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Theor Appl Genet (1998) 96: 266
— 
271
Springer-Verlag 1998
P. Vain · B. Worland · M. C. Clarke · G. RichardM. Beavis · H. Liu · A. Kohli · M. Leech · J. SnapeP. Christou · H. Atkinson
Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-I
D86)for nematode resistance in transgenic rice plants
Received: 4 August 1997 / Accepted: 22 August 1997
Abstract
We have used a genotype-independent trans-formation system involving particle gun bombardmentof immature embryos to genetically engineer rice aspart of a programme to develop resistance to nema-todes. Efficient tissue culture, regeneration, DNAdelivery and selection methodologies have been estab-lished for elite African varieties (‘ITA212’, ‘IDSA6’,‘LAC23’,‘WAB56-104’).Twenty-five transformed clonescontaining genes coding for an engineered cysteineproteinase inhibitor (oryzacystatin-I
D86,
OC
-
I
D86),hygromycin resistance (
aphI
»
) and
-glucuronidase(
gusA
) were recovered from the four varieties. Trans-formed plants were regenerated from all clones andanalysed by PCR, Southern and western blot. Detect-able levels of 
OC
-
I
D86 (up to 0.2% total solubleprotein) in plant roots were measured in 12 out of 25transformed rice lines. This level of expression resultedin a significant 55% reduction in egg production by
Meloidogyne incognita
.
Key words
Oryza sativa
·
Transformation
·
Oryzacystatin-
I
D86
·
Nematode resistance
Introduction
It is estimated that
Meloidogyne
spp. (root-knotnematodes)account for the majority of the
$
100 billion
Communicated by G. WenzelP. Vain ( )
·
B. Worland
·
H. Liu
·
A. Kohli
·
M. LeechJ. Snape
·
P. ChristouJohn Innes Centre, Colney lane, Norwich NR4 7UH, UKFax: (44) 1603 502241E-mail: vain
@
bbsrc.ac.ukM. C. Clarke
·
G. Richard
·
M. Beavis
·
H. AtkinsonCentre for Plant Biochemistry and Biotechnology,University of Leeds, LS2 9JT, UK
(US) of annual loss attributed to nematode damage incrops (Sasser and Freckman 1987). In the tropics, aver-age crop production is reduced by 11
— 
25% as a resultof damage by nematodes (Sasser 1979).
Meloidogyne
ssp. are an important component of the range of rootparasitic and foliar nematodes that can cause severecrop losses in the various rice ecosystems (Bridge et al.1990). The majority of 
Oryza sativa
cultivars are sus-ceptible to
Meloidogyne
spp. (Bridge et al. 1990).
M
.
graminicola
causes losses in both upland and lowlandrice in Asia either before flooding or in intermittentcultivation systems (Prot 1994), while
M
.
incognita
isprincipally a problem of upland rice and can reduceplant height and yield by 60% in West Africa (Plow-right and Hunt 1994; Babatola 1984). The wide hostrange of 
Meloidogyne
spp. that attack rice also placesother crops in rotation with rice at significant risk.Their control is therefore an important component inthe development of sustainable agriculturalproductionsystems. Unfortunately, chemical control is unsuitableon economic and environmental grounds. Clearly, hereis an important opportunity to use biotechnology toimprove rice production by developing resistance tonematodes. However, to date, there is no report of transgenic resistance against any nematode by anymeans in cereals.The introductionof pest-specificanti-nutritionalfac-tors into crop plants offers a promising approach forthe control of a wide range of pests. Protease inhibitorshave been used to engineer insect (Hilder et al. 1987;Johnson et al. 1989; Boulter et al. 1989; Duan et al.1996) and nematode (Hepher and Atkinson 1992; Ur-winet al. 1995; 1997)resistance.Cowpea trypsininhibi-tor expressed in transgenic potato was shown to affectfecundity and the male/female ratio of 
Meloidogyne
spp. and
Globodera
spp., respectively (Hepher andAtkinson 1992). Cysteine proteinase inhibitors (cys-tatins) represent an attractive option for a safe defencestrategy against specific pests because they are the onlyclass of proteinases not expressed in the digestive
 
system of mammals (Atkinson et al. 1995). Rice Oryza-cystatin-I(OC-I)ormaize(CC-I)cystatinshavealreadybeen introduced and constituively expressed in modelrice varieties (Hosoyama et al. 1994; Irie et al. 1996),potato (Benchekroum et al. 1995) and tobacco(Masoud et al. 1993), but their effect on nematodes wasnot evaluated. A variant of Oryzacystatin-I (OC-I
D86) produced by site-directed mutagenesis wasshown to mediate nematode resistance when expressedinto tomato hairy roots (Urwin et al. 1995) and
Ara
-
bidopsis
plants (Urwin et al. 1997). Despite these effortsthere is no report to date of proteinase inhibitor-me-diated transgenic resistance against any nematode incereals.One of the key elements in developing efficient gen-etic engineering of rice for pest, disease or herbicideresistance is the ability to genetically transform anygiven elite variety. In the work presented here we haveused a genotype-independent transformation systeminvolving particle gun bombardment of immature em-bryos (Christou et al. 1991) to genetically engineera range of elite African rice varieties. We report theintroduction and expression of a variant of Oryza-cystatin-I (OC-I
D86) into African elite rice cultivarsresulting in a significant 55% reduction in egg produc-tion by
M
.
incognita
. This strategy seeks to providea basis for concomitant control for
M
.
incognita
andother root parasites of rice.
