RESULTS

CHAPTER 4

RESULTS

4.1. Molecular phylogeny of chitinase and LTP genes

The cDNA clone Bar2chi screened from a library by Erysiphe graminis-

infected barley, (Hordeum vulgare L.) var Manchuria was 1.1 kb in size, the ORF

encodes a 318 amino acid polypeptide with a calculated molecular weight of 33.4 kDa

and iswlectric point of 8.54. Amino acid sequence alignment of barley chitinuse.

Bar2chi revealed homology with other plant chitinases (Fig. I). Barley chitinase

belongs to glycoside hydrolase family 19 chitinase domain and belongs to C~LFS I

chitinases having N-terminal cysteine-rich, chitin-binding domain which is separated

from the catalytic domain by a proline and glycine-rich hinge region. Phylogenetic

tree constructed on the basis of amino acid sequence alignment revealed the similarity

of barley chitinase with other plant chitinases. The predicted amino acid sequence of

barley chitinase BarZchi, (Hordeum vulgare, PI 1955) has 95.3% homology with rye

chitinase (Secale cereale, Q9FRV1), 79.8% with chitinase of Poa prafensis

(AAF04454), 78.5% with chitinase of Triricum aestivum (CAA53626), 74.3% with

chitinase of Musa paradisiaca (ABD47583). 71.2% with chitinase of Zea

diploperennis (AAT4@327), 68.3% with chitinase of Oryza sativa (AAL34318),

65.1% with chitinase of Nicotiana tabacum (P08252), 61.090 with chitinase of

Solanurn tuberosum (CAA32351) and 48.4% with chitinase of Hordeum vulgare

(CAA55344) (Fig. 2).

. I .. :. .,./

-1: % $ k.&; vk Er.* rn b,, eT ri- ........ 1.0.......1 I D . . .

Fig. 1 Amino acid sequence alignment of barley chitinase, Bar2chi (Hordeurn

vulgare P11955) with other plant chitinases. Secole cereule (Q9FRV1,

AAG53609). Triticum aestivum (CAA53626). Poa prurrnsi.~ (AAF044541, Oryzu

r(lriva (CAA60590, CAA82849, AAL34318). Musa purudisiaca (ABD47583) and

/ P O diploperennis (AAT40027). The conserved amino acid residues are highlighted

"lth asterisks. Conserved and semiconserved substitutions are represented by colons

dnd dots, respectively.

ABW2819 L i m n i u bicolor

AAWI895 Castawd Sativa

AM17593 G l p h

,

P36W7 P i s u s d t i v u

A M U ? W Dalcga oriantdlis

P11955 Hordeua wlgare (Bar2ohi)

AALY3lB 0r)od sa t iva

AAR18735 Blrbma o l d h a i l

Fig. 2 Phylogenetic tree of barley chitinase, Bor2chi (Hordeum vulgare, P11955) on the

basis o f amino acid sequence homology with other plant chitinases.

The cDNA clone Ltp 3FI screened from a library by Fusarium graminerrrum-

infected barley, (Triticum aestivrrn~ L.) var Sumai 3 was 348 hp in size. the ORF

encodes a 115 amino acid polypeptide with a calculated molcculnr weight of 11.1

kDa and isoelectric point of 9.35. Amino acid scquence align~nent of wheat lipid

transfer protein, Ltp 3F1 revealed homology with other monocot LTPs (Fig. 3). The

eight-cysteine motif (8 CM) appears to be a structural scaffold of conserved helical

regions connected by variable loops. The position of the eight cysteine residues are

conserved where the third and founh cysteines are consecutive in the polypeptide

chain and the fifth and sixth cysteines are separated by only one residue. Phylogenetic

tree constructed on the basis of amino acid sequence alignment revealed the similarity

of wheat LTP with other plant LTPs. The predicted amino acid sequence of Ltp 3F1

(Triticum aestivum, EF432573) has 80% homology with Hordrum vulgare

(CAA91436), 71.3% with LTP of Triticum aestivum (AAV28706). 64.2% with the

LTP of Oryza sativa (AAB18815), 62.7% with LTP of Sorghum hicolor

(CAA50660), 60.2% with LTP of Zea mays (AAB06443) and 50.4% with LTP of

Seraria italica (AAL.30846) (Fig. 4).

* : : : , . ,1* :: ~ ~ 8 0 6 6 0 S o m k r .- .... r U n O U I I L.

L t p 311 F r i t i c r . . W U $ 6 I D C & l .. MV78T06 T r i t i C l -- - m s o s r s Setuia lrWam$ m.....

ruler 1 ....... ~~........~~........IO.......A~........IO........M........~O........ 80

::*.*:* * *, ,.,..,* ****:*: .* * ,,,:: unsO660 s a m k r MB06141 L a

MVaBT06 Tri t icu M L l 0 8 4 6 S e t . L i a 2 A h - .

mler ........ 90 ....... 100.......110.......1a0......

Fig. 3 Amino acid sequence alignment of wheat lipid transfer protein, I f p 3FI

(Trificurn aestivum, EF432573) with other monocot LTPs. Sorghum hii.olor

ICAA50660); Zea muys (AAB06443): Oryzu surivo (AABl8815); Hordeurn vulgrrre

ICAA91436); Triticum arstivum (AAV28706); Seturiu irulico (AAL30846). The

conserved amino acid residues are highlighted with asterisks. Conserved and

srm~conserved substitutions are represented by colons and doh , respectively.

