.I. 1n.w~ Phyhl.
Pergamon
Vol. 40, No. IO, pp. 893-900. 1994
Science Ltd
Printed in Great Britain. All rights reserved
CopyrightCc 1994Elsewer
0022-1910(94)00047-6
0022-1910194 $7.00 + 0.00
Inhibition of Diabrotica Larval Growth by a
Multicystatin from Potato Tubers
GREGORY
L. ORR,*j.
JAMES
Received 6 Januar_v 1994; reked
A. STRICKLAND,*
TERENCE
A. WALSH*
17 March 1994
Second instar western corn rootworm larvae have a higher proportion of total proteinase activity which
is attributable to cysteine proteinase (92%) than southern corn rootworm larvae (75%). E-64, potato
multicystatin (PMC) and tryptic fragments of PMC (T-PMC) are effective inhibitors of gut cysteine
proteinase activity, in vitro. The presence of PMC in the diet causes a dose-dependent inhibition of
growth in neonate southern corn rootworm and second instar southern corn rootworm and western corn
rootworm. Neonate southern corn rootworm and second instar western rootworm have similar
sensitivity to the inhibitor (50% inhibition at 25-43.8 pg/cm*), whereas second instar southern corn
rootworm are about S-fold less sensitive. In contrast to southern corn rootworm larvae, western corn
rootworm growth is completely halted by PMC. Long-term exposure of southern corn rootworm larvae
to PMC suggests that the larvae become less sensitive to the inhibitor during development. Hen egg
cystatin (HEC) and T-PMC are unable to inhibit growth of either species but, in southern corn
rootworm, co-feeding of potato carboxypeptidase
inhibitor (PCI) with T-PMC causes growth
inhibition. Direct measurements of gut cysteine proteinase activity after feeding of the inhibitors
indicates that PMC and PC1 + T-PMC cause significant inhibition of cysteine proteinase in the gut,
whereas HEC, PC1 and T-PMC do not. These observations indicate that multicystatins such as PMC
may be effective cystatins for use in controlling larvae of Diubrotica species in transgenic plants.
Cystatin
Diabrotica
Rootworm
Growth inhibition
INTRODUCTION
A role for plant proteinaceous
proteinase
inhibitors
in
deterring phytophagous
insect pests is suggested by the
widespread occurrence of these inhibitors in plants and
their increased
production
and accumulation
induced
by insect feeding (Green and Ryan, 1972; Ryan, 1990).
Direct evidence in support of this hypothesis is limited
to observations
from diet incorporation
assays (see
below) and protection provided by these inhibitors when
expressed in transgenic
tobacco plants (Hilder et al.,
1987, 1993; Johnson et al., 1989).
In general, the proteinase classes found in the digestive
tracts of phytophagous
insect pests are either serine
which predominate
in Lepidoptera,
or
proteinases,
cysteine proteinases,
the major proteinase class found in
many Coleoptera.
Plant serine proteinase inhibitors are
effective against insect serine proteinase activity in d-0
(Hamad and Attias, 1987; Broadway,
1989; Houseman
et al., 1989; Johnston
et al., 1991) and many have
been shown to inhibit
growth and development
of
lepidopteran
larvae when incorporated
into artificial
diets (Gatehouse
et al., 1979; Gatehouse
and Boulter,
*DowElanco.
Biotechnology
Department,
Cl/306
Indianapolis,
IN 46268-1053, U.S.A.
?To whom correspondence
should be addressed.
9410 Zionsville
Rd,
Proteinase inhibitor
1983; Shukle and Murdock,
1983; Broadway
and
Duffey, 1986; Hilder et al., 1990; Johnston et al., 1993).
Expression
of cowpea trypsin inhibitor
(Hilder et al.,
1987) and tomato inhibitor II (Johnson et al., 1989) in
tobacco plants has proven effective in reducing feeding
damage by lepidopteran
larvae. Similar investigations
with coleopteran
pests are less numerous.
