Www.lge.ibi.unicamp.br Mistérios Moleculares da Vassoura de Bruxa Gonçalo Guimarães Pereira UNICAMP Salvador - Bahia 23/04/04.

Apresentação em tema: "Www.lge.ibi.unicamp.br Mistérios Moleculares da Vassoura de Bruxa Gonçalo Guimarães Pereira UNICAMP Salvador - Bahia 23/04/04."— Transcrição da apresentação:

1 www.lge.ibi.unicamp.br Mistérios Moleculares da Vassoura de Bruxa Gonçalo Guimarães Pereira UNICAMP Salvador - Bahia 23/04/04

2 www.lge.ibi.unicamp.br Basis Organism Gene Biochemistry Discover the function of the Gene A in the Organism X Genomics Discover that the Organism Y has a gene similar to the Gene A found in the Organism X A(Y) ~ A(X)

3 www.lge.ibi.unicamp.br Genomics Tools Organism Raw Sequence Putative Genes Cellular Program Sequencing BioinformaticsExpression Analysis

4 www.lge.ibi.unicamp.br Genomics Mission Genomics X Trial and Error Hypothesis for Targets Definition

5 www.lge.ibi.unicamp.br Genome Xylella fastidiosa 3 Mb 50 kb 1 a 2 kb 50 kb Sequencing ATGGGCCTTTG CTTTGCCCCCAAA TGCCCCCAAAAGGGGG AAAGGGGGAAAAG ATGGGCCTTTGCCCCCAAAAGGGGGAAAAG CCTGGGGGATGGGCCTTTGC AGGGGGAAAAGTTTGC Identification of genes ATGCT Bioinformatics

6 www.lge.ibi.unicamp.br BLAST Virtual Metabolism

7 www.lge.ibi.unicamp.br Publication

8 www.lge.ibi.unicamp.br More

9 www.lge.ibi.unicamp.br Xylella fastidiosa - Pierce's Disease Strain: Grape phytopathogen Leifsonia xyli subsp. xyli (Sugarcane phytopathogen) Leptospira Eucalyptus Coffee Caw and more....

10 www.lge.ibi.unicamp.br

11 Regional Genomes Leishmania chagasi Schistosoma mansoni Trypanossoma cruzi Paracoccidioides brasiliensis Human diseases Agriculture improvement Herbaspirillum seropedicae Gluconacetobacter diazotrophicus Eucalyptus Phytopathogen Crinipellis perniciosa

12 www.lge.ibi.unicamp.br Samba, football and..... Genomics

13 www.lge.ibi.unicamp.br Practical Results ????????????

14 www.lge.ibi.unicamp.br Cacau Production in Brazil Thousand Tons

15 www.lge.ibi.unicamp.br International Price

16 www.lge.ibi.unicamp.br Cacau Income U$ Billion

17 www.lge.ibi.unicamp.br Witche’s Broom Loses U$ Million U$ 1.380.000.000,00 300.000 jobs 100.000 ha Forest

18 www.lge.ibi.unicamp.br 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

19 www.lge.ibi.unicamp.br Symptoms - 1 Fotos - João de Cássia - CEPEC-CEPLAC

20 www.lge.ibi.unicamp.br Fotos - João de Cássia - CEPEC-CEPLAC Symptoms - 2

21 www.lge.ibi.unicamp.br Fotos - João de Cássia - CEPEC-CEPLAC Symptoms - 3

22 www.lge.ibi.unicamp.br Crinipellis perniciosa Fotos - João de Cássia - CEPEC-CEPLAC

23 www.lge.ibi.unicamp.br Life Cycle Differentiation Meiose

24 www.lge.ibi.unicamp.br Complexity X Strategy Prokaryotes Eukaryotes Fungi - Saccharomyces cerevisiae Human ATGCT ATGCT ATGCT ATGCT ~ 3 Mb 1G/1Kb ~ 20 Mb 1G/2Kb ~3.000 Mb 1G/75 Kb ATG (?)CT

25 www.lge.ibi.unicamp.br Sequencing X Coverage Singlets Contigs

26 www.lge.ibi.unicamp.br 1X 2X 303 >1000bp Gaps X Coverage

27 www.lge.ibi.unicamp.br Sequencing Strategy 20 Kb 40 reads: 500 bp/each 3 reads Genomic Shotgun

28 www.lge.ibi.unicamp.br Bahia Genomics Network UESC CEPLAC CENARGEN UNICAMP DNA Coordination UNICAMP UFBA-Farmácia UFBA- IB UEFS UCSAL Databank Bioinformática UNICAMP/UESC Interface Cacau Research Community Libraries Sequences