EF432573 Triticum aestivum Ltp3F1 97 CAA91436 k ~ d t u vulqare

I

AAC18557 Orpa sativa

AAL#)846 Se taria i t s l i c a

A M 1 8 8 1 5 Oryra sativa

AAMGSO Sorghun bicolor

CAME61 Sorghn bicolor

CAA4ZB32 a r d e a vulqare -- 1 m CAA15210 R i t i c u tatqim

Fig. 4 Phylogenetic tree of wheat lipid transfer protein, Llp 3F1 (Triticum

aestivum, EF432573) on the basis of amino acid sequence homology with other

4.2. Molecular modeling of chitinase and wheat lipid transfer protein

Sequence comparison revealed that the barley chitinase is homologous to :mother

barley chitinase whose 3D structure has been determined by X-ray diffraction d i ~ t i ~

(Song and Suh, 1996). Three-dimensional structure of barley chitinase reported in this

study was successfully stimulated by homology modeling tool, molecular operating

environment (MOE, version 2001.07) using barley seed chitinase as template. The

structure of barley chitinase sharcd 71% sequence identity with another barley

chitinase (LCNS). The validity of the model was tested using WHATCHECK and

PROCHECK (Laskowski et al., 1993). The predicted 3D structural model of barley

chitinase is presented in Fig. JA. The structure of wheat chitinase revealed the

presence of 13 a-helices, 5 P-strands and 28 loop turns and the presence of 6

conserved cysteine residues that are responsible for the formation of 3 disulphide

bridges (Cys98-CyslM), Cys172-Cys180 and Cys279-Cys311). The active site

residues Glu142 and Glu164 responsible for catalyzing the chitinase activity were

identified in the loop sequence connected to the p-strand Fig. 5B. Root mean square

deviation (RMSD) of query sequence structure and template (ICNS) was found to be

0.06797 A. The result of Rarnachandran plot for barley chitinase exhibited good

stereochemistry since it has just 0.5% residues in disallowed regions, 87.4% and

12.1% in the most favoured and additionally allowed regions respectively (Fig. SC).

Pip. 5 Predicted JD structure of barley chitinase BarZchi. ( A ) 3D \tructural n~odcl

( ' 1 the 3.5-kDa barley chit~nase wss propo\cd w ~ t h 13 11-hel~ce\. 5 [j-\trand., and 2X

1 'q~ turns. (B) The catalytic glutam~c a c ~ d revdue\ Glu142 and GIu164 re\pon\~hle

chrt~nase activity were identified in the loop sequence connected lo (3-\trand.

PROCHECK

Ramac handran Plot

Plu (depees) Plot St"tl i t lC5

Fig. 5 (C) Ramachandran plot of barley chitinase structure.

Sequence comparison revealed that the wheat lipid transfer protein is homologous to

mother maize ns-LTP whose 3D structure has been determined by 'H NMR

spectroscopy data (Gomar el al., 1996). Three-dimensional structure of wheat LTP

reported in this study was successfully stimulated by homology modeling tool,

molecular operating environment (MOE, version 2001.07) using maize ns-LTP as

template. The structure of wheat LTP shared 50% sequence identity with maize ns-

LTP (IAFH). The validity of the model was tested using WHATCHECK and

PROCHECK (Laskowski et a/., 1993). The predicted 3D structural model of wheat

LTP is presented in Fig. 6A. The structure of wheat LTP revealed the absence of P-

strands and presence of 6 a-helices, and 9 loop tums and the presence of 8 conserved

cysteine residues that are responsible for the formation of 4 disulphide bridges

(Cys29-77. Cys39-54, Cys55-97, Cys75-111). The active site residues Gly30. Pro5O.

Ala52 and Cys55 are responsible for catalyzing the reaction in lipid binding (Fig. 6B).

Root mean square deviation (RMSD) of query sequence structure and template

(IAFH) was found to be 0.932601 A. The result of Ramachandran plot for wheat LTP

exhibited good stereochemistry since it has just 3.9% residues in disallowed regions.

76.3% and 19.7% in the most favoured and additionally allowed regions respectively

(Fig. 6C).

Fig. 6 Predicted 3D structure of wheat lipid transfer protein 3F1. (A) 3D

\tructural model of the 9-kDa wheat LTP was proposed with 6 a-helices and 9 loop

'urns. (6) The active site catalytic residues GIy30, Pro50, Ah52 and Cys55 are

't~ponsible for catalyzing the reaction involved in lipid binding.

PROCHECI:

Ramachandran Plot

Fig. 6 (C) Ramachandran plot of wheat LTP structure.

4.3. Amplification of chitinase and lipid transfer protein genes

When the barley cDNA clone (Bur2chi) was used as template, the gene-

.pecific primers successfully amplified chitinase gene of 1.1 kh size (Fig. 7). The

nucleotide and amino acid sequence of harley chitinase gene is presented (Fig. R) .

When the wheat cDNA clone (L tp 3FI ) was used as template. gene-specific primer\

\uccessfully amplified wheal lipid transfer protern gene of' 348 hp size (Fig. 9). The

nucleotide and ammo acid sequence of wheat lipid transfer protein gene is presented

(Fig. 101.

Fig. '7 Amplification of DNA corresponding to the sequence encoding barley

chitinase. Agarose gel: Lane 1, I kb DNA ladder (Promega, Madison, WI USA);

Lane 2, Bur2chi gene ( I . 1 kb).

Fig. 9 Amplification of DNA corresponding to the sequence encoding wheat lipid

transfer protein. Agarme gel: Lane I . I kh DNA ladder (S~gma Chemica l \ Co . . St.

Louis, MO. USA); Lane 2, Ltp 3FI gene (348 hp).

