Antifungal Traits of a 14 kDa Maize Kernel Trypsin Inhibitor Protein in Transgenic Cotton

16 Pages·2008·3.35 MB·English

Antifungal Traits of a 14 kDa Maize
Kernel Trypsin Inhibitor Protein in
Transgenic Cotton
Kanniah Rajasekaran
Jeffrey W. Cary
Zhi-Yuan Chen
Robert L. Brown
Thomas E. Cleveland
ABSTRACT. Transgenic cotton plants expressing the maize kernel
trypsin inhibitor protein (TIP) were produced and evaluated for antifungal
traits. This 14 kDa trypsin inhibitor protein has been previously associated
with resistance to aflatoxin-producing fungus Aspergillus flavus. Successful
transformation of cotton (Gossypium hirsutum L.) and expression of
trypsin inhibitor was demonstrated by PCR and Northern analysis, respec-
tively. Proteins extracted from cottonseed and leaf tissues of transgenic
plants were separated using SDS-PAGE, and it indicated the presence of a
15-16 kDa protein in transgenic tissues as compared to control. Only
transgenic cottonseed tissue reacted with the TIP antibody, indicating the
expression in cottonseed. No cross-reaction to the TIP antibody was
K. Rajasekaran, J.W. Cary, R.L Brown, and T.E. Cleveland are affiliated with
the Food and Feed Safety Research Unit, Southern Regional Research Center,
Agricultural Research Service, United States Department of Agriculture, 1100
Robert E. Lee Blvd., New Orleans, LA 70124-4305.
Z.-Y. Chen is affiliated with the Department of Plant Pathology and Crop Physiol-
ogy, Louisiana State University Agricultural Center, Baton Rouge, LA 70803-2110.
The authors thank Kurt Stromberg, Mark Cambre, and Pamela Harris for their
excellent technical assistance.
Address correspondence to: Kanniah Rajasekaran at the above address
(E-mail: [email protected]).
Journal of Crop Improvement, Vol. 22(1) (#43) 2008
Available online at http://jcrip.haworthpress.com
© 2008 by The Haworth Press. All rights reserved.
doi:10.1080/15427520802042457 1
JOURNAL OF CROP IMPROVEMENT
detected from leaf extracts, indicating the TIP was either not expressed
or expressed at a level too low to be detected by Western blot. Crude
leaf extracts from transgenic cotton plants did not show significant
control of colonies from pre-germinated spores of A spergillus flavus or
Verticjf/jum dahljae;
however, extracts from transgenic cottonseed
tissue showed about 60% reduction of V. dahliae colonies, indicating
the antifungal nature of the maize TIP by itself. Cotton bolls inoculated
with a green fluorescent protein (GFP) expressing A. flavus strain
showed no difference among controls or transgenic cotton plants
indicating that the expression of TIP in cottonseed is not high enough to
prevent A. flavus colonization.
KEYWORDS. Aflatoxin, A.spergillusflavu.c,
disease resistance, Gossypium
hi,utum,
maize trypsin inhibitor, transgenic cotton,
Verijcj//ju,n
dahliae
ABBREVIATIONS. (TIP) trypsin inhibitor protein; (GFP) green
fluorescent protein; (GUS)
,B-glucuronidase; (PDA) potato dextrose
agar; (PDB) potato dextrose broth; (CFU) colony forming units
INTRODUCTION
Aspergillusfiavus and A. parasiticus
that produce aflatoxin in sev-
eral crop species including cotton, peanuts, tree nuts, and maize, are
not true plant pathogens but opportunistic, saprophytic fungi. Plant
defense mechanisms against phytopathogens are not specific to these
saprophytic fungi. Success in conventional breeding for resistance to
mycotoxin-producing or phytopathogenic fungi is dependent on the
availability of resistance gene(s) in the germplasm. Even when it is
available, breeding for disease-resistant crops is very time consuming,
especially in perennial crops such as tree nut crops, and does not lend
itself ready to combat the evolution of new virulent fungal races. Sev-
eral reports indicate the association between maize kernel morphology,
including wax and cutin in pericarp (Guo et al., 1995), or kernel pro-
teins (Huang, White, and Payne, 1997; Guo et al., 1998; Chen et al.,
1998; Brown et al., 2006) and resistance to aflatoxin accumulation.