However,
oryzacystatin,
a cysteine proteinase inhibitor (CPI) from
rice, hen egg cystatin (HEC) and a non-proteinaceous
cysteine proteinase inhibitor,
E-64, have been shown to
inhibit the activity of midgut cysteine proteinases in vitro
(Murdock et al., 1987; Weiman and Nielsen, 1988; Thie
and Houseman,
1990; Liang et al., 1991; Gilliken et al.,
1992). Deleterious
effects on growth and development
in Coleoptera
have been reported
for oryzacystatin
against red flour beetle (Chen et al.. 1992) and with E-64
against bean weevil (Hines et al., 1990), cowpea weevil
(Murdock
et al., 1988) and Colorado
potato beetle
(Wolfson and Murdock,
1987). On the basis of these
observations,
it has been suggested that cystatins, such
as oryzacystatin
and HEC, could provide
effective
protection
against coleopteran
pests when expressed in
transgenic cereals (Liang et al., 1991; Chen et al., 1992)
or maize (Gilliken et al., 1992).
We have characterized
(Walsh and Strickland,
1993)
and cloned (Waldron
et al., 1993) a unique cystatin
GREGORY
894
(potato multicystatin, PMC) from potato tubers consisting of eight tandem 10.8 kDa cystatin domains
linked by proteolytically-sensitive junctions. PMC can
be cleaved by trypsin into five single domains and a
three-domain cystatin that all retain inhibitory activity
in vitro. In contrast, HEC, oryzacystatin and cystatins
from other plant sources are composed of a single
I1 kDa inhibitory domain. The presence of PMC in
the peridermal cells of the tuber, its wound inducibility
in leaves and its solubility in mildly acid environments suggest it could play a defensive role against
coleopteran pests. In the present study we compare the
effects of PMC, trypsin-treated PMC and single domain
cystatins on larval growth and cysteine proteinase
activity in vivo and in vitro using southern and western
corn rootworm larvae. We also discuss the potential for
using cystatins to confer rootworm resistance in transgenie maize.
MATERIALS AND METHODS
Insect bioassay
Proteins to be tested were dissolved and diluted in
sterile water, applied (0.03 ml) to the surface of 0.25 ml
artificial diet (adapted from Rose and McCabe, 1973)
in 24-well microtiter plates and allowed to air dry in
a sterile flow hood. This technique resulted in a very
thin (approx. 1.0 mm) layer of diet which reduced
the potential for larvae to burrow through the diet
and avoid contact with the active material. We observed
little or no tunneling even with second instar larvae.
Eggs of southern corn rootworm (Diabrotica undecimpunctata
howardi)
and western corn rootworm
(Diabrotica virgijka virgfira)
were obtained from
French Agricultural
Research (Lamberton,
MN).
Individual wells were then infested with either a neonate larva hatched from surface-sterilized eggs or a
preweighed (2.5-3.0 mg) second instar southern or western corn rootworm reared on corn seedlings. The plates
were then incubated at 26°C in sterilized, sealed plastic
containers for 6 days (neonate southern corn rootworm)
or 3.5 days (second instar southern corn rootworm and
western corn rootworm) prior to final weighing. In
long-term studies, the larvae were weighed after 1 week
and then placed on fresh untreated or treated diet as
indicated.
Protein purijication
PMC was purified from the peel of tubers purchased
from a local market essentially as described by Rodis
and Hoff (1984). Typical yields were 10-50 mg pure
PMC from 10 lb of tubers. Proteolytically-cleaved PMC
(10 and 32 kDa products, T-PMC) was produced by
digestion with trypsin (Sigma Type XIII, PMC-trypsin,
20: 1, w/w) in 50 mM Tris-Cl, pH 7.5 at 37°C for
2 h. Following digestion, trypsin was inactivated by
addition of 3,4-dichloroisocoumarin
(0.1 mM final concentration)
to the digestion. Separate experiments
L. ORR et al.
showed that 3,4-dichloroisocoumarin
had no effect on
cysteine proteinase activity. Size exclusion chromatography of T-PMC was performed using a Pharmacia
(Piscataway,
NJ) Superose- 12 column
calibrated
with protein standards from BioRad. In all cases, homogeneity and complete digestion was determined by
SDS-polyacrylamide
gel electrophoresis via a Phastgel
System (Pharmacia). HEC was purchased from TaKaRa
Biochemical Inc. (Berkeley, CA), dialyzed against H,O
and stored at 4°C prior to use. Papain inhibition
titrations with HEC and T-PMC were performed (see
below) to confirm inhibitory activity before bioassay.
