Chitinase genes and insect management in crop plants

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Chitinase genes and insect management

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Chitinase genes and insect management in crop plants

  1. 1. C hitinas e genes and ins ec tmanagement in crop plants Prakash Adavi
  2. 2. Introduction• Chitin – biopolymer, structural polysaccharide• Produced by fungi, arthropods and nematodes• Insects - scaffold material (procuticle & PM up to 40%)• Involved in insect growth & morphogenesis• Insects repeatedly produce chitin synthases and chitinolytic enzymes in different tissues
  3. 3. Chitin structure
  4. 4. Chitin location in insects
  5. 5. Depicted pathway: Chitin synthesis (Merzendorfer & Zimoch, 2003)
  6. 6. Chitinases• Found in – Archaebacteria, Eubacteria, Protists, Fungi, Plants and AnimalsFunction – Digestion – Molting in arthropods – Defense mechanism – Pathogenicity (Merzendorfer & Zimoch, 2003)
  7. 7. Characteristic features• Inducible• Substrate specificity• Glycosylation• 3-D structures• Aspartic acid & glutamic acid : conserved region• Insect chitinases: 40–85 kDa• Fungi: 27-190 kDa• Plant : 25–40 kDa• Bacteria : 20–60 kDa
  8. 8. Classification • IUB Enzyme Nomenclature (1984): EC 3.2.1.x • First 3 digits : enzymes hydrolysing O-glycosyl linkages • last number : substrate and sometimes reflects the molecular mechanismChitinases EC.NoEndochitinases 3.2.1.14ExochitinaseChitobiosidases or chitin-1,4-β- 3.2.1.29chitobiosidasesβ-(1,4)-Nacetyl-glucosaminidases 3.2.1.30(GlcNAcase) or chitobiases
  9. 9. Mode of action
  10. 10. Two different families – 18 GH : Bacteria, yeast, fungi, viruses, plants & animals – 19 GH : Exclusively plantsShows multi domain architecture – Catalytic – Chitin binding domains – Non catalytic (Arakane and Muthukrishnan, 2009)
  11. 11. Manduca sexta chitinase• Blue-catalytic, red-serine/threonine-rich, green- chitin-binding domain• C-terminal deletion: studies• Chitinase gene: Single copy in genome• Molting fluid : 3-chitinases(85,62,50 kDa) (Zhu et al.,2001)
  12. 12. Model ribbon structureM. sexta chitinase (amino acidresidues 80–387)S. marsecens chitinase residues227–534sequence similarity (81%)(βα)8 barrel fold structureYellow-beta-sheet structuresRed-alpha-helical segmentsBlue-turn segmentsGreen-conserved regions(Kramer and Muthukrishnan,1997)
  13. 13. Fungal chitinase: domain organisationa: Saccharomyces cerevisiae endochitinase (CTS1)b: Rhizopus oligosporus chitinase (CHI1)c: T. harzianum chitinase(CHIT33)1: Signal peptide region 2: catalytic domain3: Serine/Theronine-rich region4: chitin-binding domain5: C-terminal extension region. (Duo-Chuan, 2006)
  14. 14. Family 18 chitinases• Largest chitin family• Undergone evolutionary modification from protists to mammalia• Most extensive expansion of chitinase have occurred in class Insecta• Exclusively endochitinases and prefer to digest β -1,4- linkages• Yield β-anomers at the reducing ends• No exochitinase activity (Arakane and Muthukrishnan, 2009)
  15. 15. Domain architecture of insect chitinases• Multi-domain structural organization• Includes1–5 catalytic domains• 0–7 cysteine-rich chitin-binding domains (CBD)• Heavily glycosylated serine/ threonine -rich linker regions• Most chitinases are predicted to have a leader peptide or a transmembrane – spanning domain• Eight unique groups (Arakane and Muthukrishnan, 2009)
  16. 16. Group I chitinases• Correspond to the enzymatically well-characterized chitinases• Contain a signal peptide• One catalytic domain• Ser/Thr- rich linker region• One C-terminal chitin-binding domain with six cysteines (Arakane and Muthukrishnan, 2009)
  17. 17. Group II chitinases• Large molecular weight chitinases• Have four or five catalytic domains• Four to five CBDs• All species• Dipterans catalytic CBD (Arakane and Muthukrishnan, 2009)
  18. 18. Group III chitinases• Contain two catalytic domains and one CBD• Typically in a - arrangement• Posseses a predicted trans membrane segment at the N-terminal region (Arakane and Muthukrishnan, 2009)