(;AT GGC CCG TTC TCC TCT TGC TCA GGT CGT GCT CGT CGC ('GT (;GI' (;(;C T(;(' TAT GCT ('('T

1 R h \ I \ \ I \ \ \ \ \ \ \ I 1 . 1 .

TGC ACT CAC GCA GCC GGC TGT ATC CTG CGG TCA GGT GAG CTC TG(' CTT CA(; CCC CTG CAT

\ V T E 1 4 \ S ( ' ( ; Q \ r i 4 \ 1 , $ l ' ( ' l

CTC CTA TGC ACG CGG CAA CGG CGC CAG CCC ATC TGC GCC CTG CTO CAI; C(;(; (:(;T TAG GAG

\ ~ 4 H ( ; ; A \ P s . \ \ ( ( 4 ( . \ H 4

TCT AGT CAG CTC AGC CCG GAG CAC CGC TGA CAA CCA AGC GGC GTG CAA GTG CAT CAA (;A(;

I \ S 4 A H S 1 \ I1 K Q A \ ( ' h ( ' I K 4

CCC TGC TGC TGG GCT CAA CGC TGC CAA GGC CGC CGG CAT CCC CAC AAA GTG CCG CGT TAG

\ I 4 K A A G I P T h ( ( ; \ 4

CGT CCC TTA CGC CAT CAG CTC TTC GGT CGA CTG CTC TAA CAT TCC

\ P \ A I S S S \ D ( ' h h I H

Fig. 10 Nucleotide and amino acid sequence of wheat lipid transfer protein, Llp 3F1 gene.

4.4. Cloning and overexpression of chitinase gene in E. coli

Barley chitinase gene. Har2chi was suhcloned into pET 28a+ and

overexpressed in E. mli. The amplified chitinase gene (1.1 kh) hav~ng Ndt.1 and

BamH1 linkers was ligated into the expression vector PET 2Ra+ (Fig. I I). The

i,verexpression of the recombinant plasmid, PET-Barlchi wah achieved at 30 "C in

LB medium with kanamycin (SO pg/ml) and induced for 5 h with IPTG (0.25 mM).

SDS-PAGE analysia revealed the accumulation of 35 kDa chlt~nnse only in the IPTG-

~nduced culture. The uninduced control did not express ch~tinahe ( k g . 12 A).

Fig. 11 Construction of expression vector, PET-BarZchi for recombinant chitinase.

4.5. Purification of recombinant chitinase

The insoluble nature of the rccombint~nt protein synthesized in E. coli

facilitated its purification. A single centrifugation of the b;~cterial lys;~te was usc~l lo

isolate the inclusion bodies in which the recombinant protein was only slightly

contaminated. In order to eliminate these contan~inating bacterial proteins that co-

purify with the recombinant protein, inclusion bodies were washed. Addition of wrnh

buffer containing 10 m M Tris HCI pH 7.5. 300 mM NaCI. 1 mM EDTA, 1% Triton

X-100 and 1 M urea, was effective and yielded high purity of recombinant protein. In

order to solubilize the recombinant protein urea was used as denaturant. After

incubation of inclusion bodies with 8 M urea solution the chitinase protein was

recovered in the supernatant fraction. To keep the protein in monomeric form. P-

mercaptoethanol (2.5 mM) was added to the urea solution. SDS-PAGE analysis of

purified chitinase (Fig LO A) and Western blot analysis with an antiserum against

barley chitinase indicated the expression of recombinant chitinase in E. roli (Fig 12

9). The protein was further concentrated by lyophilization. The yield of purified

chitinase was 38 m g ~ l .

Fig. 12 (A) SDS-PAGE analysis of overexpressed chitinase. Lane I . Protein

rnillecular weight marker (Genei, Bangalore, India); Lane 2, total cell protcins from

i~ninduced E. c ~ l i BL-21 containing PET-Bar2chi: Lane 3, total cell proteins from

~nduced E, c , o l i BL-21 containing PET-Bar2chi; Lane 4, supernatant from

centrifugation of induced sonicated cells. Lane 5, pellet from centrifugation of

Induced sonicated cells. Lane 6, purified chitinase from inclusion bodies. (B) Western

blot of purified chitinase after incubation with barley chitinase antiserum. The arrow

Indicates the chitinase enzyme (35 kDa).

4.6. Cloning and overexpression of wheat Up gene in E. coli

The wheat Lrp 3FI gene (34.5 hp) having BcrnrHI and Hit~dl l l l ~ n k e n war

, p l e d into the expreshlon vector pMAL-p?x ( F I ~ 13). The o\,ert.xpn.ha!on of the

,-~ornhinant plasm~d pMAL-LTP wa!. achleved at 37 "C in LB rned~uni w ~ l h

,,n~p~cillin (100 pglml) and induced for 5 h ul th IPTG (0.3 mM1. SDS-PAGE

.,nalyr~s revealed the accumulation 01 51 kDa LTP-MBP ( 1 1 LDa LTP + 40 LDa

\IBP) f u \ ~ o n protein only in the IPTG-induced culturc. The unlnduced cc~nlml d ~ d not

:rpre\z the LTP-MBP fu\ion proteln (Fig. 11).

fig. 13 Construction of expression vector, pMA1.-I.TP for recombinant lipid transfer

protein.

Fig. 14 SDS-PAGE analysis of recombinant 1,TP fusion protein. Lanc M. I'rotein

111i1lecular weight marker gene^. Bangalore, India): Lane I . total ccll protein from

uninduced E. coli BL-21 (DE.7) containing pMAL-LTP. L.anc 2, total cell protein

from induced E. roli BL-21 (DE3) conta~ning pMAL-LTP: Lane 3, pellcl frcrm

ccntnfugation of induced son~cated cella; Lane 4, rupernatant I'rom centrifugauon of

Induced sonicated cells: Lane 5, purified LTP-MBP I'ur~on protein. The arrow

Indicates the MBP-LTP fusion protein (51 kDa).