Benzoyloxycarbonyl - Phe - Arg - 7 - (4 - methyl)coumarylamide (Z-Phe-Arg-MCA) was purchased from Bachem
Inc. (Torrance, CA). Proteins were quantified using a
BioRad protein assay kit or determined spectrophotometrically at 280 nm using E,, = 12.0. Unless otherwise
reagents were purchased
from Sigma
indicated,
Chemical Co. (St Louis, MO).
In vitro assay of cysteine proteinase activity
Insect gut extracts were obtained from 100 individual
second instar western corn rootworm or southern corn
rootworm larvae (reared on corn roots) with average
weights of 9.35 + 0.1 or 9.15 f 0.1 mg, respectively.
Alimentary canals were dissected and placed into
an Eppendorf tube on ice containing 200 pl of assay
buffer (200 mM sodium acetate, 8 mM dithiothreitol
and 4.0 mM sodium ethylenediaminetetraacetic
acid,
pH 6.0). After dissection, the samples were vortexed
and centrifuged (13,OOOg, 2 min). The extracts (supernatants) were then recovered and the remaining pellets
were washed with 200 ,ul assay buffer and recentrifuged
as described above. The supernatants were then pooled,
protein content determined, and frozen for future
use. Cysteine proteinase inhibition by PMC, T-PMC,
E-64 and HEC was measured by determining cysteine
proteinase activity using either N-a-benzoyl-~-argininep-nitroanilide (BAPNA), FITC-casein or Z-Phe-ArgMCA as substrates.
Assays involving BAPNA as substrate were performed as follows: in wells of a microtiter plate, 50~1
of inhibitor solution at various concentrations was
mixed with either 50 pl of 100 pg/ml papain (217 pmol)
solution or 50 p-11
of 300 pg/ml insect gut protein extract
in assay buffer. After 10 min preincubation, 100 ~1 of
2 mM BAPNA in 100 mM sodium acetate, pH 6.0, was
added and allowed to incubate at 37°C for 1.Oh. The
absorbance at 405 nm was recorded using a microplate
reader.
When using Z-Phe-Arg-MCA as a substrates, 500 pl
of a 17.0 pg soluble protein/ml rootworm gut extract
was incubated with 100 ng of inhibitor and allowed to
incubate at 25°C for 30 min. After incubation, 100 pl of
this reaction mixture was added to 0.2 ml of Z-Phe-ArgMCA (20 mM final concentration).
After reacting
for 3 min, data were collected using a Fluoroscan II
fluorescent microtiter plate reader (Labsystems Oy,
Research Triangle Park, NC) linked to a Biometallics
INHIBITION
OF CORN
data collection software system with kinetic capabilities.
Excitation
and emission
wavelengths
were 380 and
460 nm, respectively. Data were collected at a single time
point. Assays using FITCcasein
as a substrate
were
performed
as follows: 200 ~1 of a 0.5% FITCcasein
solution in assay buffer was added to gut extract (7.5 pg
soluble protein) for a final reaction volume of 250 ~1.
Reactions were allowed to proceed for 20 min at room
temperature
and were then stopped by the addition of
50 ~1 of 10% trichloracetic
acid. After precipitation
for
30 min and centrifugation
(13,000 g, 5 min), 100 ~1 of the
supernatant
was removed, placed in a microtiter
plate
and 100 ~1 of 2N NaOH was added. Data were collected
at a single time point using a Fluoroskan
II fluorescent
microtiter
plate reader as described above with excitation and emission wavelengths
at 485 and 538 nm,
respectively.
61 viva q3teine proteinuse activity
In these assays, inhibitors
were applied to the diet as
described above at a final concentration
of 62.5 pgg/cm’.
The diet was placed in a sterile Petri dish and 20 second
instar southern corn rootworm
or western corn rootworm were allowed to feed for 3 h. After feeding, the
alimentary
canal
from each individual
larvae was
removed and placed in individual,
prechilled Eppendorf
tubes containing
50 ~1 assay buffer and vortexed vigorously. Samples were then frozen on dry ice and stored
at -70°C.
