chitooligosaccharides and immunity in plants€¦ · lysm3) is deeply buried in ecp6 and displays...
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Chitooligosaccharides and immunity in plants
Appa Rao PodileSenior Professor and J.C. Bose Fellow
85th Annual Meeting of Indian Academy of SciencesHost-Pathogen Interactions Symposium
November 08, 2011
17, 495 – 502, 2012
LysM domains of certain plant kinases enable the plant to recognize its symbiotic microbes like rhizobia and mycorrhizae or sense and
induce resistance against pathogenic fungi
Cladosporium fulvum overcomes PAMP(chitin)-triggered immunity by secreting LysM-domain containing effector molecules like Ecp6
eLife 2: e00790, 2013
Limpens et al. Ann. Rev. Phytopathol. 2015
9: e1003769
Ligand-induced composite binding groove (LysM1 and
LysM3) is deeply buried in Ecp6 and displays ultrahigh
(picomolar) chitin-binding affinity, which is significantly higher than that of plant immune receptors
(GlcNAc)8 induces dimerization of LysM1-2, and not LysM1-2 I122A
Hayafune et al., PNAS 2013
Hydrolysis/TG of Hydrolysis/TG o Sp of pSp ChiD from DP6p hiD from DPCh -P6 DP3 substrates 6- P3 substrates DD
DP6 DP5
DP4 DP3Transglycosylation (TG) on DP6 -DP3 substrates. Monomers (DP1) were themajor end products due to chitobiase activity. DP : Degree of polymerization
Chitooligosaccharides (DP3-DP6)
TG products(DP13-DP7)
TransglycosylationN- acetyl glucosamine
Transglycosylation (TG) by Sp ChiD
Transglycosylation
Hydrolysis
Two possible mechanisms: glycoside hydrolases and transglycosidases.
Hydrolysis occurs when the glycosyl-enzyme intermediate is broken down by water.
Transglycosylation occurs when a sugar moiety acts as the glycosylacceptor.
MALDI-TOF MS analysis of products from DP3-DP6
DP3- Up to DP7
DP4- Up to DP10
DP5- Up to DP12
DP6- Up to DP13
Purushotham, P., and Podile, A.R. 2012. J. Bacteriol.
The residues targeted for mutation
Catalytic center - M226,Y228, R284, E159 andY160
Catalytic groove -F64, F125, G119, S116and W120.
Solventaccessible region-W247
A: at 30 min, B: at 360 min
Comparision of quantifiable TG products
Group Details of Mutations
Position Mutated to Out come
Group I
Catalytic center
Met -226 Ala More TG, Less hydrolysis
Tyr - 228 Ala More TG, Less hydrolysis
Glu -159 Asp Loss of both activities
Tyr - 160 Ala Only increased hydrolysis
Group II
Catalytic groove
Phe - 64 Trp More TG, Less hydrolysis
Gly - 119 Ser More TG, Less hydrolysis
Ser - 116 Gly More TG, Less hydrolysis
Phe - 125 Ala More TG, Less hydrolysis
Trp - 120 Ala No TG, More hydrolysis
Group IIISurface exposed
regionTrp - 247 Ala More TG, Less hydrolysis
Madhuprakash et al. J. Biol. Chem. 2012
Entry and exit sites for SpChiD