29 www.lge.ibi.unicamp.br Data Treatment: “on going” annotation Shotgun Blast Pre-Annotated Read Local clustering ORFs Assembling BACs Complete Genome Representative Read Chips Molecular Mapping Inactivation Structural Analysis Proteomics Metabolic Maps

30 www.lge.ibi.unicamp.br Bioinformatics Services Serviços Labs productivity 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 GeraçãoControleAnálise Gene Project

31 www.lge.ibi.unicamp.br Gene Project* Data bank: Blasted reads Selected reads Assembled Reads Consensus Annotated Gene *Carazzolle, MF, Digiampietri, L, Araújo, MRS, Formighieri, EF, Tsukumo, F & Pereira, GAG Selection: File, Keyword, Blast, Seq. pattern, all Assembling: PPC, Saturation Blast of consensus Analysis: ORF finder, Comparison, Prediction

32 www.lge.ibi.unicamp.br Chitin Metabolism UDP-N-Acetilglicosamina CHITIN N-Acetylglicosamine-1- phosphate N-Acetylglicosamine-1- phosphate Glucosamine-6- phosphate Glucosamine-6- phosphate Glutamine Glutamate AcetylCoA CoA UTP N-Acetylglucosamine UDP 2. 7. 23 2. 7. 23 Chitobiose N-Acetylglucosamine-6-hosphate 2. 3. 1. 4 2. 3. 1. 4 5. 4. 2. 3 5. 4. 2. 3 2. 7. 1. 59 2. 7. 1. 59 pyrophosphate 2. 6. 1. 16 Chitosan 3. 5. 1. 41 3. 5. 1. 25 2. 4. 1. 16 3. 2. 1. 14 3. 2. 1. 52 Fructose-6- phosphate Fructose-6- phosphate Glucose-6-phosphate 5. 3. 1. 9 Glucosaminide 3. 2. 1. 132 Glucosamine 3. 2. 1. - 2. 7. 1. 1 ATP ADP ATPADP Nc, Sc, Sp, Cp Nc, Sc, Cp Only Nc None Goes-Neto, Priminho, et. Al UEFS

33 www.lge.ibi.unicamp.br ControlInfected 13 27 314 421 DAIPhases 535 661 7130 Biochemistry of Infection - 1 Ph.D. Work: Leandra Scarpari; Supervisor: Gonçalo Pereira; Co-supervisors: Paulo Mazzafera e Alan Pomella Acknowledge: Almirante Cacau

34 www.lge.ibi.unicamp.br Biochemistry of Infection - 2 Component > Control> InoculatedNo Diference Açucares - Solúveis totais XXX - Sacarose XXX - AmidoXXX - HemiceluloseXXX - Açúcares redutores XXX - Celulose XXX - Pectinas XXX Aminoácidos Quantitativo XXX Qualitativo ***ASP, GLU ASN Fenóis totais XXX Taninos XXX Nitrato XXX Flavonóides totaisXXX Clorofilas e carotenóidesXXX Hormônios Etileno XXX Auxina Citocinina Açucar Solúvel Hemicelulose AA - Controle AA - Inoculado AA - Total Amido Flavonóides Totais Clorofila Total

35 www.lge.ibi.unicamp.br Biochemistry of Infection - 3 C I Acido Glutâmico Asparagina The infection triggers the plant Programmed Cell Death - PCD

36 www.lge.ibi.unicamp.br Tunnel

37 www.lge.ibi.unicamp.br 1 2 3 4 5 6 7 8 9 10 1. 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 CSL Molecular Features Rincones, J, Meinhardt, L, Vidal, B & Pereira, GAG

38 www.lge.ibi.unicamp.br 3.1 2.8(2) 4.65.2 3.5(3) 29 Mb Genome Size

39 www.lge.ibi.unicamp.br Genome Variability BahiaIII Amazônicos Probably only two clones of C. perniciosa have been introduced in Bahia

40 www.lge.ibi.unicamp.br Telomeric PCR III Bahia A1-R 1018 bp 1636 bp 2036 bp 3054 bp 517 bp III Bahia A1 Meinhardt, L, Rincones, J. & Pereira, GAG

41 www.lge.ibi.unicamp.br Amazônicos Rondônia????

42 www.lge.ibi.unicamp.br Genetic Solution Creation of strict sanitary barriers between Amazonia and Bahia

43 www.lge.ibi.unicamp.br Transposons S C C L Class I Super-family Gypsy/Ty3 Marisa Queiroz - UFV M L S U 1 2 3 4 5 6 P B 394 Reverse Trancriptase S C C L Impala-like S C C L Restless-like

44 www.lge.ibi.unicamp.br Transposon Distribuition

45 www.lge.ibi.unicamp.br Variability Dynamics Homobasidiomycete Sexual Phase ??? Meioses 1. Cromossomal variations can be immediately fixed 2. Variability may raise by mutation and transposons 3. Why we do not see higher variability in Bahia????