For assay, samples were thawed on ice and
then vortexed gently. The samples were then centrifuged
( 13,000 g, 5 min) and the supernatants
were assayed for
activity. Assays were performed
in 96-well microtiter
plates. 5 ~1 of each extract was added to 50 ~1 of assay
buffer and allowed to preincubate
for IOmin at 22°C.
200 ~1 of Z-Phe-Arg-MCA
was added and initial rates
on extracts from each individual
gut were collected as
described
above. Data are expressed as fluorescence
units/min.
ROOTWORM
GROWTH
895
attributable
to cysteine
proteinases
can be derived
from the extent of maximal inhibition
by E-64. The
gut extract contained
quite different levels of soluble
protein
(1.3 fig/midgut
for western
corn rootworm.
2.9 pg/midgut
for southern corn rootworm) despite the
larvae from which the extracts were obtained being the
same size and reared on the same natural corn root diet.
The results of the titrations are therefore best compared
on a larval midgut basis.
Southern
corn rootworm
midguts contained
more
total proteolytic
activity
than western
corn rootworm midguts
(74.0 FU/midgut
cf. 41.6 FU/midgut).
Of the total activity in southern corn rootworm
larval
gut extracts, 75% was maximally
inhibited
by E-64,
whereas 92% was maximally
inhibited by E-64 in the
western corn rootworm extracts (Fig. 1). Other experiments showed that the residual proteolytic
activity
after maximal
E-64 inhibition
could be inhibited
by
pepstatin-A,
an inhibitor
of aspartic
proteinases.
Based on titrations
with E-64, southern
corn rootworm midguts contained
3.8 pmol cysteine proteinase/
midgut
which can be inhibited
with 0.74 pmol of
PMC (data not shown). Western corn rootworm larvae
contained
3.0 pmol cysteine proteinase/midgut
which
was inhibited
by 0.5 pmol of PMC (data not shown).
From these data it can be calculated
that PMC is
38% less effective than E-64 (assuming
8.0 proteinase
inhibition
sites/molecule
of PMC and 1.O/molecule
of E-64). Therefore,
although
southern
corn rootworm midguts contained
greater amounts
of cysteine
proteinases,
this activity represented
a smaller proportion of the total proteolytic activity than in western
corn rootworm.
The ability of PMC and T-PMC to inhibit the cysteine
proteinase component
of rootworm gut extracts in oitro
was compared
to that of E-64. Z-Phe-Arg-MCA
was
used as a cysteine proteinase-specific
synthetic substrate.
Statistical unalWis
Statistical analysis was performed
using the Instat”
computer
program
(GraphPad
Software,
San Diego,
CA). Statistical
significance
was declared at P values
less than 0.05. Values stated in the manuscript
reflect the
calculated level of significance.
RESULTS
In vitro inhibition of rootworm midgut proteolytic activity
by E-64 and PMC
Titrations
of proteolytic
activity
in second instar
southern corn rootworm and western corn rootworm gut
extracts with E-64 were performed using FITC-casein
as
a sensitive, generic substrate (Fig. 1). As E-64 is a potent,
specific inhibitor
of cysteine proteinases,
the absolute
amount of cysteine proteinase
within the extracts can
be quantitated
from the linear stoichiometry
of the
titration.
The proportion
of total proteolytic
activity
0
5
10
15
20
E-64 (pmol)
FIGURE
I. Titration
of total proteolytic
activity of southern corn
rootworm (m) and western corn rootworm (0) gut extracts with E-64.
The gut extracts contained
7.5 pg soluble protein, equivalent
to the
contents of 2.6 southern corn rootworm midguts and 5.8 western corn
rootworm midguts. The values are from two separate experiments.
GREGORY
896
L. ORR
et al.
Efect
of long-term
exposure to PMC
southern corn rootworm larvae
1M)
80
60
40
20
0
Control
E-64
T-PM?.
PMC
Treatment
FIGURE
2. Effect of potato multicystatin (PMC), trypsin-treated
PMC (T-PMC) and E-64 on cysteine protease activity of southern corn
rootworm
gut extracts using Z-Phe-Arg-MCA
as substrate.