  19. 19. Group IV chitinases• Constitute the largest and most divergent group• Have a signal peptide and a single catalytic domain• Most, but not all, members of this group of chitinases lack a CBD (Arakane and Muthukrishnan, 2009)
  20. 20. Group V chitinase• Chitinase -like proteins include the imaginal disk growth factors (IDGF’s)• Have a leader peptide and a catalytic domain• No CBDs• Devoid of chitinase activity (Arakane and Muthukrishnan, 2009)
  21. 21. Group VI chitinases• Larger than the group I chitinases but similar in domain structure• Have a signal peptide• N-terminal catalytic domain• One CBD• Long stretch at the C-terminus contains 25–30% Ser / Thr residues• Heavily glycosylated – resistant to proteases (Arakane and Muthukrishnan, 2009)
  22. 22. Group VII chitinases• Exhibit a domain architecture similar to that of group IV chitinases• A Signal peptide• Single catalytic domain• No CBD (Arakane and Muthukrishnan, 2009)
  23. 23. Group VIII chitinases• A catalytic domain• No CBD• A predicted trans-membrane span (Arakane and Muthukrishnan, 2009)
  24. 24. Phylogenetic analysis• T. castaneum, D. melanogaster, A. gambiae, N. vitripennis and A. mellifera (Arakane and Muthukrishnan, 2009)
  25. 25. Domain architecture of T. castaneum chitinases Blue boxes- signal peptide; pink boxes- catalytic domain; green boxes- chitin-binding domain; red boxes- transmembrane span; lines- linker regions (Arakane and Muthukrishnan, 2009)
  26. 26. Functional specialization: RNA interference • Group I: TcCHT5 - Pupal-adult • Group II: TcCHT10- L-L,L-P,P-A & egg hatch (Zhu et al., 2008)
  27. 27. Group III: TcCHT7- abdominal contraction & wingexpansion (Zhu et al., 2008)
  28. 28. Group IV: TcCHT2,4,6,8,9,11…16 feeding stages (larvae & adults)Group V: TcIDGF2 & 4.TcIDGF4- Adult Eclosion (no chitinolytic activity) (Zhu et al., 2008)
  29. 29. Approaches for insect management• Transgenic plants• Improvement in entomopathogenic fungus• Exploiting synergism with Bt
  30. 30. Presence & expression of MSCH gene Hybridization of RT-PCR and PCR products blotted and probed with the MSCH gene. Lanes: 1 Plasmid control 2 and 3, transgenic plants T-24 and T-23 with RT 4-non-transformed Kapoho control 5 and 6-transgenic plants T-24 and T-23 without RT (McCafferty et al., 2007)
  31. 31. Colorimetric assay of chitinase activity (McCafferty et al., 2007)
  32. 32. Laboratory insect assayb (McCafferty et al., 2007)
  33. 33. Field data of mite population (McCafferty et al., 2007)
  34. 34. Source of chitin-binding domainsComparison b/w BmChBD & mBmChBD (Fan et al., 2007)
  35. 35. Maps of pBARGFP :vector for B. bassiana (Fan et al., 2007)
  36. 36. A: Chitin binding abilityB: FITC-labeled Chitin-binding assay (Fan et al., 2007)
  37. 37. Chitinase activity on insoluble substrate (A) & different soluble substrates (B) (Fan et al., 2007)
  38. 38. Electron microscopy : scanning of the hydrolytic effects A: Control B:Bbchit1 C:Bbchit1-BmChBD D:Bbchit1mBmChBD (Fan et al., 2007)
  39. 39. Chitinase activity analysis in B. bassiana transformants (Fan et al., 2007)
  40. 40. Maps of pBARGFP :vector for B. bassiana Pgpd-promoter Bar-herbicide resistance gene Egfp-enhanced green fluroscent protein gene Trpc- terminator of trpc gene (Fang et al., 2009)
  41. 41. Western blot analysis A: anti Bbchit1 Lane 1:WT 2,3&4: Bbchit1-26,28,&35 5,6,&7: BbCDEP1:CHIT-4,41,&4 2 B: anti-CDEP1 Lane 2,3 &4 :BbCDEP1-1,6,& 10 (Fang et al., 2009)
  42. 42. Expression of CDEP1, Bbchit1 and CDEP1:Bbchit1in B. bassiana (Fang et al., 2009)
  43. 43. Construction of recombinant plasmid pHUAccB5 • pHT315 vector • Bt strain 4.0718 – 1Ac , terminator • pBG1112 containing chi gene • acrystalliferous Bt strain XBU001 (Ding et al., 2008)
  44. 44. Bt Transformants XBU-HUAccB5 tested by PCR • Lane1: chi gene fragment -0.91kb • Lane2: chi gene and terminator fragment-1.2kb • Lane3: The whole fusion gene fragment 5.2 kb (Ding et al., 2008)
  45. 45. PioneersDr. S. Muthukrishnan Dr. Karl Kramer Dr. Yasuyuki Arakane
  46. 46. THANK YOU

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