Considerable progress has been made in identifying resistance markers
through proteomic comparisons of differentially resistant maize variet-
ies, thus indicating protein markers that could be used in breeding or
3
Rajasekarafl et al.
gene insertion technologies to enhance resistance in crops to aflatoxin
contamination. For example, after extensive analysis of kernel protein
profiles, Chen et at. (1998) demonstrated the preponderance of a
14 kDa trypsin inhibitor protein (TIP) in relatively high concentration
in kernels of seven maize genotypes that are naturally resistant to
infection compared to none or low concentration in six
A.flavus
susceptible ones. They concluded that the trypsin inhibitor protein may
not act alone in resisting A. flavus infection in kernels. The TIP was
also demonstrated to inhibit the growth of A. flavus and several other
pathogenic fungi, possibly through the inhibitory effect of TIP on
fungal a-amylase (Chen et al., 1999a, 1999b). The objective of our
research is to evaluate the antifungal activity of maize trypsin inhibitor
in transgenic cotton. Disease-resistant cottons have been developed
with some efficacy for resistance to a limited number of foliar and
vascular fungal pathogens (Murray et al., 1999; Emani et al., 2003;
Tohidfar, Mohammadi, and Ghareyazie, 2005; Rajasekaran et at.,
2005b). Emani et al. (2003) transformed cotton plants with a cDNA
clone encoding a 42 kDa endochitinaSe from the mycoparasitic fungus,
Homozygous T2 plants of the high endochitinase-
Trichoderma virens.
expressing cotton lines showed significant resistance against a soil-
borne pathogen, Rhizoctonia solani, and a foliar pathogen, Alternaria
alternata. Expressing the Talaromyces flavus glucose oxidase (GO)
gene in transgenic cottons resulted in a limited antifungal activity
against the root pathogen, V. dahliae (Murray et al., 1999). However,
these authors also discovered that the expression of GO in cottons resulted
in phytotoxicity and reduced yield. There are no known naturally resistant
among cotton cultivars. The availability of
varieties to Aspergillus
transgenic varieties with antifungal traits will be extremely valuable in
cotton breeding. Rajasekaran et al. (2005b) demonstrated the efficacy of
A. flavus,
transgenic cottons expressing a synthetic peptide in controlling
Fusariurn vertjcillioides; and• Thielaviopsis basicola. Thus, disease-
resistant transgenic crops would not only control mycotoxinprodUcing
organisms such as A. flavus, A. parasiticus, and Fusarium spp., but also
several other microbial (fungal, bacterial, and viral) diseases which
cause significant economic losses in crop production. Although
is often a polygenic trait in maize (Paul et al.,
resistance to A. flavus
2003; White et at., 1999), we conducted this study to evaluate th
individual contribution of the 14 kDa trypsin inhibitor protein from
maize kernels in controlling mycotoxinproducing organisms such as
and other microbial pathogens in transgenic cotton.
A. flavus
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MATERIALS AND METHODS
Construction of TIP Binary Vector and Agrobacterium
Transformation of Cotton
The maize TIP gene (Wen et al., 1992) was subcloned into a pVC-
based plasmid vector that placed it under the control of the CaMV double
35S
RNA-TMV 5' untranslated RNA leader promoter and the nopaline
synthase transcriptional terminator (
pUC-05SQ-nos) (Cary et al., 2000).
The TIP coding sequence including the N-terminal 28 amino acid signal
sequence was PCR amplified from maize genomic DNA using the prim-
ers 5' Ncol TIP,
S'-CCATGGCGTCGTCGTCTAGCAGCAGC 3' and 3'
Sacl TIP,
S'GAGCTCTFACTFGGAGGGCATCGnCCGc 3' and Pfu
polymerase (Stratagene). The amplified TIP gene was subcloned into
pCR-Script Cam (Stratagene) and the sequence confirmed by DNA
sequencing. The TIP coding region was ligated into
NcoI-SacI digested
pUC-d35SQ-nos vector following NcoI-SacI digestion of the pCR
-
Script-TIP vector to generate the vector
pUC-05SçT1P05 The
CaMVd355ç .. Jp
DNA region was introduced into the plant binary
vector pBIl 21 (referenced in Cary et al., 2000) by
Hind III-Sacl digestion
and ligation into Hind III-Sad digested pBIl21 to generate the vector
pBI-CaMV 05SQ-TIp
nos The binary vector was introduced into
Agrobacterium tumefaciens
strain LBA 4404 (Gibco-BRL, Bethesda,
Maryland) as described previously (Cary et al., 2000).