4.7. Purification of recombinant protein

A cost-effective two-step purification prolocol \\.;IS uaerl to purify the

recombinant LTP in which, initial fr;lc~ion:ll precipit:~ti~,n uxing ammonium sulphate

frnclionation w ; ~ s followed by dialysis and gel pcrmc:~tir>n chrom:1togr:1phy. In or~lcr

to eliminate the contaminating bacterial proteins th:ll co-purify with the recombinant

protein, the supernatant after sonication kind centrifi~gati~ln was fraclloniited using

50% ammonium sulphate. The precipitate was dissolved in phosphate huffered saline

(PBS) and dialyzed using PBS huffer (pH 7.2). The dialyzed protein samples were

slze fractionated by Sephadex G-75 column using the same hul'fcr. The fract~ona

containing fusion protein was p i e d (Fig. 15). The protein was filnher concentrated

hy lyophilization. The yield of purified LTP fusion protein w u 20 tngl.

4.8. Determination of protein solubility and Factor Xa cieavnge n s ~ y

When Wilkinvon and Harrison model (1991) for theoretical calculation was

employed, wheat Lrp 3FI showed 82.7% chance of insolubility when overexpressed

in E. coli. Therefore, pMAL-p2x expression system wllr used to enhance the

solubility of LTP fusion protein. Purified fusion protein was subjected to Factor Xa

cleavage and the cleavage products were analyzed by SDS-PAGE. Complete cleavage

of the fusion protein was observed after 6 h digestion at 4 'C, with Factor Xa.

However, after complete digestion, no protein appeared with an eleclrophoretic

mobility corresponding to the 11 kDa LTP. This was due to a rapid proleolysis of

released protein probably caused by bacterial protease activity that co-purify with the

LTP fusion protein.

1 4 7 10 13 16 19 2225 2831 34 3740

Fraction number -

Fig. 15 Gel filtration of MBP-LTP fusion protein on Sephadex <;-75 column,

previously equilibrated with phosphate buffered saline (PBS) pH 7.2, eluted with

same buffer at a flow rate of 0.3 mumin. and measured by absnrbance at 280 nm.

The fractions containing fusion protein were pooled and concentrated.

4.9. Antifungal assays of purified chitinase

Antifungal assays were performed using major phytopathogenic fungi. The

purified chitinase showed a broad-spectrum antifungal activity at a concentration of

100 pg (0.42 U) md 300 pg (1.2 U) against phytopathogenic fungi such as Rorrytis

cinereo (blight of tobacco), Pestulotia rheue (leaf spot of tea). Bipoloris oryzue

(brown spot of rice), Alternuria sp. (grain discoloration of rice). Curvrrlario lunuru

(leaf spot of clover) and Rhizoctonia solani (sheath blight of rice) (Fig. 16). The SEM

image from the zone of inhibition plate of P. rhrar revealed the lysis and

fragmentation of fungal mycelium and inhibition of myceliill branching, whereus thr

control showed highly-branched and well-developed mycelium (Fig. 17). When rlic

~nycelium of B. oryzue from the periphery of the zone of inhibition produced by thc

purified chitinase was examined under a light microscope (~400) . the hyphi~r.

appeared to have clear cut mycelinl deformations such as lysis and fmgmentation.

whereas the mycelium from the control plate was normal and intact without any

distortion (Fig. 18). Similarly. the treated mycelia of B. cinererr appeiued to have

*uppression of spomlalion and deformations where as the contn~l hliowed hpxul;~~ion

and intact mycelia (Fig. 19).

4.10. Effect of purified chitinase on germination olsclerotia

Sclerotial germination &.says revealed the inhibition of germination of R.

solani sclerotia after 2 days of chitinwe (300 pg) treatmenl, while the control

sclerotia showed germination by 2 days. Even after 4 days of incubation the chitinau

treated sclerotia did not germinate (Fig. 20).

Fig. 16 Inhibitory activity of purified chitinase towards (1) Bolryds cinerea, (11)

Pestolotio theoe, (111) Bipolaris oryzae, (IV) ANernaria sp. ( A ) Control 3(X) pg af

protein extracts of induced E roli containing pET28 a+ without ch i t inue insert (B)

700 pg chitinase and (C) 100 pg chitinare.

Fig. 17 Scanning electron microscopy (SEM) ( A ) lniage ol Pc,.srol~rrtcr rlic~oc~

hhc~wing well developed and branching niycel~a from the ctlnlrnl (6 ) image ol'

deformed mycelia showing lysis and fregnientat~on with poor myccl~lrl develi~plncnt

due to chitinase treatment.

A B -- - - * @ " " .Y - - . + - .; -1 , f. iiLJ ;-4. 0 0+

ie; a b - - ',+$ -." - ---.- $$ lip? .*>=

;-j; : i " F

Fig. I 8 Light microscopic observation of control and treated fungal mycelin of

Bipoloris oryzae. (A) Control B. oryzae showed normal branched mycelium (B)

Chitinase-treated hyphae showed deformed mycelium with lysis and fragmentation.

Fig. 19 Light microscopic observation of control and treated fungal mycelia of

Hotrytis cinerea. ( A ) Control B. cinrr<uz rhowcd normal well developed ntycel~urn

u.~th spomlation. (9) Chitinahe-treated hyphae showed deforniot~on In thc rnycrlial

fragment\ without sporulation.

Fig. 20 EtTect of purified chitinase on germination of sclerotia of Rhizoclonia

rohni. (A) Control sclerotia germinated in 48 h (B) Chitina\e-treated sclerotia did not

eerminate even after 4 days.

4.11. Antifungal assays of purified LTP

The purified LTP fusion protein showed a broad-spectrum antifung:~l :~ctivity

at a concentration of 100 and 300 pg against illrrrri~rrirt sp. (grain ~liscolor:~tion of

rice), R. soloni (sheath blight of rice) (Fig. 21). C I~ttioro (Icaf spot of clover). 0.

oryzoe (brown spot o f rice). Cylindrocludittrn scopnriurii (root necrosis of h;lnana) ;~nd

Sorocludium oryzue (sheath rot of rice). When the mycelium of C. irrr~~tru m d

Airemuria sp. from the periphery of the zone of inhibition produced by purified LTP

fusion protein was examined under light microscope ( ~ 4 0 0 ) . the hyphne appeared to

have clear cut mycelial deformations such as poorly developed mycelium with

swollen margin, whereas the mycelium from the control plate was normill, branched.

well-developed and intact (Fig. 22 A and B).

