Increased resistance to fusarium wilt in transgenic tobacco lines co-expressing chitinase and wasabi...

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Plant Pathology (2010) Doi: 10.1111/j.1365-3059.2010.02352.x

Increased resistance to fusarium wilt in transgenictobacco lines co-expressing chitinase and wasabidefensin genes

V. O. Ntui, P. Azadi, G. Thirukkumaran, R. S. Khan, D. P. Chin, I. Nakamura

and M. Mii*

Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo,

Chiba 271-8510, Japan

Marker-free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces

griseus strain HUT 6037 were produced by Agrobacterium-mediated transformation. One marker-free transgenic line,

TC-1, was retransformed with the wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed

shoots, 37% co-expressed the ChiC ⁄WD genes, as confirmed by western and northern analyses. Southern blot analysis

showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic

lines either expressing ChiC or WD, or co-expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae

(Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co-expressing both genes had

significantly enhanced protection against the fungus (infection indices 0Æ0–1.Æ2) compared with corresponding isogenic lines

expressing either of the genes (infection indices 2Æ5–9Æ8). Whole-plant infection indices in transgenic lines were significantly

related (r = 0Æ93, P < 0Æ01) to the extent of root colonization of the host, which ranged from 2Æ1% to 11Æ3% in lines

co-expressing both genes, and from 16Æ8% to 37Æ7% in lines expressing just one of the genes (compared with 86Æ4% in

non-transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal

abnormalities.

Keywords: Fusarium oxysporum f.sp. nicotianae, gene-stacking, marker-free, MAT vector, Nicotiana tabacum,

retransformation

Introduction

Chitinases are glycosyl hydrolases catalyzing the degra-dation of chitin, an insoluble linear b-1,4-linked polymerof N-acetylglucosamine. They are produced by a widerange of organisms, and have been shown to play impor-tant physiological and ecological roles. Plant chitinasesact as a defence against chitin-containing fungal patho-gens by inhibiting spore germination and germ-tube elon-gation and degrading hyphal tips (Collinge et al., 1993;Graham & Sticklen, 1993). Transgenic plants harbour-ing chitinase (ChiC) genes have been produced in severalspecies. In carrot, the tobacco class I ChiC gene conferredresistance to Botrytis cinerea (Punja & Raharjo, 1996).Transgenic tobacco transformed with bean class I ChiCexhibited enhanced resistance to Rhizoctonia solani(Broglie et al., 1991), whereas transgenic potato express-ing the chitinase (ChiC) gene conferred enhanced resis-tance to Alternaria solani (Khan et al., 2008).

*E-mail: miim@faculty.chiba-u.jp

ª 2010 The Authors

Plant Pathology ª 2010 BSPP

The Japanese horseradish, wasabi (Wasabia japon-ica), which has a protective role against microorganismsand is used as a food ingredient, is considered to containsome antimicrobial substances. Secondary metabolitesof wasabi, such as wasalexin and 6-methylsulfonylhexylisothiocyanate, were reported to have antifungal andantibacterial activity, respectively (Ono et al., 1998;Pedras et al., 1999). An antimicrobial protein-encodinggene (WT1) was isolated from wasabi (Saitoh et al.,2001) and named the wasabi defensin (WD) gene byKanzaki et al. (2002). The antimicrobial protein codedfor by WT1 showed a strong expression and inhibitoryeffect on fungal and bacterial growth in transgenicNicotiana benthamiana (Saitoh et al., 2001). Further-more, Kanzaki et al. (2002) reported that growth of riceblast fungus was inhibited in transgenic rice over-expressing the wasabi defensin gene, whilst Khan et al.(2006a) reported partial resistance to grey mold (B. cine-rea) in transgenic potatoes expressing the wasabi defen-sin peptide. Therefore, expression of the chitinase andwasabi defensin genes in a single plant speciescould greatly increase the level of resistance to fungalpathogens.

2 V. O. Ntui et al.

Fusarium wilt, caused by the fungus Fusarium oxyspo-rum f.sp. nicotianae (Fon) is one of the most persistentand important diseases of tobacco (Lamondia, 2001). Itcauses yellowing of foliage which precedes wilting. How-ever, sometimes a sudden wilt occurs without any yellow-ing of foliage. The primary means of managing fusariumwilt is through the use of resistant cultivars. Therefore,generating resistant cultivars will significantly combatFon infection and increase tobacco production.

The objectives of this study were to (i) produce marker-free transgenic tobacco plants containing the chitinasegene via the MAT vector method, (ii) retransform themarker-free tobacco plants with the wasabi defensingene, and (iii) evaluate the level of resistance of transgeniclines expressing either of the antimicrobial genes (ChiCor WD) and the retransformed transgenic tobacco plantsco-expressing ChiC ⁄ WD after inoculation with Fon as amodel. Although retransformation has been achievedin tobacco, this is the first report using the MAT vector toretransform tobacco with two antifungal genes.

