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