4.12. Effect of LTP on germination of spores

Spore germination assays revealed the fungistatic effects of LTP against

phytopathogenic fungi such as M. griseu, M. phuseolinu. Fusurium sp., A. i.inereu, S.

uryzue and B. oryzae. Fungicidal activity was observed against Alrernuriu sp. and C.

lunatu. The control spores showed germination but LTP fusion protein treated spores

did not show germination (Fig. 22 C and D).

Fig. 21 Inhibitory activity of LTP fusion protein towards Rhizocfonia solani. (A1

Control containing 300 pg maltose hinding pro t r~n; ( B l 300 pg of LTP fus~on protein

and (C) 100 pg of LTP fusion proteln.

CONTROL TREATED CONTROL TREATED

-. C D -

CONTROL TREATED TREATED - Fig. 22 Effect of LTP fusion protein on mycelial growth and sporulation. Light

micro\copic oh\ervation of control and treated fungal mycelia nf ( A ) C'rrr~~rrluri~r

lro~uta and (B) Alternoria sp. Control hhowed normal well developed rnycelia wtth

\porulation and treated mycelia \bowed por~rly devcl(~ped mycclium wirh hwollen

niugin without sporulation. Effecl of LTP fusion prote~n on germination of fungal

\pores. Light microscopic observation of control and treated spores of IC) C'urvulurio

lrrr~uru and ( D ) AIternoriu sp. Control spores showed gerrnination and treated \pores

d ~ d not show germination.

4.13. Cloning and expression of cyanamide hydratase (cah) marker gene

4.13.1. Site-directed rnutagenesis in calr gene

Sequencing data of mutated crrh indict~ted the single hilse suhst~tution, G to A

ot nucleotide position 81 or T to C at nucleotide position 81 of ccrl~ gene (Fig. 23). The

one base substitution does not change [he amino acid it encodes since both AAG

(wild) and AAA (cah MutK mutant) encode lysine and the codons. CTT (wild) and

CTC (cuh MutL mutant) encode leucine.

4.13.2. Cloning and overexpression of wild and mutant type cah genes in E. coli

Recombinant plasmids, pQ-cah (wild-type), pQ-rahMutK and pQ-i.ohMutL

(mutant- type) were constructed through cloning of amplified mutated rah genes into

pQE-60 (Fig. 24). IPTG induction of E. coli carrying wild-type pQ-ruh as well as

mutants, pQ-cahMutK and pQ-cahMutL showed overexpression of rah as his-tag

fusion protein.

4.13.3. Purification of enzyme and cyanamide hydratase assay

Cell-free crude enzyme preparations were purified using ~ i ' ' NTA agarow

column. Crude enzyme preparations of pQ-cah, pQ-cahMutK and pQ-cuhMutL

showed Cah enzyme activity of 1090, 1060 and 1000 Ulmg protein respectively.

Purified enzyme exhibited 5-fold higher activity than the crude enzyme preparation

(pQ-cah. 5200; pU-cahMutK, 5200 and pQ-cahMutL 5100 Ulrng protein).

I \TG TCT TCT TCA CAA GTC AAA GCC AAC GCA TGG ACT GCC GTT CCA GTC ACC (;CA AAG CCC

( \ I S S S E l 4 \ T 4 P \ 4 4 h \

61 4TT GTT GAC TCC CTC GGA GGT GAT GTC TCC TCA TAT T(T CTG GAA GAT ATC (;CG

! , I \ D S I . ( ; b L ( ; U \ S h \ S \ F I ) I h

121 TTC CCT GCG GCA GAC AAA CTT GTT GCC GAG CCA CAG GCC lTT GTG AAC GCC CCA TTG ACT

J I I ' E T H I F I \ I 4 W K

181 CCC GAA ACC TAC AAT CAC TCC ATC CGC GTT TTC TAT TGC C(;A ACT (;TC ATC (;CG ACA CCT

, 1 1 1 I. P A 4 l ) h : I . \ \ t : \ Q \ F \ h \ R I .

241 I'TA CTT CCC GAG CAA GCT AAA GAT TTC TCT CCA ACT ACA TGC GCA CTC ACA TGT CTT CTC

n l \ I. P E Q \ h I l 1 . h P 4 I \ \ \ I 'I ( ' I , l

1111 (.4T CAT GTT CGT ACT GCC GAG GTA T.4C TTT ACA TCT A('A C(;A AT(; TCC TT(' (;AT ATT TAC

1 ,1111 1) \ C T h t 4 \ t ' l S I U \I 5 k 1) I \

M I I;(;T GCC ATTAAG CCT ATG GAG CTC; CTC AAC GTC CTT (;(;(; ACT A(;C A('C(;Ac'CA(; (;('T (;A(;

I I h a \I t \ I h \ I (; r 4 'I I ) ~ J \ t

121 (;CT GTT GCC GAG CCC ATC ATT CCT CAT CA(; CAT (:TC CC(; (;TA (;AT (;(;(' AA(' AT('A('A TTl'

I \ 4 I \ I I H I 1 b 1 ) 1 1 ; \ I > ( , \ I ' I t

481 ("I C GCT CAC TTG ATC CAC CTC GCT ACC CTT TAT (;AC AAT (:TC (;C(; <;C<'TA(' (;AT G(;(; AI'T

I I I 0 I 1 'r I I ) \ I ( ; \ \ I ) ( , I

541 ( A T CAT TTT GGT AGC TCG GTT CAT CAC ACC AC4 C(;('AA(' ACT ATC AA(' A('(; (;('A TT(' C('A

h l I F ( 5 M \ I ) I ) 'I I U \ \ I \ I \ t 1'