Materials and methods

Plant material, transformation vector andAgrobacterium strain

Leaves from in vitro-grown tobacco (Nicotiana tabacumcv. Havana) were used in this study. For production ofmarker-free transgenic plants expressing the ChiC gene,Agrobacterium tumefaciens strain EHA105 harbouring aMAT vector, pMAT21, containing ChiC (isolated fromStreptomyces griseus strain HUT 6037, Ohno et al.,1996) and the removable cassette in the T-DNA region,was used. Escherichia coli strain DH5-a containing theMAT vector plasmid was kindly provided by NipponPaper Industries, and the plasmid was transferred intoA. tumefaciens by tri-parental mating (Ditta et al., 1980).For retransformation and production of transgenic plantsexpressing the wasabi defensin gene alone, A. tumefaciensstrain EHA101, which harbours the binary vector,pEKH1-WD containing the chimeric defensin gene(approximately 0Æ5 kb), isolated from W. japonica (Sai-toh et al., 2001), and kanamycin and hygromycin resis-tance genes, was used.

Production of transgenic plants expressing eitherchitinase or wasabi defensin gene

A single colony of A. tumefaciens EHA105 harbouringpMAT21, or EHA101 harbouring pEKH1-WD, was cul-tured overnight on a rotary shaker (120 cycles min)1) at28�C in 50 mL liquid LB medium containing 50 mg L)l

kanamycin (Wako Pure Chemical Industries) and25 mg L)l chloramphenicol (Sigma-Aldrich). Agrobacte-rium tumefaciens strain EHA101pEKH1-WD was addi-tionally supplemented with 100 mg L)l spectinomycin.The bacterial suspension was centrifuged for 10 min(3000 g) and then resuspended in inoculation medium:MS (Murashige & Skoog, 1962) medium containing

100 lM acetosyringone (3,5-dimethoxy-4-hydroxy-ace-tophenone; Sigma-Aldrich), 30 g L)l sucrose, to a finaldensity of OD600 = 0Æ5.

Newly formed expanding leaves were excised fromin vitro-grown plants, cut into portions of 5–8 mmsquare and inoculated with A. tumefaciens strainEHA105pMAT21 or EHA101pEKH1-WD for 10 minwith gentle shaking. After inoculation, the explants wereblotted dry and co-cultivated for 3 days in the dark onphytohormone-free MS medium supplemented with100 lM acetosyringone and solidified with 8 g L)l agar,pH 5Æ8.

To produce marker-free plants expressing the chitinasegene alone, the explants infected with A. tumefaciensstrain EHA105pMAT21 were washed three times withsterilized distilled water after co-cultivation and culturedon M1 [MS medium containing 30 g sucrose, 8 g agarand 20 mg meropenem trihydrate (Meropen; DainipponSumitomo Pharma; Ogawa & Mii, 2004) L)l to eliminatebacterial carry over], without any selective chemicalagent. The explants were subcultured at 2-week intervalsto fresh medium. One month after co-cultivation, adven-titious shoots were excised and transferred to the samefresh medium for production of the ipt (isopentenyl trans-ferase)-shooty phenotype. Selected ipt-shooty masseswere subcultured to fresh medium every 2 weeks andobserved for generation of phenotypically normalmarker-free transgenic shoots. The phenotypicallynormal shoots were transferred to medium of thesame composition for rooting. The cultures weremaintained at 25 ± 1�C, with a 16-h photoperiod and30–40 lmol m)2 s)1 cool white fluorescent light.

To produce transgenic plants expressing the wasabidefensin gene alone, after co-cultivation, the explantsinfected with A. tumefaciens strain EHA101pEKH1-WDwere transferred to M1 supplemented with 1 mg BA,0Æ1 mg NAA and 100 mg kanamycin L)1 to select trans-formed tissues. When adventitious shoots inducted fromthe explants reached 2–3 cm in height, each shoot wasexcised and transferred to the same medium, but withoutphytohormones, for rooting.

Retransformation

For retransformation, leaves from the first in vitro mar-ker-free transgenic tobacco plant expressing the ChiCgene (TC-1) were infected with EHA101pEKH1-WDand cultured as described above.

Molecular analysis of transgenic plants

Genomic DNA was extracted from young leaves ofthe non-transformed control, ipt, normal shoots, trans-genic and retransformed plants using a modified cetyltrimethyl ammonium bromide (CTAB) method (Rogers& Bendich, 1988). PCR amplification was performedusing genomic DNA of each plant as a target and oligonu-cleotide primers for ipt, ChiC, nptII and WD. The PCRproduct was expected to be 0Æ8 kb for the ipt gene, 0Æ7 kb

Plant Pathology (2010)

Transgenic tobacco resistance to fusarium wilt 3

for the nptII gene, a 0Æ9-kb fragment for the ChiC and0Æ5 kb for wasabi gene (Khan et al., 2006b, 2008; Thiruk-kumaran et al., 2009).

For Southern blot, 15 lg genomic DNA was digestedovernight with HindIII, EcoR1 and Xba1 for the ChiCgene and Xba1 for the WD gene, separated on a 0Æ8%(w ⁄ v) agarose gel, and subsequently transferred to anylon membrane (Immobilon-Ny+ Transfer Membrane;Millipore Co.). For northern blot analysis, total RNAwas extracted from the leaves of non-transgenic andtransgenic lines as described by Krapp et al. (1993), withslight modification, and 30 lg RNA was fractionated on0Æ8% agarose gel. Southern and northern blotting wereperformed according to the instruction manual of theDIG Labelling and Detection System (Roche Diagnos-tics). Hybridization with the DIG-labelled probes wasperformed at 41�C for 16 and 18 h for ChiC and WD,respectively, for Southern blot. For northern blot, prehy-bridization, hybridization and washing temperatureswere set at 50�C for both genes. For detection of hybrid-ization signals, membrane was exposed to a detectionfilm (Lumi-Film Chemiluminescent Detection Film;Roche Diagnostics) for 15–20 min at room temperature.