The region of TrpTTTTTTTTTTTTTTTTTTTTThhhhhhhhhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeeeeeeeeeeee rrrrrrrrrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeeeeeeeeeeeeggggggggggggggggggggggggggggggiiiiiiiiiiiiiiiiiiiiiiiiiiiiioooooooooooooooooooonnnnnnnnnnnnnnnnnnnnnnnnnnnnnn oooooooooooooooooooooofffffffffffffffffffffffffffff TTTTTTTTTTTTTTTTTTTTTTTTTrrrrrrrrrrrrrrrrrrrTTTTTTTTTTTTTTTTTTTT -rrrrrrrrrrrrrrrrrrrrppppppppppppppppppppppp 120 and Glypppppppppppppppppppp------------------------ 222222222222222222222200000000000000000000 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaannnnnnnnnnnnnnnnnnnnnnnddddddddddddddddddddddddddd GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGlllllllllll1111111111111111111111222222222222222222222211111111111111111111111111111 -lllllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyy 119 was selected as probable entry site and the yyyyyyyyyyyyyyyyyyyyyyyyy----------------------- 11111111111111111111111111119999999999999999999999999999 wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaasssssssssssssssssssssssssssssssss ssssssssssssssssssssssssssssssseeeeeeeeeeeeeeeeeeeeeeeeeeeeelllllllllllllllllllllllllleeeeeeeeeeeeeeeeeeeeeeeeeeeeeeccccccccccccccccccccccccccccccccccttttttttttttttttttttttttttttttttteeeeeeeeeeeeeeeeeeeeeeeeeeeeeddddddddddddddddddddddddddddddddd aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaasssssssssssssssssssssssssssssssss pppppppppppppppppppppppppppppprrrrrrrrrrrrrrrrrrrrrrrrrrrrrooooooooooooooooooooooobbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbaaaaaaaaaaaaaaaaaaaaaaaaaaaaaabbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbllllllllllllllllllllllllleeeeeeeeeeeeeeeeeeeeeeeeeeeeee eeeeeeeeeeeeeeeeeeeeeeeeeeeeennnnnnnnnnnnnnnnnnnnnnnnnnntttttttttttttttttttttttttttttttttrrrrrrrrrrrrrrrrrrrrrrrrrrrrryyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy ssssssssssssssssssssssssssssssssiiiiiiiiiiiiiiiiiiiiiiiiiiiiitttttttttttttttttttttttttttttttttttteeeeeeeeeeeeeeeeeeeeeeeeeeeeeee aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaannnnnnnnnnnnnnnnnnnnnnnnnnnnndddddddddddddddddddddddddddddddddd ttttttttttttttttttttttttttttttttthhhhhhhhhhhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeeeeeeeeeeeee 111111111111111111111111111111111111111111111111111111111111111111side where GlyTTTTTTTTTTTTTThhhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeeee rrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeggggggggggggggggggggggiiiiiiiiiiiiiiiiiooooooooooooonnnnnnnnnnnnnnnnnnnnn ooooooooooooooooofffffffffffffffff TTTTTTTTrrrrrrrTTTTTTssssssssssssssssssssssssssiiiiiiiiiiiiiddddddddddddddddddeeeeeeeeeeeeeeeeeeeeeeeeeeee wwwwwwwwwwwwwwwwhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeeeeeeeeeeerrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeeeeeeeeeeee GGGGGGGGGGGGGGGGGGGGGGGGGGGlllsssssssssssssssssssssssssssssiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiddddddddddddddddddddddddddddddeeeeeeeeeeeeeeeeeeee wwwwwwwwwwwwwwwwwwwwwwwwwwwwwhhhhhhhhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeerrrrrrrrrrrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeee GGGGGGGGGGGGGGGGGGGGGGGGGGGlllllllsssssssssssssssss -
f TTTTTTTTrrrrrrrlllllllllyyyyyyyyyyyyyyllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyy 201 is present as exit site.