M l l ('(;A CAT GCT TGG TGT TCT TGC TTT CCC TG(' AC(; (;TT CCT AA(; (;AA CAA A(iT AA(' AAG ('('I'

2111 K I I <; M ( ' L H t \ (' 'I \ W h I, 1, 4 \ h I'

lhl T(;G TGC CAC ACA AC(; CAT ATC CCT CAC TTC CAT AAA CA(; AT(; (;AA <;('<; AA(' ACT TTG AT(;

I \ ( I 1 T H I P 0 t II h 0 \ I t \ I I hl

721 \4<; CCT TGG GAG

! I l h P M t

Fig. 23 Nucleotide and amino acid sequence of cyanamide hydratase (coh) gene.

Ihder l ined sequence indicates the H b ~ d l l l site. In order to eliminate the Hindl l l 11e .

In cah Mut K mutant [he single base substitution, G Lo A and in cah Mul L mutant [he

w g l e base substitution, T to C wa5 generated without the change of a m m o acld

q u e n c e .

Col El L-j' Col El k--/--' Fig. 24 Construction of recombinant plasmids. ( A ) pQ-cah (wild type).

1 B) pQ-cahMutK and ( C ) pQ-cahMutL for overexpression of rub in E.ccheric.hiu rol i .

4.13.4. SDS-PAGE and Western blot analyses

SDS-PAGE and Western blot analyses of the cell extracts of E coli JM109

with pQ-cuh (wild-type), pQ-rrrlzMutK i~nd pQ-c<rllMutL (inutants) grown only in thc

presence of IPTG ( I mM) showed the expression of Cah cnzyme (Fig 25) . Tlie

purified enzyme showed 27.7 kDa Cah enzyme (Fig. 26 A) :tnd the Western hlol

analysis of the purified enzyme prepatations with urh antihody (1:1000 dilution)

indicated the presence of 27.7 kDa Cah proteins (Fig 26 B).

Fig. 25 SDS-PAGE analysis of cyanamide hydratase enzyme. Lane I . Protein

molecular weight marker (Genei. Bangalore, India); Lane 2, total ccll protclns from

uninduced E. roli containing pQ-cub; Lanea 3-5, total ccll proteins from induced E.

i oli containing pQ-cab, pQ-cuhMutK and pQ-cuhMutL. The arrow indicates the Cah

enzyme (27.7 kDa).

Fig. 26 SDS-PAGE and Western blol analysis of purified Cnh. ( A ) Lanes 1-3.

purified cah protein from overexpressed ~Q-L.(I/I, pQ-c,uhMutK and pQ-c,ulrMutL. (Bl

Western blot after incubation with the cah antiserum. Lanes 1-3, purified protein of

pQ-cuh, pQ-cahMutK and pQ-cahMutL. The arrow ~ndicates the Cah enzyme (27.7

kDa).

4.14. Construction of gene cassettes

1.14.1. Construction of gene cassette with cah as nrarkcr gene

The mutated cuh gene devoid of Hindlll site was successfully cloncd along

with Ubil promoter and nos terminator into pCAMBIA 1300 veclor and the

recombinant plasmid. pCAMBIA-calf was constructed (Fig. 27 A).

1.14.2. Construction of gene cassettes with cah marker, Chi and Idp genes

The recombinant plasmid, pCAMBIA-cuh-Lrp 3FI (Fig. 27 B) was

successfully constructed by three fragment ligation of the vector (pCAMBIA-cuh)

digested with HindIII. Ubil promoter (Hindlll-BarnHI fragment) and Ltp 3F1 gene

(BamH1-Hind111 fragment). Similarly, pCAMBIA-cah-Bur2chi (Fig. 27 C) was also

constructed by three fragment ligation of vector @CAMBIA-cuh) digested with

HbldIIl, Ubil promoter (HindIII-BamH1 fragment) and chitinase gene (BurnH1-

HbldIII fragment) (Fig. 28). The ligated plasmids (pCAMBIA-cuh-Ltp 3FI and

pCAMBIA-cah-BarZchi) were transformed and maintained in E. coli JM109 cells.

Fig. 27 Genetic map of gene cassettes. (A) pCAMBIA-wh. (B) pCAMB1A-cuh-Ltp

3F1 and (C) pCAMB1A-cah-Bur2chi.

Hindlll Barn HI Barn HI Hind ill EcoRl Hind IN 5' 3' 5 ' 3' 5' 3'

Ubiprornoter Gene (Ltp 3F 1 /BwZcM) Cah+mir

I Ligation I Hind Ill Barn HI Barn HI Hindltl

5' 3' 5' 3'

J Hind Ill I ClAP Hind Ill Barn HI

Three-Fragment Ligation + Hlnd Ill

H i n d IL:, EcoRl

1 EcoRl pcmnein -w

PCAMBIA -cahL@ 3F 1 / Bw2chi

Fig. 28 Construction of gene cassettes for plant transformation. Three-fragment

ligation was performed using pCAMBlA 1300 to construct the gene cassettes.

pCAMBIA-cah-Ltp 3FI and pCAMBI.4-cah-BarZchi.

4.15. Transformation of gene cassettes harboring cah, Chi and Ltp genes in

tobacco

4.15.1. Development of tobacco plnntlets

Under sterile conditions. tobacco plnntlets were regenerated using 0.1% FlgC12

surf~e-sterilized seed on MS medium. The aseptically grown plants were used to

prepare leaf discs for transformation.