Western blot analysis was performed to determine theexpression of ChiC and WD gene proteins. Total proteinswere extracted from 50 mg leaf tissues from non-trans-genic, marker-free and retransformed transgenic plant-lets, ground in six volumes (w ⁄ v) of sample buffer[62Æ5 mM Tris–HCl (pH 6Æ8), 2% SDS, 10% glycerol and2% 2-mercaptoethanol]. Detection of the ChiC and WDproteins was performed using an ECL Advance WesternBlotting Detection Kit (Amersham Bioscience). Antise-rum was raised against a synthetic peptide correspondingto part of the ChiC protein (Ohno et al., 1996) or WDprotein (Kanzaki et al., 2002). Detection of the 31-kDa ofChiC protein or 5-kDa WD protein was carried out withgoat-anti-rabbit IgG conjugated to horseradish peroxide(HRP) at 1:100 000 v ⁄ v as secondary antibody.

Disease resistance bioassays

In vitro regeneration bioassayFusarium oxysporum f.sp. nicotianae (Fon) was pro-vided by the Plant Pathology Laboratory, Chiba Univer-sity, Japan. It was harvested from a 7-day-old culturegrown on potato dextrose agar (PDA; Difco) at 25�C.Spore suspension of inoculum was obtained by floodingthe Petri dish with sterile water, agitating the surfacegently with an inoculation loop, and filtering and adjust-ing the resulting spore suspension to 2 · 106 spor-es mL)1. Tobacco leaf explants (approximately 8 mm2)from the non-transformed control and transgenic linesexpressing ChiC or WD or ChiC ⁄ WD were placed (sixexplants per plate) on antibiotic-free M1 supplementedwith 1 mg BA and 0Æ1 mg NAA L)1. The spore suspen-sion (50 lL) was inoculated into the centre of the agarplate so that each explant was 1 cm from the suspensionand 1 cm from the next explant. The explants were co-cultivated with the fungus in a growth room for 3 weeks

at 25 ± 1�C, with a 16-h photoperiod and 30–40 lmol m)2 s)1 cool white fluorescent light. Evalua-tion of tissue resistance was based on the ability toremain green, form callus and regenerate shoots in thepresence of the pathogenic fungus. Disease severity wasrecorded after 2 weeks of co-cultivation with the fungusand expressed on a scale of 0–3 (0: explants with shoots;1: explants with callus; 2: explants green but withoutcallus or shoots; and 3: dead explants).

Infection indices were calculated according to Krishna-moorthy et al. (2003) as follows:

I.I ¼P

ðN � 1Þ Tð1Þ

where n is the number of explants in each grade, bthe grade, N the number of grades used in the scaleand T the total number of explants cultured.

In vitro bioassay of whole plant, mock inoculationand fungal colonizationFor the whole-plant bioassay, tobacco plants were sub-cultured onto fresh MS medium without antibiotics inculture bottles (one plantlet in each bottle) and allowed toroot. Then, 1 mL fungal suspension was injected into themedium with a sterile needle so that the fungus touchedthe roots. Mock-inoculation was done by spraying sterilewater onto the medium. The plants were then incubatedat room temperature for 6 weeks. The plants wereobserved daily for disease development and scored on ascale of 0–3 with respect to the time taken for lesion todevelop (0: plants without symptoms; 1: 5–6 weeks; 2: 3–4 weeks; and 3: 1–2 weeks for symptoms to appear). Theexperiment was repeated three times. Infection indiceswere calculated using the method described by Benadaet al. (1981) as follows:

I.I ¼ ðn1 þ 2n2 þ 3n3Þ 100

3 ðn0 þ n1 þ n2 þ n3Þð2Þ

where n is the number of plants in each grade (0–3) withrespect to the time taken for symptoms to appear.

Plants with a disease index of 0% were considered asnot susceptible, those with a disease index <25% ashaving high resistance, those with a disease index of25Æ1–50% as having moderate resistance, those with adisease index of 50Æ1–75% as susceptible, and those witha disease index of 75Æ1–100% as highly susceptible.

To determine the level of fungal colonization, non-transgenic and transgenic lines were inoculated with thefungal suspension as described above. Seven days afterinoculation, roots were collected, washed to remove alladhering gelling materials and surface-sterilized by dip-ping in 70% ethanol for 1 min followed by immersion in1Æ5% sodium hypochlorite for 3 min. The roots werethen washed three times with sterile distilled water, blot-ted dry, cultured on PDA containing 200 mg streptomy-cin L)1 at room temperature (25 ± 1�C) and scored forthe presence or absence of F. oxysporum as described byGao et al. (1995). The level of colonization of individualplants was quantified as the percentage of roots with thepathogen.

Table 1 Segregation of ipt shoots in tobacco (Nicotiana tabacum) explants

infected with Agrobacterium tumefaciens harbouring the pMAT21 vector

containing the chitinase (ChiC ) gene

No. (%)

Explants infected 60

Adventitious shoot lines isolated (4 WAC) 55

Adventitious shoot lines forming ipt shoots (7 WAC) 37 (67Æ3)

Adventitious shoot lines forming

normal shoots (7 WAC)

18 (32Æ7)

Normal shoots from ipt shoot lines (4 MAC) 25 (67Æ6)

Ipt shoot lines with ipt + (PCR) (4 MAC) (%) 15 (40Æ5)

Ipt shoot lines with chitinase + (PCR) (4 MAC) (%) 15 (40Æ5)

Well-rooted normal shoots containing

chitinase gene after ipt excision (14 MAC) (%)

6 (16Æ2)

WAC: weeks after co-cultivation; MAC: months after co-cultivation.