100ng
inhibitor
was incubated
with gut extracts
containing
8.5 pg total
protein. Data arc expressed as percentage
of control containing
no
inhibitor.
on growth
on
To determine
the long-term
effects of exposure to
PMC, diet was treated with 125.0 pg PMC/cm* and
neonate southern corn rootworm were allowed to feed
for 1 week, They were then weighed and placed onto
fresh diet (with or without PMC) and monitored
again
after a second week to determine the effect on growth
(Table 1). After 1 week the growth inhibition with PMC
was 68%. Replacing the PMC diet with control diet after
the first week resulted in a recovery in growth over the
second week such that the larvae were not significantly
different in size from those not exposed to PMC (14.4 vs
18.8 mg, respectively). Those insects which were continuously challenged with PMC over the 2-week period did
not remain stunted but increased in size L&fold (from 1.4
to 10.6 mg) over the second week. However, these larvae
were significantly
smaller than controls. No mortality
Figure 2 shows that both PMC and T-PMC are effective and complete
inhibitors
of rootworm
cysteine
proteolytic
activity, and equivalent
to E-64. In vitro
titrations of total proteolytic activity by PMC and HEC
using FITC+asein
inhibited
the same proportion
of
activity as E-64 (data not shown). These data indicate
that all E-6Csensitive
cysteine proteinases in the midgut
are sensitive to these cystatins in vitro.
0
EfSect of PMC on southern corn rootworm
corn rootworm larval growth.
20
40
60
and western
Dose-response
experiments
were performed to determine the potency and effectiveness of ingested PMC on
neonate
and second instar rootworm
larval growth.
Studies could not be done with neonate western corn
rootworm
as an adequate
artificial diet which would
support growth was not available. PMC caused a dosedependent
inhibition
of neonate
southern
corn rootworm growth [Fig. 3(A)] with 50% growth reduction
seen at approx.
25.0Z~gg/cm* (approx.
0.02% w/w).
Maximal growth inhibition (about 70%) was observed at
31.25 pgg/cm2 and did not increase at doses as high as
125.0 pgg/cm2 (0.1% w/w). With second instar southern
corn rootworm,
a much reduced response to PMC was
observed [Fig. 3(B)]. A 50% reduction in larval size was
not seen until a dose of approx. 125.0 pg PMC/cm* diet
was used. This represents a 5-fold reduction in effectiveness between these stages. Interestingly,
second instar
western corn rootworm were similar to neonate southern
corn rootworm in their sensitivity to PMC (50% growth
inhibition at 43.75 pg/cm2). Although western corn rootworm growth was completely arrested at 125.0 pg/cm*
diet, no larvae died within the time frame tested. Higher
doses of PMC did not induce mortality in either species
(data not shown).
80
100
120
140
PMC (pg/cm*)
._
- 5.0
14
12
- 4.0
10
- 3.0
8
- 2.0
6
4
-1.0
2
- 0.0
0
_0
2-o
40
60
80
100
120
140
PMC (vglcm*)
FIGURE
3. Effect of potato multicystatin
(PMC) on the growth of
corn rootworm larvae. (A) Neonate southern corn rootworm. Control
growth was 3.63 + 0.23 mg. Values are the mean f SEM for observations obtained in 2 separate experiments. (B) Second instar southern
(SCR) and western (WCR) corn rootworm.
Control
growth was
14.73 k 0.59 and 6.67 f 0.34 for southern
and western corn rootworm, respectively. Values are the mean & SEM for observations
from
2 separate experiments.
INHIBITION
OF CORN
ROOTWORM
TABLE
I. Effect of long-term
exposure
to potato
multicystatin
(PMC) on growth of neonate southern
corn rootworm
larvae
TABLE
Treatment
Treatmentd
Weight
Week I”
Control
PMC’
(mg)
Control
PMC’
T-PMC
PC1
T-PMC
HEC
4.21 + 0.16 (22)”
1.36 k 0.09 (49)d
Week 2
Control
PMC to control
PMC
18.83 * 0.97 (22)
14.35 k 2.16 (21)
10.56 + 1.76 (22)
diet
in those
insects
continously
EfSect of single domain cystatins
exposed
on rootworm
to
growth
The ability
of T-PMC
to inhibit
gut proteinase
activity in vitro suggested that this form of PMC may
also be effective in inhibiting
larval growth
when
ingested by the larvae. Feeding studies indicated
that
this was not the case (Table 2). Ingestion
of T-PMC
caused only limited effects in either neonate southern
corn rootworm or second instar western corn rootworm
(4.8 and 18.7% inhibition,
respectively).