4.15.2. Agrobacferium-medint transformation of gene cassettes in tobncro

Freeze-thaw method facilitated the transformation of gene cassettes (pCAMBIA-coh.

pCAMB1A-cah-BarZchi and pCAMBI.4-rah-Llp 3F1) into A#robacrerirrrn LBA4404

and tobacco leaf-disc method was found to be efficient for gene transfer.

Untransformed leaf disc when cultured on tobacco shoot selection media with

cyanamide failed to regenerate and revealed 5 mM cyanamide as LDso and 10 mM as

LD,,,. Of 150 co-cultivated leaf discs, 77 leaf discs produced shoots after 2 weeks on

MS medium amended with 5 mM cyanamide. Shoots were then transferred to 10 mM

cyanamide to further eliminate the escapes if any. Hence, about 6.5% - 10.6%

regeneration was achieved after selection using 10 mM cyanamide and about 46.6% -

66.6% were identified as escapes. Transformants efficiently formed root system on

rooting medium containing 10 mM cyanamide within 2 weeks (Fig. 29). All plants

showed normal phenotype with no indication of cytotoxicity due to the expression of

transgenes in To and T I generation. Six independent lines of coh expressing plants. 5

lines of Chi expressing plants and 8 lines of Ltp expressing plants were selected and

TI transgenic plants for each line were analyzed further for gene integration and

expression.

Fig. 29 Transformation of pCAMBIA-cah into tobacco. Selection and

regeneration of transformants. (A) Shooting of transformed leaf-disc on shoot

\election medium amended with 5 mM cyanamide. (B) Regenerated shoot transferred

on rooting medium containing 10 mM cyanamide. (C) Transgenic tobacco plant with

normal phenotype.

4.153. Cyanamide tolerance leaf dip assay

When thirty day old transformants were bubjected to cyanamide leaf dip assay

dong with the non-transformed control plants by applying 5% cyilnwlide solulion as

. d a c e spray. Transformed plants alone retained thetr healthy grren appearance.

uhere as leaves of susceptible untransformed control plant\ scorched within 72 h of

cyanamide treatment. The treated plant\ grew to maturity and set heeds U I ~ ~ O U I any

~pparent side effects due to cyanamide treatment. T h e e results ~ n d ~ c i ~ t e d the fact that

cyanamide can be employed as a solution or in granulc l'nrrn f'or effcct~vc mean* of

~drntifying transgenic plants from the non-transgenic plants In a populat~on of

\egregating progeny plants (Fig. 30).

Fig. 30 Cyanamide tolerance assay. Cyanamide tolerance depicted after foliar

application of 5% cyanamide. (A) Untransformed control and (8) transformed plant

after treatment.

4.15.4. Cyanamide hydralase assay

Cyanamide hydrates assays confirmed the presence of enzyme activity in

transgenic plants showed reduced color inlcnsity and reduction in absorption at 530

nm ranged from 0.348 to 0.414 O.D. over unlrnnsfornied ct~ntrol (Tnhle 6).

Table 6 Cyanamide hydratase enzyme activity of transgenic lobacco plants.

Reduction in absorbance a t 530 nm in colorimetric assays containing 0.5 mM

cyanamide salt with leaf extracts from tobacco plants.

Experiments with extracts (0.5 mM cyanamide + 100 rng tissue)

Reduction in O.D. (530 nm) over control due to enzyme activity

Blank

Control

Untransformed plant

Cah expressing transgenic plants

Plant 1

Plant 2

Plant 3

Plant 4

Plant 5

Plant 6

4.155. Molecular analyses of transgenic plants

4.15.5.1. PCR, Southern and Western blot analyses of call tronsfor~nonts

When the genomic DNA of cyanamide tolerant tr:~nsforni:mts was uhcd :IS

template, gene-specific primers amplified 732 hp of coh gene in the 6 transfor~~i;~nts

and positive control (pCAMBIA-cah). Conversely, no such hand was observed in the

untransformed control under identical conditions (Fig 3 IA).

Southern blot analysis was carried out on a11 the tronsfur~uants using cah

coding sequences as probes. Two transformants (plant I and 3) displayed one

hybridizable band and 4 transformants (plant 2,4 . 5 and 6) exhibited two hyhridizable

bands for rah probe from the total DNA of transformants indicating the slahle

integration of marker gene rah in transgenic tobacco plants (Fig. 31B).

Transgenic tobacco plants were studied for the expression of cyanamide

hydratase. Cyanamide hydratase antisemm detected expression of 27.7 kDa

polypeptide in all 6 transformants, whereas such expression was absent in the

unuansfonned control plant (Fig. 3 1C).

Fig. 31 PCR, Southern blot and Western blot analyses of transgenic tobacco

plants containing pCAMBIA-cah. (A) Detection of' r .uh gene using gene-specific

prlrners (732 hp). Lane M, I kh DNA ladder; Lane 1-6, transformants showing

amplification of the desired sized gene fragments. Lane 7. untransfc>rnied control

(negative control) and Lane 8, plasmid pCAMBiA-cull (pos~tive control). (B)

Southern blot analysis of cuh gene in transfbrrnants. Lane C , positive control.

pCAMBIA-cuh; Lanes 1-6, transgenic plants. 1-6. Plant I (lane I ) and 3 (lane 3)

\bowing one hybridizable band. Other plants 2. 4-6 (lanes 2, 4-6) showing two

hyhridizable bands for cah gene. Lane 7. Untransformed control plant. (C) Western

blot showed expression of cyanamide hydratase 27.7 kDa polypeptide in all the 6

rransformants, whereas such expression was absent in the untransformed control

plant.

4.1552. PCR, Southern and Western blot analyses of Chi transformants

When the genomic DNA of transformants was used :is template, gene-s(wcilic

primers amplified 1.1 kb of chitinase gene in the 5 transfornlants and positi\rc control

(pCAMBIA-cah-Bor2chi). Conversely, no such h:ind was observed in the

untransformed control under identical conditions (Fig 32 A).