Agrobacjerjz4m
cultures for transformation experiments were initiated in 50 mL of YEB
liquid medium using frozen glycerol stocks (500 j.IL) as inoculum.Fhese
cultures were grown overnight for about 18 hat 26 ± 2°C on a gyratory
shaker. The optical density (A600) values were adjusted to 0.6 - 0.8 in
liquid MS medium prior to use. The A
transforma-
tion of seedling explants of cotton
(Gossypium hirsutum L. cv. Coker
312) was carried out using G418 (Agri Bio, Bay Harbor, FL) selection
according to the published protocols (Rajasekaran, 2004; Rajasekaran
et al., 1996, 2005b). Plants transformed with the PBId35SuidA..nos
construct (CGUS) served as controls. All the primary transformants and
their progenies were grown in a greenhouse until maturity.
PCR Analysis of Transgenjc Plant DNA
To determine if the CaMV 05SI-TJp region was integrated into
the cotton genome, a 620 bp segment of DNA
encompassing the
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Rajasekaran et aL
CaMV d35SQ promoter-TIP coding region was PCR amplified from
genomic DNA of transgefliC plants. Genomic DNA was isolated from
the C801 and C804 transgenic cottons and the cotton CGUS negative
control plant by extracting leaf disks using the REDExtract-N-Amp
Plant PCR Kit (Sigma, St. Louis, MO). Oligonucleotide primers
designed to an internal region of the CaMV d35SQ promoter, 5'-
ATGACGCACAATC CCACTATCCTTC -3' and to the 3' end of the
TIP gene, SGTTACTTGGAGGGT TT CCGC-3' were used to
amplify cotton plant genomic DNA using AmpliTaq Gold polymerase
(Applied Biosystems). The following thermocycler (PTC-100, MJ
Research Inc., Watertown, MA) parameters were used: I cycle of
95°C, 10 mm; 65°C, 30 sec.; 72°C, 30 sec.; 34 cycles of 95°C, I mm.;
65°C, 30 sec.; 72°C, 30 sec.; and a final extension of 72°C for 7 mm.
Presence of the nptll gene in cotton tissue was also determined by
PCR using the primers NPTII internal, 5'TTGCCGAATATT
GTGGAAAATGGCC3' and NPTII/nos term, 5'-GATAATCATCGC
AAGACCGGCAACAGG3. The expected product was 670 bp.
Therriocycler parameters were the same as above except annealing
was performed at 68°C. The pBICaMV35SQ-TIPfl05 plasmid DNA
was used as a positive control template. PCR products obtained were
analyzed by I% agarose gel electrophoresis followed by ethidium bro-
mide staining. Validity of PCR products was determined by DNA
sequencing.
Northern Hybridization
Total RNA was purified using the method of Bugos et al. (1995).
Ten micrograms of each RNA sample were electrophoreSed on a 2.2M
formaldehyde/l% agarose gel in 1X MOPS buffer followed by blot-
ting to nylon membranes (Nytran Plus nylon membranes, Schleicher
and Schuell Inc., Keene, NH) by vacuum transfer. The nylon mem-
branes were hybridized with a random-primed (Rediprime II Kit;
32P-dCTP-labelled 470 bp
Amersham Biosciences; Piscataway, NJ),
PCR fragment representing the TIP coding region. Hybridization was
performed overnight at 42°C in ULTRAhyb Buffer (Ambion Inc.,
Austin, TX) followed by two 5 min washes in 2 X SSC/0.l% SDS at
42°C, and a final wash for 15 min in 0.IX SSC/0.1% SDS at 50°C.
Nylon membranes were placed on Kodak X-OMAT AR autoradiogra-
phy film (Eastman Kodak, Rochester, NY) and allowed to expose
overnight with intensification at —80°C.
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DNA Sequencing
PCR products were subcloned into plasmid vector pCR-Script Cam
(Stratagene). The nucleotide sequence of the subcloned PCR products
was determined by non-radioactive sequencing using the ABI PRISM 377
Automated DNA Sequencer (Applied Biosystems, Foster City, CA) using
standard —21 M13 forward and M13 reverse primers. Sequence informa-
tion was analyzed using DNAMAN (Lynon Biosoft: Quebec, Canada)
analysis software.
Gel Electrophoresis and Western Blotting
Cotton leaf or immature seed (28 dpa) tissues of both transgenic and
control (2 g) were ground with liquid nitrogen and extracted with 10 mL
of extraction buffer (0.25 M NaCl, 50 mM Tris-HCI pH 8.0, 14 mM
,B-mercaptoethanol) for 2 h at 4°C. The supernatant was recovered by
centrifugation at 17,000 g. Protein concentration in the supernatant was
determined according to Bradford (1976). Then, 10 jig of each superna-
tant was separated on two SDS-PAGE gels (Laemmli, 1970). One was
stained with Coomassie R-250. The other was Western-blotted to nitro-
cellulose membrane and probed with the 14 kDa maize trypsin inhibitor
antibody (at 1:1000 dilution) raised in rabbit against the purified protein.