Although
the
effect on western corn rootworm larvae was marginally
significant
statistically,
it is clearly much less active
than intact PMC at this dose (60.7% inhibition).
HEC,
TABLE
2. Effect of cystatins on growth of neonate
second instar western corn rootworm
Weight
Treatment”
Southern
corn rootworm
Control
PMC’
T-PMC’
PCI’
T-PMC + PC1
HEC’
3.30+0.11
2.26 f 0.14
3.14 _+ 0.19
3.60 IO.14
2.15+0.15
3.11 LO.17
(136)b
(72)d
(54)
(67)
(67)’
(61)
southern
and
gain (mg)
Western
corn rootworm
6.99
2.75
5.68
5.62
5.13
5.71
& 0.31
& 0.33
+ 0.33
f 0.51
* 0.39
k 0.52
897
3. Comparison
of cysteine protease activity in southern
rootworm
larval midgut following feeding of cystatins
+ PC1
Protease
(fluorescence
284.2
97.6
228.6
216.4
156.9
238.6
activity
units/min)
k
i_
k
+
+
f
21 .9h
10.9d
22.0
20.8
14.9d
22.8
corn
% of control
100
34
80
76
55
84
“Additions to the diet were 62.5 pg/cm’.
‘Values are the mean + SEM for data from 3 separate
experiments.
‘T-PMC, trypsin-treated
potato multicystatin
(10 and 32 kDa fragments combined); PCI, potato carboxypeptidase
inhibitor; HEC,
hen egg cystatin.
dSignificantly
different
from control,
P < 0.01 (Dunnet
Multiple
Comparison
Test).
“Insects were placed on diet for 1 week, weighed and
placed on fresh treated or untreated diet for another
week and weighed again.
‘Values are the mean + SEM for the number of determinations in parentheses
from a single experiment.
‘PMC was used at 125 pg/cm’ diet.
‘Significantly
different from control,
P < 0.0001 (unpaired t-test).
Significantly
different from control. P < 0.05 (Dunn’s
Multiple Comparison
test).
was observed
PMC.
GROWTH
(54)
(54)d
(52)
(41)
(40)d
(33)
“All treatments
were 3 1.25 pg/cm’ diet.
%alues
are the mean f SEM for the number
of observations
in
parentheses
derived from 6 separate experiments.
‘PMC = potato multicystatin.
T-PMC, trypsin-treated
potato multicystatin (for the southern corn rootworm
this was 10 kDa fragments only, for the western corn rootworm this was 10 and 32 kDa
fragments
combined);
PCI. potato
carboxypeptidase
inhibitor;
HEC, hen egg cystatin.
dSignificantly
different
from control,
P < 0.01 (Dunnet
Multiple
Comparison
Test).
Significantly
different
from control,
P < 0.05 (Dunnet
Multiple
Comparison
Test).
a single domain
cystatin
molecule,
also had limited
activity in both species although
it was an effective
inhibitor
of gut proteolytic
activity in vitro. Similar
results were seen with another single domain cystatin,
oryzacystatin,
isolated from rice (data not shown). The
lack of growth inhibitory
activity seen with these proteins may be due to their inability
to be effective in
the environment
of the midgut. Inclusion
of potato
carboxypeptidase
inhibitor (PCI) with T-PMC resulted
in restoration of the growth inhibitory effect on southern
corn rootworm.
PC1 alone had no significant effect on
the larvae. Although statistically different from control,
combining
PC1 with T-PMC did not restore growth
inhibition
in western corn rootworm larvae (the effects
of PCI, T-PMC and PC1 + T-PMC were not significantly different). Other proteinase
inhibitors
(pepstatin
and aprotinin)
had no significant synergistic effect (data
not shown).
In vivo measurement of cysteine proteinase
southern corn rootworm larval midguts
activity
in the
Given the growth inhibitory
effects observed above
and the in vitro activity of PMC, T-PMC and HEC
against gut-derived cysteine proteinase activity. it was of
interest to determine
if these observations
correlated
with changes in proteolytic
activity in the larval gut.