Materials and methods
Plant tissue preparationTen-to fifteen-day-old immature seeds from cvs ‘ITA212’, ‘IDSA6’,‘LAC23’ and ‘WAB56-104’ (Provided by WARDA, Ivory Coast)wereharvestedfromgreenhouse-grownriceplants.Seedsweresteril-izedwith2.5% (w/v) sodiumhypochloritefor 5 min and rinsed threetimes with sterile distilled water. Glumes were removed under a dis-secting microscope, and the seeds were sterilized again for 10 minand rinsed as described above. The embryos were aseptically re-moved and plated on CCM3 medium for 2 days before bombard-ment.TheCCM3mediumcontainsCCbasalsalts(Hartkeand Lorz1989), 20 g/l sucrose, 15 g/l mannitol, 15 g/l Sorbitol, 2 mg/l 2,4-D(2,4 dichlorophenoxyacetic acid), 1 g/l casein hydrolysate, 100 ml/lcoconut water (SIGMA C-5915) and 6 g/l agarose type I (SIGMAA-6013). The final pH was adjusted to 5.7 with KOH before auto-claving. Filter-sterilized CC vitamins were added after autoclaving.Plasmids and particle bombardmentPlasmids pHyg-OcI
D86 (CaMV35Spromoter::AMVleader::
OC
-
I
D86::NOSpolyA
#
CaMV35Spromoter::
aphI
»
::SpolyA) andpJIC200 (ubi-5
region::
gusA
::NOSpolyA) were used for trans-formation (
aphI
»
provides hygromycin resistance;
gusA
codes for
-glucuronidase). The
OC
-
I
D86 gene was designed by proteinengineering to improve the Ki and efficacy of native rice OC-Iprotein against nematodes;
OC
-
I
D86 differs from
OC
-
I
by onlyone amino acid (Asp86 deleted, Urwin et al. 1995). Rice immatureembryos were osmotically conditioned for 4 h prior and 16 h afterbombardment by transfer to CCM3 medium containing 55 g/lmanitol,55 g/lsorbitol(Vainetal.1993).Particlebombardmentwascarried out as described previously (Christou et al. 1991).Selection and regeneration of transformed plantsTwo days after bombardment, immature embryos were subculturedonto CCM3 medium supplemented with 50 mg/l of hygromycin(CCM3H50). After 10 days, embryogenic calli initiated from thescutellum were dissected into small pieces and transferred to freshhygromycin-containing medium, with subculture at 10-day inter-vals. After 6
— 
8 weeks, plants were regenerated by transferring thehygromycin-resistant embryogenic calli onto CCM3H50 mediumwithout2,4-D(CCR2H50).Plantlets werefirst germinatedonplates,then grown in tubes on MSR6H50 medium prior to transfer to thegreenhouse. The MSR6H50 medium contains 40% (v/v) MS basalsalts (Murashige and Skoog 1962), 10 g/l sucrose, 2 g/l phytagel(SIGMAP-8169),4 g/lbactoagar(Difco),pH5.7.Filter-sterilizedB5vitamins (Gamborg et al. 1968) and hygromycin (50 mg/l) wereadded after autoclaving.Polymerase chain reaction (PCR)DNA was prepared using a rapid extraction protocol. Leaf samplesweregroundina1.5-mlEpendorftubeusingahandpestle,sand andliquid nitrogen. Two hundred microliters of extraction buffer(500 m
M
NaCl, 100 m
M
TRIS-HCl pH 8 and 50 m
M
EDTA; Ellis1994) and 20
l 20% (w/v) SDS were added to the powder beforefurthergrinding.After centifugation(5 min at 12000 rpm) the super-natent was removed and added to an equal volume of isopropanolfor DNA precipitation. After centrifugation, the DNA pellet waswashed with 70% ethanol and suspended in TE buffer. PCR wasperformed in a total volume of 25
l containing 25 ng rice genomicDNA, 10 m
M
TRIS
-HCl pH 8.3, 2.5 m
M
MgCl
, 50 m
M
KCl
,0.05% Noridet p40, 200
M
dNTPs, 0.4
M
of each primer and1 unit of 
taq
polymerase. DNA was denatured at 95
°
C for 1 minfollowed by 30 cycles of amplification (30 s at 95
°
C, 30 s at 60
°
C,1 min at 72
°
C) and by 10 min at 72
°
C.GUS (
-glucuronidase) activityLeaftissue from 5-week-old rice plants was assayed fluorometricallyfor
-glucuronidase activity according to Jefferson (1987).Western blot hybridization for OC-I activityTotal protein was isolated from roots of rice plants. Ten microgamsof total root protein and standard quantities of OC-I protein wereloadedon SDS PAGE gelsusing the BioRadmini system.The OC-Iproteinwas recoveredfroman
E
-
coli
expressionsystem(Urwin etal.1995). The gel was blotted according to BioRad protocols andprobed with a polyclonal antibody raised againstOC-I (Urwinet al.1995). The bands were visualized using an image analyser systemconsisting of a monochrome CCD camera (Sony) plus a framegrabber and appropriate software (Quantimet 500, Leica).Bioassays with nematodesFour-week-old rice plants were potted into a sand/loam mix infec-ted with
Meloidogyne incognita
(an African population from theWest Africa Rice Development Association). Plants were harvested42 days after exposure to nematode-infested soil, and the root mass267
 
Table 1
Presence and expression of 
aphI
»
,
gusA
and
OC
-
I
D86 genes in transgenic rice plantsClone no. Plasmid used
aphI
»
gusA OC
-
I
D86pHyg-OcI
D86(
aphI
»
