquira Alvim José Antonio Peters José Bonadie MarquesJosé Luis Pires Júlio Cézar de Mattos Cascardo Leandro Lopes Loguércio Luana Mahé Costa Gomes Milton Macoto Yamada Mônica Menezes Passinho Mônica Rosa Bertão Reinaldo Figueiredo dos Santos Rita de Cássia Siqueira Bahia de Assis Uilson Vanderlei Lopes Wilson Reis Monteiro

BIOQUÍMICA Paulo Mazzafera

SISTEMÁTICA DE FUNGOS Aristóteles Goes Neto

SISTEMÁTICA VEGETAL Cíntia Schultz Coimbra FISIOLOGIA VEGETAL Marco Antonio Galeaas Aguilar Raúl René Meléndez Valle

FITOPATOGIA Carlos Priminho PirovaniEdna Dora M. N Luz Givaldo Rocha Niella Jadergudson Pereira João de Cássia do Bomfim Costa João Louis Pereira José Lauereano José Luis Bezerra Karina Peres Gramacho Marival Lopes de OliveiraStela Dalva M. N. Silva

MICROSCOPIA ELETRÔNICAStela Silva e Paulo Joazeiro

Team - 2


Funding

Fapesp


Economical Perspectives

Amex Biotech x Nasdaq x Dow Jones

-

200

400

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800

1.000

1.200

1.400

1.600

jan/90 jan/91 jan/92 jan/93 jan/94 jan/95 jan/96 jan/97 jan/98 jan/99 jan/00 jan/01

AMEX Biotech Nasdaq Dow Jones


Biotech Process

Alcohol Fermentation

mistérios moleculares da vassoura de bruxa

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