To ascertain this, we fed second instar southern corn
rootworm
larvae various inhibitors
and monitored
the
resulting changes in gut cysteine proteinase
proteolytic
activity
using a fluorescence-based
assay (Table 3).
Consistent
with its proposed
mode of action, PMC
caused a 72% reduction
in gut cysteine proteinase
activity when ingested. T-PMC, PC1 and HEC did not
cause a significant
reduction
in proteolytic
activity.
However, combining T-PMC with PC1 resulted in a 45%
reduction
in proteinase
activity. Although
this is less
inhibition than seen with PMC, the increase in inhibition
is consistent
with the observation
of enhanced growth
suppression
with co-feeding of PC1 and T-PMC in first
instar larvae.
GREGORY
898
DISCUSSION
Cysteine proteinase activity is the primary proteolytic
mechanism in the rootworm larval midgut (Murdock
et al., 1987; Purcell et al., 1992; Gilliken et al., 1992).
On the basis of in vitro inhibition of gut proteinases, it
has been proposed that CPIs similar to E-64 (Murdock
et al., 1987) or cystatins such as HEC (Gilliken et al.,
1992) could be useful in controlling rootworm if a
delivery mechanism could be established. In the present
study we have confirmed previous work (Purcell et al.,
1992; Gilliken et al., 1992) by showing that cysteine
proteinases comprise a large proportion of total proteinase activity in second instar rootworm gut and, in
addition, we demonstrate that PMC is as effective as
E-64 against rootworm cysteine proteinase in vitro.
Our previous work (Walsh and Strickland, 1993) established that the cystatin fragments produced by tryptic
digestion of PMC (5 single-domain cystatins and a
three-domain cystatin) are potent inhibitors of papain
(a model cysteine proteinase) and we have now demonstrated that these inhibitors are also effective against
rootworm digestive cysteine proteinases in vitro. These
data would suggest that PMC, its tryptic fragments and
HEC would all be active inhibitors of cysteine proteinase
in vivo and should have deleterious effects on the
larvae when ingested. Although methodological differences make a direct comparison of the present study to
the cited work difficult, PMC would appear to be among
the most potent growth-impairing proteinase inhibitors
reported to date. Indeed, the growth inhibitory effect
produced by PMC appears substantially more potent
against southern corn rootworm larvae than recombinant oryzacystatin against red flour beetle (Chen et al.,
1992) and is comparable to the activity of E-64 against
bean weevil and cowpea weevil (Hines et al., 1990;
Murdock et al., 1988). PMC is also comparable in
potency to a variety of serine proteinase inhibitors (e.g.
cowpea trypsin inhibitor, soybean trypsin inhibitor and
potato proteinase inhibitor II) assayed against lepidopteran larvae (Gatehouse et al., 1979; Gatehouse and
Boulter, 1983; Spates and Harris, 1984; Broadway and
Duffey, 1986).
Maximal inhibition of first instar southern corn rootworm growth (approx. 70%) is seen at 3 1.25 pgg/cm’ diet,
but increasing the concentration to 125 pg/cm* has no
additional effect. This suggests that the larvae have
a population of proteolytic enzyme(s) not sensitive to
PMC that are able to generate sufficient amino acids to
sustain limited growth. Consistent with this hypothesis
is the 5-fold reduction in sensitivity to PMC observed in
second instar larvae as compared to neonates. Such a
direct measurement of a stage-dependent shift in proteinase inhibitor susceptibility has not previously been
reported and may reflect an alteration in proteinase
complement during development which makes second
instar larvae less reliant upon PMC-sensitive cysteine
proteinases. This is confirmed by in vitro studies showing
a significant proportion of total proteinase activity
L. ORR et al.
which is insensitive to E-64 and PMC in second instar
larvae. Targeting of this residual proteolytic activity
could synergize the effects of PMC and result in a more
complete growth inhibitory and/or lethal effect.