(a)M P C 1 2 3 4 5 6 7 8 9 10

4 V. O. Ntui et al.

Antifungal activity of leaf extract

The method developed by Yevtushenko et al. (2005) wasadopted with some modifications where necessary. Threedays after inoculation with Fon, leaves from non-trans-genic control and selected transgenic lines (TC-1, TWD-3and TCWD-9) were collected and ground to a fine pow-der in liquid nitrogen and extraction buffer added.Ground tissues were centrifuged at 13 148 g for 10 minat 4�C. Of the supernatant, 200 lL were collected andmixed with 2 lL protein inhibitor cocktail (P9599;Sigma-Adrich). Conidial suspension of Fon was thenmixed 1:9 with the leaf extract and spread evenly onpotato dextrose agar (PDA) plates, then incubated in agrowth room at 25 ± 1�C with a 16-h photoperiod. Spo-rangia alone were used as controls. Fungal growth, i.e.number of colonies formed per cm2 (CFU cm)2), wasrecorded after 48 and 72 h of incubation. In addition,hyphal structure was determined using an inverted micro-scope (Olympus IMT-2; Olympus optical Co., Ltd). Theexperiment was performed three times.

M P C 1 2 3 4 5 6

7 8 9 10

Statistical analysis

The experiments on disease resistance, fungal coloniza-tion and antifungal activity of the leaf extract were eachset up to a completely randomized design with three repli-cations, and repeated three times. Data collected weresubjected to analysis of variance. Means were comparedusing the least significant different (LSD) test. Dataexpressed as percentages were subjected to arcsine trans-formation before statistical analysis. The relationshipbetween fungal colonization and infection index wasdetermined by correlation analysis.

(d)M P C 1 2 3 4 5 6

Figure 1 PCR analyses of marker-free tobacco (Nicotiana tabacum)

lines expressing the chitinase gene ChiC. PCRs were performed as

described in Materials and methods. (a) ipt (800-bp) fragment from

ipt-shooty phenotype (lanes 1–10). (b) ChiC (900-bp) fragment from

ipt-shooty phenotypes (lanes 1–10). (c) Excision of the ipt (800-bp)

fragment in normal shoots appearing from ipt-shooty lines (lanes

1–6). (d) ChiC (900-bp) fragment from an excision event in normal

shoots appearing from ipt-shooty lines (lanes 1–6). M: molecular

size marker (ø174 ⁄ HaeIII); P: positive control (plasmid DNA); C:

non-transgenic plants.

Results

Genetic transformation and regeneration oftransgenic plants

Tobacco explants were successfully transformed withA. tumefaciens strain EHA105 harbouring a MAT vec-tor, pMAT21, producing the ipt-shooty phenotype7 weeks after co-cultivation (Table 1). After 14 monthsof culture, six normally rooted marker-free plants inwhich the ipt gene had been excised were produced. In theipt-shooty lines, PCR analysis amplified the predicted800-bp ipt fragment (Fig. 1a) and 900-bp ChiC fragment(Fig. 1b), but neither fragment was amplified in the con-trol plants. When the R ⁄ RS (hit and run cassette) systemwas eliminated (11–14 months after co-cultivation), theexpected 800-bp ipt fragment was not amplified (Fig. 1c)and the expected 900-bp ChiC fragment was amplified insix out of 15 ipt-shooty lines containing the chitinase gene(Fig. 1d). This result indicates that these six lines weremarker-free plants.

To produce transgenic plants expressing the WD genealone, 25 leaf explants from in vitro-grown cv. Havanaplants were infected with A. tumefaciens EHA101-

pEKH1-WD. The presence of the nptII (Fig. 2a) and WDgenes (Fig. 2b) in transgenic lines was confirmed by PCRanalysis.

For retransformation, leaf explants from the first mar-ker-free transgenic line TC-1 expressing the ChiC genewere infected with the WD gene as shown above. A DNAfragment corresponding to the WD gene (Fig. 2c) orthe ChiC gene (Fig. 2d) was amplified by PCR from the

M P C 1 2 3 4 5 6 7 8 9

M P C 1 2 3 4 5 6

M P C 1 2 3 4 5 6 7 8 9 10

Figure 2 Integration and expression of the wasabi defensin (WD)

transcript in transgenic tobacco (Nicotiana tabacum) lines, and the

chitinase gene (ChiC) in retransformed plants. PCR amplification of

the 700-bp nptII (a) and the 500-bp WD gene (b) in transgenic

tobacco lines expressing the WD gene alone (lanes 1–9).

(c) Amplification of the 500-bp WD gene in retransformed

transgenic lines co-expressing ChiC ⁄ WD (lanes 1–10). (d)

Amplification of the 900-bp ChiC gene in retransformed transgenic

lines co-expressing ChiC ⁄ WD (lanes 1–10). M: molecular size

marker (ø174 ⁄ HaeIII); P: positive control (plasmid DNA); C: non-

transgenic plants.