The trypsin inhibitor levels were quantified using gel densitometer.
In Vitro Analysis of Antifungal Activity of Plant Extracts
to A. flavus, F. verticillioides, and V. dahliae
The inhibitory activity of extracts from cotton plants transformed with
maize TIP gene was assessed in vitro following the method of Cary et al.
(2000). Briefly, conidial suspensions were prepared from cultures grown
on potato dextrose agar (PDA; Difco, Detroit, Ml) slants for seven days at
30°C (Aspergillus flavus) or 22°C (Verticillium dahliae). Conidial sus
pensions in I % (w/v) potato dextrose broth (PDB; pH 6.0) were adjusted
to a density of 105 conidialmL and were germinated in PDB for 8 h at
30°C (A.flavus) or overnight at 22°C (V. dahliae) prior to assay.
Plant homogenates were prepared by directly grinding cotton leaves or
immature cottonseeds (28 dpa) into a fine powder in liquid nitrogen with
no buffer added. Ground tissues were then centrifuged at 8200 x g for 10
min at room temperature and extract collected from each sample. Conic!--
ial suspensions (25 I.tl) were then added to 225 sl of plant extract, mixed,
and incubated for I h at 30°C (A.flavus) or 22°C (V. dahliae). Three 50 j.tl
Rajasekaran el al.
aliquots from each sample were then spread onto PDA plates and incu-
bated at 30°C or 22°C for 24-48 h and fungal colonies counted. One-
way ANOVA was used to determine the significance of the effect of
transgenic plant extracts on germinating conidia. Mean separations
were performed using the method of Tukey (Sokal and Rohif, 1981)
using the GraphPad Prism software (GraphPad Software Inc., San
Diego, CA).
In Planta bioassays
Construction and Transformation of the GFP Expression Vector
The EGFP gene (ClonTech) was placed under control of the constitu-
tively expressed A. nidulans glyceraldehyde phosphate dehydrogenase
(gpdA) gene promoter and the A. parasiticus nmt-1 gene transcriptional
terminator. All of these elements were subcloned into the plasmid vector
pBlueScript-SK (Stratagene) to produce the vector gpd-EGFP (Rajaseka-
ran et al. 2005b, 2008). Plasmid gpd-EGFP was cotransformed with the
A. parasiticus niaD gene into the niaD
vector 'pSL82 harboring the
mutant of A. flavus 70. One isolate stably expressing high levels of GFP,
designated A. Jlavus 70-GFP, was used in all experiments.
Boll Inoculation with A. flavus-70 GFP Strain
Cotton boils were inoculated with A. flavus 70-GFP according to the
published procedures (Rajasekaran et at., 2005b, 2008). Six to eight cotton
bolls (25-28 dpa) in each of greenhouse-grown controls and transgenic
plants were wounded in the center of one of the locuies to a depth of 5-10
mm with a 3 mm diameter cork borer. A small aliquot (10 ilL) of the
70-GFP suspension (104 conidialmL) was dropped into the hole,
A. flavus
and the fungus was allowed to coloniie boils for three weeks. Seeds from
each boll (50 dpa) were harvested separately and ground in phosphate
buffer (pH7.2; 50 mM), vortexed and centrifuged for 10 mm. at 10,000
RPM with a swing bucket rotor. One hundred Ill_ aliquots of supernatant
from each sample were placed in a 96-well Black Viewplate (Packard
Instrument Company, Meriden, CT), and the fluorescence was measured
535
at an excitation wavelength at 485 nm and an emission wavelength of
nm using the Perkin-Elmer HTS 7000 fluorometer. Relative fluorescence
values were obtained for each sample and were subjected to non-parametric
ANOVA using the GraphPad Prism software. GFP fluorescence output
due to A. flavus 70-GFP was expressed per g of seed cotyledon tissue.
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RESULTS AND DISCUSSION
Traizsforn,atjo,, of Cotton
There was no difference between controls and the transgenic cotton
plants with regard to plant morphology, flowering, and seed set. Three
putatively transformed cotton lines with the maize trypsin inhibitor gene
(C801, C802, C804) were extensively analyzed for disease resistance
along with the CGUS negative control plants, transformed with the visual
marker gene only (CGUS). A minimum of 10 RI plants was used in each
transgenic line and control for all the assays. All these plants were regen-
erated from callus cultures subjected to selection at a sublethal level (10
g/mL) of the antibiotic G418.