A consequence of the decreased sensitivity in later
stage larvae is seen in long-term studies that show the
larvae’s ability to recover from acute exposure to PMC
and adapt to the inhibitor over time. In the field, the net
result of this adaptation may be a delay in developmental time which may or may not translate into a significant
reduction in crop damage. The ability of colepteran
species to overcome continuous exposure to dietary
CPIs has been reported (Hines et al., 1990) although
high levels of E-64 and oryzacystatin can induce mortality in bean weevil (Hines et al., 1990), cowpea weevil
(Murdock et al., 1988) and red flour beetle (Chen et al.,
1993). These observations point out the potential
difficulties in delivering sufficient PMC (or other CPIs)
to produce meaningful growth inhibitory or lethal effects
in certain colepteran larvae. However, with southern
corn rootworm larvae, simultaneous delivery of multiple
proteinase inhibitors (including PMC and other CPIs)
with different specificities could produce a lethal and/or
long-term effect.
Second instar western corn rootworm larvae are
similar to neonate southern corn rootworm in sensitivity
to PMC but differ in that growth can be halted at doses
of 125 yg PMC/cm* diet. Technical problems make
studies with neonate western corn rootworm difficult but
it is likely that neonate western corn rootworm are
at least as sensitive to CPIs as second instar larvae.
This enhanced effect of PMC in western corn rootworm
may be attributable to the larger proportion of cysteine
proteinase in the gut and the apparent inability of the
less predominant proteinase species to provide sufficient
amino acids to maintain growth in the presence of high
levels of PMC. This greater dependency on a single class
of proteinase may be related to the oligophagous nature
of western corn rootworm larvae, whereas southern corn
rootworm can utilize tissue from a variety of plants and
might therefore require a more diverse suite of enzyme
specificities. Dependency on a single form of digestive
proteinase could result in an increased susceptibility
to attack by a CPI alone and would suggest that
western corn rootworm may be less able to overcome
chronic exposure to PMC (or other CPIs). In contrast to
southern corn rootworm, these characteristics make a
PMC gene an ideal candidate for incorporation and
expression in transgenic corn to provide resistance to
western corn rootworm.
Feeding studies with PMC, T-PMC, and HEC oryzacystatin in both southern corn rootworm and western
corn rootworm indicate that potency in in vitro assay
systems does not always translate directly to in tko
growth inhibition. As described above, PMC is an
effective growth inhibitor in both species, but despite
their in vitro activity, T-PMC, oryzacstatin and HEC
had little or no growth inhibitory effect when ingested.
It is possible that these single domain (or fragmented in
INHIBITION
OF CORN
the case of T-PMC) cystatins are more susceptible
to
proteolytic degradation.
This is supported by the observation that co-feeding
of PC1 with T-PMC produces
growth inhibitory
effects in southern
corn rootworm.
This synergism is not seen with western corn rootworm
larvae indicating
that another proteinase or mechanism
may be responsible for inactivating
these proteins in this
species. These observations
are consistent with studies of
oryzacystatin
which is a very effective inhibitor of the gut
cysteine proteinases
of red flour beetle in vitro (Liang
et al., 1991) but very high levels (10% w/w) are required
to inhibit growth (Chen et al., 1992). Direct analysis of
gut cysteine proteinase activity in second instar southern
corn rootworm
larvae following ingestion
of cystatin
molecules
shows that PMC is an effective inhibitor
within the insect gut, thus supporting its proposed mode
of action. In contrast, T-PMC and HEC cause only a
slight inhibition of cysteine proteinase activity. Inclusion
of PC1 with T-PMC increases the level of inhibition
to 45% which, based on feeding studies, appears to be
adequate to compromise first instar growth rates. Therefore, single domain cystatins would seen to require some
form of protection
to be active inhibitors
of cysteine
proteinase in viva and produce growth inhibition.
Multicystatins,
such as PMC, may be less susceptible
to
metabolism
and can function alone in both rootworm
species.
The differential
activity of single and multicystatin
molecules reported here suggests that gene(s) encoding
multicystatins
may be the most appropriate
cystatin
candidates
for producing
transgenic
maize resistant to
corn rootworm.
It should be noted that the feeding
studies performed
in this study used an artificial diet
containing
casein as the main dietary protein, and it is
possible that the relative effectiveness of PMC may vary
with dietary proteins
of differing digestibility
and/or
quality,
such as would be found in the corn root.
Expression
of PMC in transgenic
maize plants will be
required to assess its effect on rootworm
larvae when
delivered in the natural diet.
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Acknowledgements-We
are grateful
to Marty
Arrington,
Brent
Boyce, Larry Alward and Kevin Denno for technical assistance.