Southern blot analysis was carried out on all the transformants using Chi

coding sequences as probes. Three transform~nts (plant 1. 3 and 5 ) displayed one

hybridizable band and 2 transformants (plant 2 and 4) exhibited two hyhridiz;~hle

hands for Chi probe from the total DNA of transformants indicating the stable

integration of marker gene Chi in transgenic tobacco plants (Fig. 328).

Transgenic tobacco plants were studied for the expression of chitinace. Barley

chitinase antiserum detected expression of 35 kDa polypeptide in all the 5

transformants, whereas such expression was absent in the untransfonned control plant

(Fig. 32C).

Fig. 32 PCR analysis of transgenic tobncco plants containing pCAMRIA-cah-

BarZchi. (A) Detection of Chi gene using gene-\peciSic primers (1.1 kh). Lane M. I

kh DNA ladder; Lane 1-5, transformants showing amplificauon c11' the de\ired si/.ed

gene fragments. Lane 6, untransformed control (negative control) and Lane 7. plasmid

pCAMB1A-cah-Bar2chi (positive control). (B) Southern blot analysis of Chi in

transformants. Lane C. positive control, pCAMBIA-r.uh-Lrp 3FI, Lanes 1-5.

transgenic plants. Plant I (lane 1). plant 3 (lane 3) and plant 5 (lane 5) showing one

hybridizable band and other transformants, plant 2 and 4 (lanes 2 and 4) showing two

hybridizable bands for Chi gene. Lane 6, untransformed control plant. ( C ) Western

blot showed expression of chjtinase 35 kDa polypeptide In all the 5 transformants,

whereas such expression was absent in the untransformed control plant.

4.15.5.3. PCR, Southern and Western blot analyses of U p transformants

When the genomic DNA of tnnsformants was used as template, gcne-specific

primers amplified 348 bp of Ltp gene in the 8 trmsforrn:lnls and positive control

(pCAMBIA-coh-Ltp), Conversely, no such hand was observed in the unt~lnsfornmed

control under identical conditions (Fig 33A).

Southern blot analysis was carried out on all the transformants using Ltp

coding sequences as probes. Two transformants (plant 2 and 4) displayed one

hybridizable band and 6 transformants (plant 1. 3. 5-8) exhibited two hybridirtble

bands for cah probe from the total DNA of transformants indicating the stable

integration of marker gene cah in transgenic tobacco plants (Fig. 338).

Transgenic tobacco plants were studied for the expression of cyanamide

hydratase. Wheat LTP antiserum detected expression of 9 kDa polypeptide in all the 8

transformants, whereas such expression was absent in the untransformed control plant

(Fig. 33C).

Fig. 33 PCR analysis of transgenic tobacco plants containing pCAMBIA-cah-Up

3F1. (A) Detection of Lrp gene using gene-rpec~iic primers (348 hp). Lane M, I kh

DNA ladder; Lane 1-8, transformants showing amplification of the desired s~zed gene

fragment$. Lane 9, untransfbrmed control (negative control) and Lane 10, plasmid

pCAMBIA-cuh-Lrp 3F1 (positive control). (B) Southern blot analysis of 1.11, in

transformants. Lane C, positive control, pCAMB1A-cuh-Lfp 3FI. Lanes 1-8,

transgenic plants, plant 2 (lane 2) and plant 4 (lane 4) showing one hybridizahle band

and other 6 transformants, plant 1, 3. 5-8 (lanes I . 3 . 5-8) showing two hybr~dizable

hands for Lrp gene. Lane 9, untransformed control plant (C) Western blot showed

expression of wheat LTP 9 kDa polypeptide in all the 8 transformants. where& such

expression was absent in the untransformed control plant.

4.1555. Transgenic resistance of tobacco leaves

To study the response of Chi or Lrp-expressing tobacco plants lo fung:~l

infection, detached leaf assays were performed. The results of the fung;~l infec~ion

assay using leaves from the control (non-transgenic) and transgenic plant with single

copy of the gene revealed the expression of significant resistance in chitinase or LTP

expressing tobacco plants to fungi (Fig 34).

When observations were made 5-10 days afier inoculation of fungi, Bipolcrris

oryzrre, Cylindrocladium scopnrium and Alfernaria sp. the leaves from both non-

transgenic control plants and pCAMB1A-cah transgenic plants showed similar level

of severe infectibn (diameter of lesion 2-3 cm) with more yellowing around the

inoculated mycelial ,plug. The leaves from chitinase or lipid transfer protein

expressing transgenic plants showed restricted infection (diameter of lesion 0.8-1.2

cm) around the site of contact with the fungus contuning plug (Fig. 34. Table 7).

Fig. 34 Transgenic resistance of tobacco leaves expressing chitinme (Chi) or lipid

transfer protein (Up). (I) Detached leaf from control tobacco plant showing

spreading lesions due to ( A ) Bipolaris o r y u e . ( B ) Cylindrorladium scoparium and

(C) Allernaria sp. infection (11) Detached leaf from transgenic tobacco plant showing

limited infection and smaller lesion. Observation was made 10 days after inoculalion.

Table 7 Resistance of transgenic tobacco expressing chitinase (Chi) or lipid

transfer protein (Up)

Tobacco plant Fungus Di;tn,stcr o f lesion (cm) (* S.E.)

Control Bipoluris oryzae 3.0 0.2

Transgenic (Chi) B. oryzae 1.2 * 0 . 2

Control Cylindrocludium scoporium 2.5 i 0.3

Transgenic (Llp) Cy. scopurium 1.3 k 0 . 3

Control Alrernuri(~ sp. 2.0 +. 0.2

Transgenic (Ltp) Alrernariu sp. 0.8 i 0.2

Data represents the average of three replications.