PCR Analysis
The presence of the CaMV d35SQ promoter-TIP coding region in
DNA isolated from cotton leaf tissue was determined by PCR (Figure 1).
Analysis of DNA isolated from Ri transgenic cotton tissues and control
DNAs showed that the expected PCR product of 620 bp representing the
CaMV d35SQ promoter-TIP DNA region was present in tissues from
transgenic plants C801 and C804, as well as the pBI-CaMVd35SçJp
plasmid positive control DNA, but not in C802, an escape. No PCR prod-
uct was detected in CGUS, transformed with p13I121 or the C802 DNA.
The presence of a 670 bp PCR product representing an internal region of
the nptll gene was also confirmed by PCR in all samples except the C802.
This indicated that cotton C802 was a non-transformed escape.
Northern Analysis
Northern hybridization analysis of total RNAs isolated from RI cotton
plants C801, C802, C804, and cotton CGUS was performed with p32_
labeledTIP gene probe (Figure 2). Hybridization signals at the expected
size of about 0.55
kb were detected for C801 and C804 while the cotton
p131121 and C802 (a non-transformed escape) samples did not demon-
strate transcripts that hybridized with the probe as expected.
Gel Electrophoresis and Western Blotting
Proteins were extracted from cottonseed and leaf tissues, and separated
using SDS-PAGE (Figure 3). A 15-16 kDa protein was visible in the
transgenic cottonseed, but missing in the control seed. A protein of similar
Rajasekarafl et al.
FIGURE 1. PCR analysis of genomic DNA isolated from cotton leaf
tissues. DNA from putative transgenic cotton and control DNAs were
nptll
analyzed for the presence of the TIP coding region as well as the
gene (see Materials & Methods). Note: PCR results showed that both
cotton plants C801 and C804 were transgenic, while the C802 sample did
not generate PCR products with either primer pair indicating that this
plant was a non-transformed escape. Plasmid=pBI-CaMV d35SQ-TIP-
nos DNA; No DNA sample had no template DNA added to the PCR
reaction.
Z
Z U U U U
TIP
6?() bp
Nfl II
7O bp
size was also visible in all leaf protein extracts (including the control leaf
extract). In order to determine whether this protein band in the leaf tissue
is the maize trypsin inhibitor protein or the small subunit of ribulose-1,5-
biphosphate carboxylase/oxygeflaSe (which has a molecular mass of
about 15.5 kDa), a duplicate SIDS-PAGE gel was blotted to a nitrocellu-
lose membrane and probed with an antibody raised against maize trypsin
inhibitor protein (Figure 3). Only the protein extracts from the transgenic
cottonseed tissues reacted with the TIP antibody, indicating the expres-
sion of maize TIP in RI cottonseed. The presence of two bands in the
Western may indicate the presence of two forms of TIP: unprocessed TIP
10
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FIGURE 2. Northern hybridization of cotton leaf total RNA with
radiolabelled maize TIP probe. Equivalent amounts of total RNA (1 0ig) of
each sample were loaded in each lane. This was confirmed by ethidium
bromide staining (data not shown). The membrane was probed with a
P32-labeled DNA fragment representing the TIP coding region. The
expected transcript of about 0.55 kb was observed for cotton C801 and
C804 samples, while no transcript was detected for the C802 escape and
CGUS samples as expected.
kb
U U U
27
1.35
0.55
0.24-
with the leader sequence (top band) and the mature TIP, after removal of
the leader sequence (lower band). No cross-reaction with the TIP antibody
was detected from leaf extracts, indicating the TIP was either not expressed
or expressed at a level too low to be detected by Western blot.
In Vitro Assay of AntifungalActivity
There was no significant antifungal activity from crude leaf extracts of
the transgenic RI cotton plants compared to controls expressing the GUS
marker gene only when assayed against pre-germinated conidia
(Figure 4) or V. dahliae (Figure 5). However, extracts from seed ocfo Aty.lJelrdno'unss
from the RI transgenic cotton plants C801 and C804 showed significant
reduction (an average of 60%) against V. dahliae compared to CGUS
controls and the non-transformed escape C802 (Figure 6).
V. dahliae is one
of the very sensitive pathogens (for example, against a synthetic peptide,
[Rajasekaran et al., 20011), and it appears that it is susceptible to maize
trypsin inhibitor protein as well.