ChiC + WD

Kb21·2

5·15·04·3

3·62·0

M C TC-1 1 2 3 4

M C 1 2 3 4 5 6 7 8 9 10(d)

ChiC + WD

Figure 3 Southern blot analyses of retransformed tobacco

(Nicotiana tabacum) lines expressing chitinase (ChiC) and wasabi

defensin (WD) genes. For the ChiC gene, genomic DNA from

transgenic and non-transformed controls was digested with

HindIII (a), EcoR1 (b) and Xba1 (c), and hybridized with a

genomic DNA of all regenerated plant clones rooted onhormone-free MS medium.

DIG-labelled fragment containing the ChiC gene. Lane TC-1,

marker-free transgenic tobacco line expressing ChiC alone; lanes

1–4, independent transgenic tobacco lines co-expressing ChiC ⁄ WD

genes. For the WD gene, genomic DNA from retransformed

tobacco lines was digested with Xba1 (d). Lanes 1–10, independent

transgenic tobacco lines co-expressing ChiC and WD. M: DIG-

labelled molecular weight marker III; C: non-transformed control.

Transgenic lines used in lanes 1–4 in (a–c) are the same lines used

in lanes 2, 4, 9 and 10, respectively, in (d).

Molecular analysis of transgenic plants

Southern blot analysis confirmed the integration of thechitinase and wasabi defensin transgenes in all PCR-posi-tive plants analysed. For the chitinase gene, genomicDNA digested with HindIII (Fig. 3a), EcoR1 (Fig. 3b) andXba1 (Fig. 3c) revealed that the hybridization patterns ofthe first (lane TC-1) and the second (lanes 1–4) transfor-mations were similar, indicating that none of the retrans-formed tobacco lines examined had undergonerearrangement of the ChiC insertion. For the WD gene,genomic DNA from transgenic lines expressingChiC ⁄ WD digested with Xba1 showed that the analysedplants had undergone one to five integration events(Fig. 3d, lanes 1–10), indicating that the transgenic clonesoriginated from independent transformation events.Northern hybridization revealed that all the transgeniclines had detectable levels of ChiC (Fig. 4a) with varyingexpression. Similarly, the wasabi defensin gene wasexpressed in all five retransformed lines tested (Fig. 4b).Its expression was more pronounced in lines TCWD-10,TCWD-9 and TCWD-1, moderate in line TCWD-2 andlow in transgenic line TCWD-4. The 31-kDa peptide ofthe ChiC protein was detected as a single band in the

leaves of transgenic clones (Fig. 4c). Also, the 5-kDa pep-tide corresponding to the processed form of wasabi defen-sin was detected in the total protein fraction extractedfrom seven lines (Fig. 4d). No hybridization bands weredetected with DNA, RNA or proteins from non-trans-formed control plants. All retransformed transgenic lineshad the normal phenotype of tobacco, with no indicationof cytotoxicity as a result of expression of the ChiC ⁄ WDgenes.

Evaluation of in vitro regeneration bioassay forpathogen-resistant explants

To screen transgenic lines for resistance to pathogens, asimple in vitro approach was adopted based on the ability

C TC-1 TC- 2 TC- 3 TC- 4 TCWD-1 TCWD-7 TCWD-9

C 1 2 3 4 5 6 7 8 9 10

◄5 kDa

◄31 kDa

ChiC ChiC + WD

WD mRNA

Total RNA

C TCWD-1 TCWD-2 TCWD-4 TCWD-7 TCWD-9

Total RNA

ChiC mRNA

Figure 4 Northern blot (a, b) and western blot (c, d) analyses of

accumulation of chitinase (ChiC) and wasabi defensin (WD) genes

in transgenic tobacco (Nicotiana tabacum) plants. (a) Northern blot

analysis of ChiC gene in marker-free transgenic tobacco plants

expressing ChiC (lanes TC-1–TC-4) and transgenic lines co-

expressing ChiC and WD (lanes TCWD-1, TCWD-2, TCWD-7 and

TCWD-9). (b) Northern blot analysis of WD in transgenic tobacco

lines co-expressing ChiC and WD (lanes TCWD-1, TCWD-2, TCWD-

4, TCWD-7 and TCWD-9). Northern hybridization was carried out

using the DIG-labelled DNA probe of the ChiC or WD gene. (c)

Western hybridization of ChiC gene in leaves of marker-free

transgenic lines expressing ChiC (lanes TC-1–TC-4) and transgenic

lines co-expressing ChiC and WD (TCWD-1, TCWD-7 and TCWD-9).

(d) Western blot analysis of the expression of wasabi defensin

protein in leaves of retransformed tobacco lines co-expressing ChiC

and WD (lanes 1–10) Note the varying level of expression among

different clones. Lane C, non-transformed control plant.

6 V. O. Ntui et al.

of resistant explants to remain green, form callus andregenerate in the presence of the pathogen. After 3 days,the fungus quickly grew and mycelia spread completelyover the medium in all the Petri dishes. After 1 week, theuntransformed (control) explants were completely over-taken by the fungus with no sign of callus formation, anddied within 10 days of co-cultivation (Fig. 5a). In con-trast, the transgenic explants remained green and formedcallus and shoots within 2 weeks of co-cultivation(Fig. 5b–d). The rate of regeneration was higher in trans-genic lines co-expressing both the ChiC and WD genesthan in isogenic lines expressing a single transgene.Consequently, the infection indices in the transgenic linesco-expressing ChiC ⁄ WD were lower (ranging from 0Æ0 to1Æ2) than those of transgenic lines expressing either ChiC

or WD (which ranged from 2Æ5 to 9Æ8) (Table 2). In plantsexpressing ChiC ⁄ WD, one line (TCWD-9) conferredcomplete resistance as all the explants regenerated shootsin the presence of the fungus (Table 2).