46 www.lge.ibi.unicamp.br Mitochondria

47 www.lge.ibi.unicamp.br Senescence Kalilo - like 109 kb

48 www.lge.ibi.unicamp.br Senescence Waves Hugo

49 www.lge.ibi.unicamp.br Alternative Oxidase C. perniciosa Alternative Oxidase

50 www.lge.ibi.unicamp.br 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

51 www.lge.ibi.unicamp.br Acidocalcisome ??? Nuclei ???

52 www.lge.ibi.unicamp.br Homeostase Ca

53 www.lge.ibi.unicamp.br Necrosis Factor Phytophthora Crinipellis C. perniciosa VV VS G M RT-PCR do gene indutor de etileno e necrose ~ 4 paralogs No introns Secreted UESC & UNICAMP Expressão, Estrutura e Regulação

54 www.lge.ibi.unicamp.br Fátima Alvim - UESC Necrosis Induction

55 www.lge.ibi.unicamp.br Dr. Lyndel Mainhardt Plant X Pathogen BT NC

56 www.lge.ibi.unicamp.br Biotrophyc Hyfas

57 www.lge.ibi.unicamp.br First Global Expression

58 www.lge.ibi.unicamp.br Ethylene Formation

59 www.lge.ibi.unicamp.br 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

60 www.lge.ibi.unicamp.br “Omics” Era Genomics Metabolomics TranscriptomicsProteomics Economics

61 www.lge.ibi.unicamp.br Malaria Nature 419, 498 - 511 (2002)

62 www.lge.ibi.unicamp.br The Regress

63 www.lge.ibi.unicamp.br Price Reaction Fonte: Thomas Hartman Início.

64 www.lge.ibi.unicamp.br www.lge.ibi.unicamp.br/vassoura Homepage

65 www.lge.ibi.unicamp.br Team COORDENAÇÃO Gonçalo Amarante Guimarães Pereira - Júlio Cascardo COORDENADORES DE CENTROS CEPLAC Raúl René Meléndez Valle e Karina Gramacho EMBRAPA - Dário Gratapaglia UESC -Júlio Cascardo e Dário Ahnert UNICAMP - Gonçalo Guimarães Pereira UFBA - FC - Marilda Gonçalves UFBA - IB – Luciana Veiga UEFS - Aristóteles Goes Neto e Carlos Priminho Pirovani UCSAL - Luzimar Gonzaga GERÊNCIA E ASSESSORIA JURÍDICA Eliane Laranja Dias e Leonardo Grecco COORDENADORAS DNA Carolina Cotomacci - Dirce Maria Carraro CONSULTORES DNA Anderson Ferreira da Cunha e Helaine Carrer PLANTA X PATÓGENO Leandra Scarpari, Allan Pomella e Lyndel Meinhardt BIOINFORMÁTICA Marcos Renato Araujo, Luciano Digiampetri, Marcelo F. Carazzolle e Eduardo Formigheri SEQÜENCIAMENTO Carolina Cotomacci, Raquel, Anrdéa de Assis, Camilla Ramos Vitor Almeida, Robson, Monica, Luana, Reimar Maíza Alves Lopes e Bruno Motta Oliveira MAPEAMENTO MOLECULAR Johana Rincones, Lyndel Maynhardt CICLO SEXUAL Carolina Cotomacci ECOLOGIA MOLECULAR / MITOCÔNDRIA Naiara Sardinha Pinto, Eduardo Formigheri EXPRESSÃO GLOBAL Maricene Sabha e Leandra TRANSFORMAÇÃO DO FUNGO Marisa Queiroz

66 www.lge.ibi.unicamp.br GENÉTICA VEGETAL Acássia Benjamim Leal Pires Alana Maria Cerqueira de Oliveira Fábio Gelape Faleiro Fátima Cerquira Alvim José Antonio Peters José Bonadie Marques José 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 Pirovani Edna 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 Oliveira Stela Dalva M. N. Silva MICROSCOPIA ELETRÔNICA Stela Silva e Paulo Joazeiro Team - 2

67 www.lge.ibi.unicamp.br Funding Fapesp

68 www.lge.ibi.unicamp.br Economical Perspectives

69 www.lge.ibi.unicamp.br Biotech Process Alcohol Fermentation

70 www.lge.ibi.unicamp.br