222222222222200000000000000 aaaaaaaaaaaaaaaaaaaaaaaannnnnnnnnnnnnnddddddddddddddddddd GGGGGGGGGGGGGGlllllllll111111111111112222222222222211111111111111 llllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy 1111111111111111999999999999999 wwwwwwwwwwwwwwwwwwwwaaaaaaaaaaaaaaaaaaaassssssssssssssss ssssssssssssssseeeeeeeeeeeeeeeellllllllllllllllllleeeeeeeeeeeeeee111111111111111111111111111111111111pppppppppppppppppppppppppTTTTTTTTTTTTTTTTTTrrrrrrrrrrrrrrrrpppppppppppppppppppyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy-------------------- 000000000000000000111111111111111111111111 iiiiiiiiisssssssssssssssssssssssss pppppppppppppppppprrrrrrrrrrrreeeeeeeeeeeeeeeeeeeeeeeeeeeeessssssssssssssssssssssssseeeeeeeeeeeeeeeeeeeeeeeeeeeennnnnnnnnnnnnnnnnnttttttttttttttttttttttt aaaaaaaaaaaaaaaaaaaaaasssssssssssssssssssssssssss eeeeeeeeeeeeeeeeeeeeeeeeeeeeexxxxxxxxxxxxxxxxxxxiiiiiiiiiiiitttttttttttttttttttttt ssssssssssssssssssssssssssiiiiiiiiiiiitttttttttttttttttttttteeeeeeeeeeeeeeeeeeeeeeeeeeeee....................00000000001111111 iiiiiiiiiiiiiiiiiiiiiiiiiiissssssssssssssssssssssssssssss pppppppppppppppppppppppprrrrrrrrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeesssssssssssssssssssssssssseeeeeeeeeeeeeeeeeeeennnnnnnnnnnnnnnnnnn0000000001111111111111110000000000000000000011111111111111111111111111 nnnnnnnnnnnnnnnnnntttttttttttttttttttttttt aaaaaaaassssssssssssssssssssssssss eeeeeeeeeeeeeeeeeeeexxxxxxxxxxxxxxxxxxxxxxxxxxxxxiiiiiiiiiiiiiiiiiiiiiiiiiiittttttttttttttttttttttttttttttttt ssssssssssssssssssssssssssiiiiiiiiiiiiiiiiiiiiiiiiittttttttttttttttttttttttttttttttteeeeeeeeeeeeeeeeeeettttttt aaaaaaaaaaassssssssssssssssssttttttttttttttttttttttttttt aaaaaaaaaaaaaaaaaaaaaaaaaaaaa22222222222222222220000000000000022222222222222222222222200000000000000002222222222222222222222222222222222222
Both the ‘Gly’ residues were mutated to corresponding ‘Trp’sssssssssssssiiiiiiiiiiiiiiiiiiiddddddddddddddddddddddeeeeeeeeeeeeeeeee wwwwwwwwwwwwwwwwwwwwhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeerrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeee GGGGGGGGGGGGGGGllllllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy 0000000000001111111111111111111 iiiiiiiiiiiiiiiissssssssssssssss pppppppppppppppppppppprrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeessssssssssssssseeeeeeeeeeeeeeeennnnnnnnnnnnnnnnnnnnnttttttttttttttttt aaaaaaaaaaaaaaaaaasssssssssssssssss eeeeeeeeeeeeeeeeeexxxxxxxxxxxxxxxxxxxxxxxxxiiiiiiiiiiiiiittttttttttttttttttt ssssssssssssssssiiiiiiiiiiiiiiiitttttttttttttttttttteeeeeeeeeeeeeeee..................22222222222220222222222222BBBBBBBBBBBBBBBBBBBBBBBoooooooooooooooooooooottttttttttttttttttttttttttthhhhhhhhhhhhhhhhhh ttttttttttttttttttttttttthhhhhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee GGGGGGGGGGGGGGGGGGGGGGGGGGGGGllllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyyyyy rrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeeeeeeeeeeessssssssssssssssssssssssssssiiiiiiiiiiiiiiiiiiiiddddddddddddddddddddddddduuuuuuuuuuuuuuuuuuuuuuueeeeeeeeeeeeeeeeeeeeeeeeeeeeessssssssssssssssssssssssssssss wwwwwwwwwwwwwwwwwwwwwwwweeeeeeeeeeeeeeeeeeeeeeeeeeeerrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeeeeeee mmmmmmmmmmmmmmmmmmmmmmmmmuuuuuuuuuuuuuuuuuuuuuuuuttttttttttttttttttttttttttaaaaaaaaaaaaaaaaaaaaaaaaaattttttttttttttttttttttttteeeeeeeeeeeeeeeeeeeeeeeeeeeeeddddddddddddddddddddddd ttttttttttttttttttttttttttoooooooooooooooooooooo cccccccccccccccccccccccccccccooooooooooooooooooooorrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeeeeeeeeeessssssssssssssssssssssssssssssssspppppppppppppppppppppppppppppppppoooooooooooooooooooooonnnnnnnnnnnnnnnnnnnnnnnnnnnndddddddddddddddddddddddddddddddiiiiiiiiiiiiiiiiiiiiiiiinnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnngggggggggggggggggggggggggggg ‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrppppppppppppppppppppppppppppp’’’’’’’’’’’’’’’’’’’’’’’’’’’BBBBBBBBBBBBBBBBBBBBBoooooooooooooooooooottttttttttttttttttttttttttttttthhhhhhhhhhhhhhhhhhhhhhhhhhhhh tttttttttttttttttttttttttttttthhhhhhhhhhhhhhhhhhhhhhhhhhheeeeeeeeeeeeeeeeeeee ‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘GGGGGGGGGGGGGGGGGGGGGGGGGGGGllllllllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyyyyyy’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’ rrrrrrrreeeeeeeeeessssssssssssssssssssssiiiiiiiiiiiiiiiiiiiiiiiiiiiiiddddddddddddddddddddddddduuuuuuuuuuuuuuuuuuuuuuuuuueeeeeeeeeeeeeeeeeeeeesssssssssssssssssssssssss wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwweeeeeeeeeeeeeeeeeeerrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeee mmmmmmmmmmmmmmmmmmmmmmmmmmuuuuuuuuuuuuuuuuuuuuuuuuutttttttttttttttttttttttttttttttaaaaaaaaaaaaaaaaaaaaaaaaaaattttttttttttttttttttttttttttteeeeeeeeeeeeeeeeeddddddddddddddddddddddddddddddd tttttttttttttttttttttttttttttttooooooooooooooooooo cccccccccccccccccccccccccoooooooooooooooooooorrrrrrrrreeeeeeeeesssssssssssssssssssrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrreeeeeeeeeeeeeeeeeeeBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
Madhuprakash et al. 2014 BBA General Subjects
HPLC analysis
Fusion approach
PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin
CBP21 strongly promotes hydrolysis of crystalline -chitin by chitinases.
CBP+ChiD
ChiD+PKDPKD+ChiD
ChiD+CBP
CBP+ChiD+PKD (CDP) PKD+ChiD+CBP (PDC)
GH18CBP21 CBP21GH18
GH18PKD GH18 PKD
CBP21 GH18 PKD PKD CBP21GH18
Madhuprakash et al. PLoS One 2015
Structure of the substrate binding cleft
The polypeptide chain of SpChiD folds into two distinct partsbetween which the substrate binding cleft is formed. Lower N-terminal part starts from Ala18-Glu220 and the upper C-terminalpart starts from Met221-Glu420.A loop from Asn30-Asp42, intrudes into the substrate binding cleft.
Mutations based the crystal structure
Residue Targeted Mutated to
Tyrosine-28 Ala
Valine-35 Gly
Phe
Threonine-36 Gly
Phe
Glutamate-153 Ala
Deletion of loop Asn30-Asp42
Madhuprakash et al. Sci. Rep. 2015
24Madhuprakash et al. 2014 (Biores. Technol.)
HPTLC analysis of fractions collected ffrom chitosans DA61% and 35% with
SpChiD and W114A
90 and 30 min for degradation of chitosan DA 61% with SpChiD andW114A were selected, respectively.
90 min for degradation of chitosan DA 35% with both SpChiD andW114A was selected 25
DP2DP3DP4DP5DP6DP7DP8DP9
DP10DP11DP12
SpChiD with DA61%AD, A2
AD2, A2D, A3A2D2, A3D
A2D3, A3D2A2D4, A3D3, A4D2
A2D5, A3D4, A4D3, A5D2A3D5, A4D4, A5D3
A4D5, A5D4A4D6, A5D5
A5D6A2D10
W114A with DA61%AD, A2
AD2, A2DAD3, A2D2, A3D
A2D3, A3D2A2D4, A3D3, A4D2A2D5, A3D4, A4D3A2D6, A3D5, A4D4
A3D6, A4D5A4D6, A5D5
A5D6
DP2DP3DP4DP5DP6DP7DP8DP9
DP10
SpChiD with DA35%AD, A2
AD2, A2DD4, AD3, A2D2, A3D
A2D3, A3D2A2D4, A3D3,A2D5, A3D4A2D6, A3D5A2D7, A3D6
W114A with DA35%AD, A2
D3, AD2, A2DD4, AD3, A2D2, A3D, A4
AD4, A2D3, A3D2D6, AD5, A2D4, A3D3
AD6, A2D5, A3D4A2D6, A3D5A2D7, A3D6
A3D7
MALDI-TOF-MS analysis of crude
with DA61% W114
mixtures
26
Dose-dependent elicitation of oxidative bburst in rice cell suspension cultures
27
Chitosan crude mixtures producedby the mutant W114A were moreactive than SpChiD, though therewere subtle differences seen in theMALDI-TOF-MS analysis.