Resistance of transgenic lines to Fon

In the whole-plant bioassay, the mock-inoculated plantsshowed no morphological or developmental abnormali-ties under the period of study (Fig. 6a–c). The non-trans-formed control plants were readily penetrated by thefungal mycelia and died 10 days after inoculation(Fig. 6d–f). In contrast, transgenic lines expressing eitherChiC (Fig. 6g–i) or WD (Fig. 6j–l) or co-expressingChiC ⁄ WD (Fig. 6m–o) remained green and continuedgrowth through the fungal hyphae with no sign of diseasedevelopment. Four weeks after inoculation, most of thetransgenic lines expressing a single transgene started dis-playing browning and necrosis symptoms, resulting inwilting 5 weeks after inoculation. By contrast, mostof the transgenic lines co-expressing ChiC ⁄ WD did notdisplay any wilting symptoms, even 5 weeks after inocu-lation (data not shown), indicating significantly enhancedresistance to fusarium wilt. Based on the calculated infec-tion indices, transgenic line TCWD-9 co-expressingChiC ⁄ WD conferred complete resistance to the fungus,as no symptoms were observed under the period of study(Table 3). These observations were in line with the in vi-tro regeneration bioassay and reflected the expressionlevels of ChiC and WD genes in the northern and westernblot analyses.

The pathogen, Fon, was isolated from 86Æ4% of theroots of non-transformed controls, but only from a fewroots of the transgenic lines (Table 3). In the roots oftransgenic lines, the extent of colonization was lowest(2Æ1%) in line TCWD-9 and highest (37Æ7%) in line TC-4(Table 3). The relationship between infection index andthe level fungal colonization was assessed by correlationanalysis. A highly significant relationship (r = 0Æ93,P < 0Æ01) between root colonization and infection indexwas obtained.

Antifungal activity of leaf extract from non-transgenic and transgenic lines

Leaf extracts of transgenic lines significantly reduced thegrowth of Fon compared to non-transgenic plants(Fig. 7a). The leaf extract of the transgenic line co-expressing ChiC ⁄ WD (line TCWD-9) was the most effec-tive in inhibiting mycelia growth of Fon, resulting in morethan 90% reduction in the number of fungal colonies.Hyphal growth abnormalities were observed with leafextracts of transgenic lines. Abnormal hyperbranching ofgerminating spores and condensed hyphal aggregates ofonly a few cells in length, compared with thin, elongatedwell-extended mycelial growth in controls, were noted(Fig. 7b). However, the leaf extract of the transgenic lineco-expressing ChiC ⁄ WD had a more severe effect onhyphal morphology (Fig. 7b-iv) than the extracts from

(a) (b)

(d)(c)

Figure 5 In vitro plant regeneration bioassay

of tobacco (Nicotiana tabacum) explants

challenged with Fusarium oxysporum f.sp.

nicotianae (Fon). Leaf explants were

co-cultured with Fon on tobacco

regeneration medium without antibiotics. (a)

Leaf explants of non-transformed control. (b)

Leaf explants of marker-free transgenic

tobacco line TC-1 expressing chitinase

(ChiC) gene alone. (c) Leaf explant of

transgenic tobacco line TWD-3 expressing

wasabi defensisn (WD) gene. (d) Transgenic

tobacco line TCWD-9 co-expressing

ChiC ⁄ WD genes. Pictures were taken

2 weeks after cultivation with the fungus. Bar:

0Æ25 cm.

Table 2 Infection indices of in vitro plant regeneration bioassay of non-

transgenic and transgenic tobacco (Nicotiana tabacum) explants

inoculated with Fusarium oxysporum f.sp. nicotianae

Disease

indexa Resistance ratingChitinase

Wasabi

defensin

Control ) ) 37Æ5 ± 0Æ5h Highly susceptible

TC-1 + ) 3Æ9 ± 0Æ1e Moderately resistant

TC-2 + ) 6Æ2 ± 0Æ15f Susceptible

TC-3 + ) 3Æ6 ± 0Æ12e Moderately resistant

TC-4 + ) 9Æ8 ± 0Æ25g Susceptible

TC-5 + ) 2Æ6 ± 0Æ1d Moderately resistant

TCWD-1 + + 0Æ5 ± 0Æ15ab Highly resistant

TCWD-2 + + 1Æ0 ± 0Æ15bc Highly resistant

TCWD-4 + + 1Æ2 ± 0Æ11c Highly resistant

TCWD-9 + + 0Æ0 ± 0Æ0a Not susceptible

TWD-1 ) + 4Æ2 ± 0Æ2e Moderately resistant

TWD-3 ) + 2Æ5 ± 0Æ2d Moderately resistant

TWD-4 ) + 3Æ9 ± 0Æ12 Moderately resistant

TWD-6 ) + 6Æ5 ± 0Æ2f Susceptible

TWD-9 ) + 6Æ8 ± 0Æ15f Susceptible

aInfection indices were calculated using Eqn 1 in Materials and

methods. Lines with a disease index of 0 were considered as not

susceptible, those with a disease index <2 as having high

resistance, those with a disease index of 2Æ1–5 as being moderately

resistant, those with a disease index of 5Æ1–10 as susceptible, and

those with a disease index >10 as highly susceptible, under the

period of study. Data presented are means ± SE from three

independent experiments. Means followed by the same lower-case

letter are not significantly different at (P > 0Æ05).

transgenic lines expressing either of the genes (Fig. 7b-ii, -iii).These results are in agreement with the results of the plantresistance bioassays.

