Chitinase genes and insect management

1. C hitinas e genes and ins ec t
management in crop plants
Prakash Adavi

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. Chitin structure

4. Chitin location in insects

6. Depicted pathway: Chitin synthesis
(Merzendorfer & Zimoch, 2003)

7. Chitinases
• Found in – Archaebacteria, Eubacteria, Protists,
Fungi, Plants and Animals
Function
– Digestion
– Molting in arthropods
– Defense mechanism
– Pathogenicity
(Merzendorfer & Zimoch, 2003)

8. 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

9. 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
mechanism
Chitinases EC.No
Endochitinases 3.2.1.14
Exochitinase
Chitobiosidases or chitin-1,4-β- 3.2.1.29
chitobiosidases
β-(1,4)-Nacetyl-glucosaminidases 3.2.1.30
(GlcNAcase) or chitobiases

10. Mode of action

11. Two different families
– 18 GH : Bacteria, yeast, fungi, viruses, plants
& animals
– 19 GH : Exclusively plants
Shows multi domain architecture
– Catalytic
– Chitin binding domains
– Non catalytic
(Arakane and Muthukrishnan, 2009)

12. 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)

13. Model ribbon structure
M. sexta chitinase (amino acid
residues 80–387)
S. marsecens chitinase residues
227–534
sequence similarity (81%)
(βα)8 barrel fold structure
Yellow-beta-sheet structures
Red-alpha-helical segments
Blue-turn segments
Green-conserved regions
(Kramer and Muthukrishnan,
1997)

14. Fungal chitinase: domain organisation
a: Saccharomyces cerevisiae endochitinase (CTS1)
b: Rhizopus oligosporus chitinase (CHI1)
c: T. harzianum chitinase(CHIT33)
1: Signal peptide region 2: catalytic domain
3: Serine/Theronine-rich region
4: chitin-binding domain
5: C-terminal extension region. (Duo-Chuan, 2006)

15. 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)

16. 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)

17. 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)

18. 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)

19. 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)

20. 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)

21. 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)

22. 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)

23. 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)

24. Group VIII chitinases
• A catalytic domain
• No CBD
• A predicted trans-membrane span
(Arakane and Muthukrishnan, 2009)

25. Phylogenetic analysis
• T. castaneum, D. melanogaster, A. gambiae,
N. vitripennis and A. mellifera
(Arakane and Muthukrishnan, 2009)

26. 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)

27. Functional specialization: RNA interference
• Group I: TcCHT5 - Pupal-adult
• Group II: TcCHT10- L-L,L-P,P-A & egg hatch
(Zhu et al., 2008)

28. Group III: TcCHT7- abdominal contraction & wing
expansion
(Zhu et al., 2008)

29. 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)

30. Approaches for insect management
• Transgenic plants
• Improvement in entomopathogenic fungus
• Exploiting synergism with Bt

32. 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)

33. Colorimetric assay of chitinase activity
(McCafferty et al., 2007)

34. Laboratory insect assay
b
(McCafferty et al., 2007)

35. Field data of mite population
(McCafferty et al., 2007)

37. Source of chitin-binding domains
Comparison b/w BmChBD & mBmChBD
(Fan et al., 2007)

38. Maps of pBARGFP :vector for B. bassiana
(Fan et al., 2007)

39. A: Chitin binding ability
B: FITC-labeled Chitin-binding assay
(Fan et al., 2007)

40. Chitinase activity on insoluble substrate (A)
& different soluble substrates (B)
(Fan et al., 2007)

41. Electron microscopy : scanning of the
hydrolytic effects
A: Control
B:Bbchit1
C:Bbchit1-BmChBD
D:Bbchit1mBmChBD
(Fan et al., 2007)

42. Chitinase activity analysis in B. bassiana
transformants
(Fan et al., 2007)

44. 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)

45. 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)

46. Expression of CDEP1, Bbchit1 and
CDEP1:Bbchit1in B. bassiana
(Fang et al., 2009)

48. 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)

49. 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)

50. Pioneers
Dr. S. Muthukrishnan
Dr. Karl Kramer
Dr. Yasuyuki Arakane

53. THANK YOU