and
OC
-
I
D86)pJIC200(
gusA
)PCR Resistance tohygromycinPCR Expression
Expression
ITA212 X1
# ! # # ! !
NDITA212 15
# ! # # ! !
NDITA212 X5
# ! # # ! !
ND or
(
0.1%ITA212 X4
# ! # # ! !
0.014%ITA212 X3
# ! # # ! !
0.1
— 
0.2%ITA212 4
# ! # # ! !
0.1
— 
0.2%ITA212 17
# ! # # ! !
0.02
— 
0.03%ITA212 45
# ! # # ! !
NDITA212 1
# # # # #
28958
(
0.1%IDSA6 Y1
# ! # # ! !
ND or
(
0.1%IDSA6 20
# # # # #
119 ND or
(
0.1%IDSA6 9
# # # # ! ! (
0.1%IDSA6 Y2
# # # # #
11474
(
0.25%IDSA6 2
# # # # #
0 ND or
(
0.1%IDSA6 3
# # # # #
255 ND or
(
0.1%IDSA6 4
# # # # #
54767 ND or
(
0.1%LAC23 6
# ! # # ! !
0.012%LAC23 3
# # # # #
0
(
0.25%LAC23 1
# # # # #
0 ND or
(
0.1%LAC23 2
# # # # #
0
(
0.1%LAC23 3b
# # # # #
22202
(
0.1%WAB56-1041
# ! # # ! ! (
0.25%WAB56-10424
# # # # #
7820 ND or
(
0.2%WAB56-10426
# # # # ! !
ND or
(
0.1%WAB56-10438
# # # # #
26 ND or
(
0.1%
#
, plasmid used;
!
, plasmid not used
In pmoles MU/min per mg protein after substraction of background level (37 pmoles MU/min per mg protein)
Inpercentageoftotalsolubleproteins.Eachwesternanalysiswasrepeatedtwice.ND
"
non-detected,
(
0.1%
"
detectedasinferior tothelower standard (here 0.1%) but not quantifiedwas cleaned thoroughly in water and blended for 10 s in a Waringblender with sodium hypochlorite diluted to give 1% availablechlorine. Nematode eggs were washed from the blended roots intoa large volume of water using a 1-mm sieve. The eggs were collectedon an open sieve, resuspended and counted in 1-ml aliquots usinga Peter’s counting slide (Southey 1986).
Results and discussion
Selection and regeneration of transformed plantsThe methodology described in this report allowed theproduction of highly embryogenic rice tissue and thesubsequent transformation of more than 30 African,South American and Asian elite varieties (Christouet al. 1991; Vain et al. 1997). Sixto eight weeks followingparticle bombardment, clear differential growth wasobserved between transformed (hyg
#
) and non-trans-formed (hyg
!
) calli during proliferation, regenerationand germination. Twenty-five independently trans-formed hyg
#
clones were selected from the fourAfrican genotypes tested in this study (Table 1).Transformation of the hyg
#
clones was further con-firmed by GUS, PCR, Southern and western blot ana-lyses of regenerated plants.Transformation frequenciesof up to 8% were obtained from all of the elite Africangenotypes tested.Analysis of transformed plantsThe regenerated plants (Fig. 1) were first screened byPCR using two primers (ACTCACCGCGACGTCTG-TCG forward 5
and GATCTCCAATCTGCGGGATCreverse 3
) amplifying a 1.3-kb fragment containing the
aphI
»
gene. All the plants regenerated from the 25independently transformed hyg
#
clones were PCR
#
for this set of primers. Southern blot analyses of regen-erated plants confirmed the integration of the pHyg-OcI
D86 plasmid into the rice genomic DNA (Vainet al. 1997).When both pHyg-OcI
D86 and pJIC200 (the
gusA
-containing plasmid) were used for transformation, theregenerated plants were also screened by PCR using
268
of 00

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