Discussion

The MAT vector system is considered a novel and effec-tive system to stack transgenes into plants using a singleselectable marker (ipt) gene (Sugita et al., 2000). Previousstudies showed that the ipt gene is efficient as a dominantand visual selectable marker for Agrobacterium-medi-ated plant transformation because transgenic plants withabnormal phenotype can be generated on a hormone-freemedium (Ebinuma et al., 1997; Sugita et al., 1999; Thir-ukkumaran et al., 2009). The abnormal phenotype subse-quently reverts into normal marker-free transgenicshoots with target transgene as a result of the excision ofthe ipt gene by the function of hit-and-run cassette sys-tem, allowing the MAT vector to be used again in anotherround of transformation. In this study, marker-free trans-genic lines in which the ipt gene had been excised wereproduced when tobacco leaf explants were inoculatedwith pMAT21 containing the ChiC gene. The ipt-typeMAT vector, pMAT21, is equipped with the R ⁄ RS sys-tem. The latter and other site-specific recombination sys-tems mediated the excision of a DNA fragment betweentwo directly oriented recombination sites in plant cells(Lloyd & Davis, 1994; Onouchi et al., 1995), allowingthe elimination of non-useful genes, including the iptgene, after they have completed their role.

Defence proteins have been the focus of numerousstudies aiming to develop genetically engineered fungalresistance in plants. Enhanced expression in variousplants of chitinase (Broglie et al., 1991; Khan et al., 2008)and small cysteine-rich plant defensins (Terras et al.,1995; Gao et al., 2000) have successfully increased resis-tance to pathogenic fungi and bacteria. However, theexpression of individual antimicrobial proteins in trans-genic plants is not always effective in conferring completeresistance, and often only confers partial resistance. This

(l) (o)

(n)(h)

Figure 6 In vitro evaluation of resistance against Fusarium oxysporum f.sp. nicotianae (Fon), conferred by transgenic tobacco (Nicotiana

tabacum) lines expressing either the chitinase or wasabi defensin gene, or co-expressing both genes. Three-week-old well-rooted in vitro non-

transformed control and transgenic lines were inoculated with 1 mL fungal suspension by injecting the suspension into the medium with a

sterile needle so that the fungus touched the roots. Mock-inoculation was done by spraying sterile water onto the surface of the medium.

Plants were cultivated for 6 weeks at room temperature. Upper row: start of co-cultivation with Fon; second row: 1 week after co-cultivation;

third row: 2 weeks after co-cultivation. (a–c) mock-inoculated control, (d–f) non-transgenic control plants, (g–i) marker-free transgenic tobacco

line TC-1 expressing chitinase gene, (j–l) transgenic tobacco line TWD-3 expressing wasabi defensin gene, (m–o) transgenic tobacco line

TCWD-9 co-expressing chitinase and wasabi defensin genes. Bar: 1 cm.

8 V. O. Ntui et al.

indicates that expression of two or more antimicrobialproteins is required to establish effective disease resis-tance in transgenic plants.

From this point of view, it was assumed that the com-bined effect of ChiC ⁄ WD would lead to enhanceddestruction of hyphal cell walls and therefore improveresistance against fungal attack. In this work, transgenictobacco lines either expressing ChiC or WD, orco-expressing ChiC ⁄ WD, were generated and testedfor resistance against Fon. Assessment of plant survivalin the presence of Fon showed that transgenic linesco-expressing both genes were highly resistant to the fun-gus, more so than transgenic lines expressing a single gene(Figs 5 and 6; Tables 2 and 3), suggesting the synergisticprotective interaction of the co-expressed antifungalproteins. Chen et al. (2009) clearly demonstratedenhanced growth inhibition of B. cinerea by combinedoverexpression of a chitinase and a defensin gene in trans-genic tomato. Synergistically enhanced protectionagainst fungal attack by co-expression of chitinase andglucanase in planta was also demonstrated by Zhu et al.(1996).

To efficiently screen transgenic lines for resistance topathogens, a quick and simple in vitro bioassay based onthe ability of resistant explants to regenerate in the pres-ence of the pathogen was adopted. In tissue culture, plantregeneration is seriously affected by unfavourableconditions such as changes in medium composition, pH,temperature or the presence of pathogens in the medium.Therefore, it was assumed that if transgenic lines express-ing a single transgene or co-expressing ChiC ⁄ WD couldform calli and shoots in the presence of Fon, they wouldhave an advantage in morphogenetic capacity oversusceptible lines when subjected to fungal infection. Mostof the transgenic lines screened formed shoots, unlikecontrols. However, the rate of shoot formation was sig-nificantly higher in transgenic lines co-expressingChiC ⁄ WD than in lines expressing a single transgene,and was concomitant with the expression level of thegenes in the tissues of transgenic lines, and with the resultsof the whole-plant bioassay.