Probable reason is the mutantW114A is more active andtransformed much of the polymericfraction into oligomeric fraction.
HPTLC and SEC analysis of products oobtained from chitosan DA 61%
28
SpSpChiDSpSpChiDChiDCC W114AW114AW114A
100 mg of chitosan DA 61% substrate was used for degradation withSpChiD or W114A and purified the CHOS
FractionNumber
Type of oligosaccharides Degree of polymerizationSpChiD W114A
12 A4D7, A5D6, A6D5, A7D4 A3D8, A4D7, A5D6, A6D5 11
13 A4D6, A5D5, A6D4 A3D7, A4D6, A5D5, A6D4 10
14 A3D6, A4D5, A5D4, A6D3 A3D6, A4D5, A5D4 9
15 A3D5, A4D4, A5D3, A6D2 A3D5, A4D4, A5D3 8
16 A2D5, A3D4, A4D3, A5D2 A2D5, A3D4, A4D3 7
17 A2D4, A3D3, A4D2, A5D A2D4, A3D3, A4D2 6
MALDI-TOF-MS analysis of fractions collected through SEC
Fractions 12 to 17 generated by SpChiD and W114A, with chitosan DA61%, were analyzed through MALDI-TOF-MS
29
Dose-dependent elicitation of oxidative bburst in rice cell suspension cultures using
purified oligomeric fractions
30
Purified oligomeric fractions (DP8, DP9 & DP10) from SpChiD were more active thanfrom W114A. the DP of oligomers produced from both the enzymes is same– may be the pattern of acetylation (PA) is influencing elicitor activity - yet to beconfirmed through MS2 analysis.
CCHOS treatment
Cont. DP6 DP7 DP5 SA
Root dip treatment Foliar spray treatment
Oryza sativa cv. BPT 5204
Rice seedlings were grown in ½ MS media. Seedlings at 3rd leaf stage were used for treatments.Oligomers [DP5 (BBEPP), DP6 & DP7] and Salicylic acid (positive control) at 10 μg/mLconcentration were used for activity studies. For foliar application, 0.01% Tween-20 used assurfactant.
Foliar treatment induced elicitation response early and sustained till longer time31
1. Amino groups of chitosan are crucial for binding to a family 32 carbohydrate binding module of a chitosanase from Paenibacillus elgii, Journal of Biological Chemistry, 2016
2. Transglycosylation by a chitinase from Enterobacter cloacae subsp. cloacae generates longer chitin oligosaccharides, Scientific Reports, 2017
3. HarpinPss encapsulation in chitosan nanoparticles for improved bioavailability and disease resistance in tomato, Carbohydrate Polymers, 2018
4. Applicability of endochitinase of Flavobacterium johnsoniae with transglycosylation activity in generating long-chain chitooligosaccharides, Int J Biol Macromol. 2018
5. Key residues affecting transglycosylation activity in family 18 chitinases: Insights into donor and acceptor subsites, Biochemistry, 2018
6. Carboxy-terminal glycosyl hydrolase 18 domain of a carbohydrate active protein of Chitinophagapinensis is a non-processive exochitinase, Int J Biol Macromol. 2018
7. Structural and thermodynamic signatures of ligand binding to the enigmatic chitinase D of Serratia proteamaculans. J Phys Chem B. 2018
8. A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki. Int J Biol Macromol. 2019
9. A transglycosylating exochitinase from Chitiniphilus shinanonensis efficiently hydrolyzes chitin in a processive manner, Int J Biol Macromol, 2019
Prof. G. Lalita Prof. B. MoerschbacherProf. T.P. Singh
We gratefully acknowledge
Department of Biotechnology- Research Projects - TATA Innovation Fellowship
“Nano3Bio” FP7, EUROPEAN UNION
Department of Science and Technology- J C Bose Fellowship- SERB Grant