The whole-plant resistance bioassay revealed differentlevels of resistance in transgenic lines expressing eitherChiC or WD or co-expressing the ChiC ⁄ WD genes.

Table 3 Infection indices of whole plant bioassay and level of fungal colonization in non-transgenic and transgenic tobacco (Nicotiana tabacum) lines

challenged with Fusarium oxysporum f.sp. nicotianae

Disease indexa (%) Roots with pathogen (%) Resistance ratingChitinase Wasabi defensin

Control ) ) 93Æ2 ± 1Æ5i 86Æ4 ± 2Æ3l Highly susceptible

TC-1 + ) 33Æ8 ± 0Æ5e 22Æ1 ± 0Æ4fg Moderately resistant

TC-2 + ) 52Æ3 ± 1Æ0g 29Æ3 ± 0Æ8i Susceptible

TC-3 + ) 34Æ3 ± 1Æ0e 21Æ2 ± 0Æ3f Moderately resistant

TC-4 + ) 60 ± 0Æ0h 37Æ7 ± 1Æ2k Susceptible

TC-5 + ) 28 ± 1Æ3d 17Æ4 ± 0Æ5e Moderately resistant

TCWD-1 + + 9Æ3 ± 1Æ0b 7Æ9 ± 0Æ2b Highly resistant

TCWD-2 + + 17Æ7 ± 1Æ1c 10Æ5 ± 0Æ2d Highly resistant

TCWD-4 + + 18Æ5 ± 1Æ8c 11Æ3 ± 0Æ3d Highly resistant

TCWD-7 + + 9Æ8 ± 1Æ5b 8Æ7 ± 0Æ4b Highly resistant

TCWD-9 + + 0Æ0 ± 0Æ0a 2Æ1 ± 0Æ1a Not susceptible

TCWD-10 + + 10 ± 1Æ7b 9Æ2 ± 0Æ5c Highly resistant

TWD-1 ) + 34Æ5 ± 1Æ2e 21Æ5 ± 1Æ1f Moderately resistant

TWD-3 ) + 25Æ8 ± 0Æ8d 16Æ8 ± 0Æ7e Resistant

TWD-4 ) + 42Æ2 ± 2Æ1f 22Æ6 ± 1Æ4g Moderately resistant

TWD-6 ) + 48Æ5 ± 1Æ8g 27Æ8 ± 1Æ2h Moderately resistant

TWD-9 ) + 58 ± 2Æ4h 35Æ6 ± 1Æ6j Susceptible

aInfection indices were calculated using Eqn 2 in Materials and methods. Resistance rating is based on the calculated infection indices.

Plants with a disease index of 0% were considered as not susceptible, those with a disease index <25% as having high resistance, those with

a disease index of 25Æ1–50Æ00% as being moderately resistant, those with a disease index of 50Æ1–75Æ00% as susceptible, and those with a

disease index of 75Æ1–100% as highly susceptible, under the period of study. The level of colonization of individual lines was quantified as the

percentage of roots with the pathogen. Data presented are means ± SE from three independent experiments. Means followed by the same

lower-case letter are not significantly different at (P > 0Æ05).

48 h 72 hHours after inoculation

–2(l

TCWD-9

iviiiiii

Figure 7 Antifungal activity of leaf extracts of non-transgenic control and transgenic tobacco (Nicotiana tabacum) lines expressing either

chitinase (ChiC) or wasabi defensin (WD) genes or co-expressing ChiC ⁄ WD. (a) Effect of leaf extracts on the growth of Fusarium oxysporum

f.sp. nicotianae after 48 and 72 h of incubation. Bars represent means and standard errors and those with same lower-case letter in a given

period are not significantly different at (P > 0Æ05) by least significant different (LSD) test. (b) Hyphal growth abnormalities as affected by leaf

extract of transgenic and non-transgenic lines (i, NT), (ii, TC-1), (iii, TWD-3) and (iv, TCWD-9). Photographs taken after 48 h of incubation, Bar:

100 lm. NT: non-transgenic control; TC-1: transgenic line 1 expressing chitinase ChiC; TWD-3: transgenic line 3 expressing WD; TCWD-9:

transgenic line 9 co-expressing ChiC ⁄ WD.

10 V. O. Ntui et al.

Transgenic lines expressing the highest level ofChiC ⁄ WD showed significantly higher levels of protec-tion against the fungus than lines having low expressionlevels. This result suggests that transcriptional or post-transcriptional silencing could have affected the level ofdisease resistance. Silencing of transgenes has beenreported in many plant species (Chareonporwattanaet al., 1999). Also, a positional effect in the genome maycause differences in gene expression (Meyer, 1995) andthus different levels of resistance to pathogenic micro-organisms.

The frequency of fungal colonization was based onpathogen recovery from roots of non-transgenic andtransgenic lines. The highly significant correlation(r = 0Æ93, P < 0Æ01) between infection indices and thelevel of fungal colonization demonstrated that the lowerthe level of colonization, the lower the severity of the dis-ease. Thus, the enhanced resistance found in transgeniclines was associated with a reduction in root fungal colo-nization (Gao et al., 1995; Shaw et al., 2010), caused bythe antimicrobial peptides.

Acknowledgement

The authors would like to thank Pulp and PaperResearch, Nippon Paper Industries, Tokyo for providingthe MAT vector construct.

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