Fractionated extracts of Russian wheat aphid eliciting defense responses in wheat.

Abstract

It is hypothesized that the interaction between aphids and plants follows a gene-for-gene model. The recent appearance of several new Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), biotypes in the United States and the differential response of wheat, Triticum aestivum L., genotypes containing different resistance genes also suggest a gene-for-gene interaction. However, aphid elicitors remain unknown. This study was conducted to identify fractionated Russian wheat aphid extracts capable of eliciting differential responses between resistant and susceptible wheat genotypes. We extracted whole soluble compounds and separated proteins and metabolites from two Russian wheat aphid biotypes (1 and 2), injected these extracts into seedlings of susceptible wheat Gamtoos (dn7) and resistant 94M370 (Dn7), and determined phenotypic and biochemical plant responses. Injections of whole extract or protein extract from both biotypes induced the typical susceptible symptom, leaf rolling, in the susceptible cultivar, but not in the resistant cultivar. Furthermore, multiple injections with protein extract from biotype 2 induced the development of chlorosis, head trapping, and stunting in susceptible wheat. Injection with metabolite, buffer, or chitin, did not produce any susceptible symptoms in either genotype. The protein extract from the two biotypes also induced significantly higher activities of three defense-response enzymes (catalase, peroxidase, and beta-glucanase) in 94M370 than in Gamtoos. These results indicate that a protein elicitor from the Russian wheat aphid is recognized by a plant receptor, and the recognition is mediated by the Dn7-gene product. The increased activities of defense-response enzymes in resistant plants after injection with the protein fraction suggest that defense response genes are induced after recognition of aphid elicitors by the plant.

Full text

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PLANT RESISTANCE
Fractionated Extracts of Russian Wheat Aphid Eliciting Defense
Responses in Wheat
NORA L. V. LAPITAN,1,2YOU-CHUN LI,1JUNHUA PENG,1AND ANNA-MARIA BOTHA3
J. Econ. Entomol. 100(3): 990Ð999 (2007)
It is hypothesized that the interaction between aphids and plants follows a gene-for-
genemodel.TherecentappearanceofseveralnewRussianwheataphid,Diuraphisnoxia(Kurdjumov)
(Homoptera: Aphididae), biotypes in the United States and the differential response of wheat,
Triticum aestivum L., genotypes containing different resistance genes also suggest a gene-for-gene
interaction. However, aphid elicitors remain unknown. This study was conducted to identify frac-
tionatedRussianwheataphidextractscapableofelicitingdifferentialresponsesbetweenresistantand
susceptible wheat genotypes. We extracted whole soluble compounds and separated proteins and
metabolites from two Russian wheat aphid biotypes (1 and 2), injected these extracts into seedlings
of susceptible wheat Gamtoos (dn7) and resistant 94M370 (Dn7), and determined phenotypic and
biochemicalplantresponses.Injectionsofwholeextractorproteinextractfrombothbiotypesinduced
thetypicalsusceptiblesymptom,leafrolling,inthesusceptiblecultivar,butnotintheresistantcultivar.
Furthermore, multiple injections with protein extract from biotype 2 induced the development of
chlorosis,headtrapping,andstuntinginsusceptiblewheat.Injectionwithmetabolite,buffer,orchitin,
did not produce any susceptible symptoms in either genotype. The protein extract from the two
biotypes also induced signiÞcantly higher activities of three defense-response enzymes (catalase,
peroxidase,and?-glucanase)in94M370thaninGamtoos.Theseresultsindicatethataproteinelicitor
from the Russian wheat aphid is recognized by a plant receptor, and the recognition is mediated by
the Dn7-gene product. The increased activities of defense-response enzymes in resistant plants after
injection with the protein fraction suggest that defense response genes are induced after recognition
of aphid elicitors by the plant.
ABSTRACT
KEY WORDS
Duraphis noxia, Triticum aestivum, biotype, elicitor, plantÐaphid interaction
The Russian wheat aphid, Diuraphis noxia (Kurdju-
mov) (Homoptera: Aphididae), is a serious pest of
small grain cereals, particularly wheat, Triticum
aestivum L., and barley, Hordeum vulgare L.. Russian
wheat aphid occurs in most major cereal production
regions of the world, and it causes signiÞcant damage,
especially in areas where it is newly introduced, such
as South Africa and North America (Halbert and
Stoetzel 1998). The Russian wheat aphid penetrates
leaf tissue intercellularly until it reaches the phloem,
whereplantsapisingested(Fouche ´ etal.1984).Once
the stylet enters a phloem sieve tube, watery saliva is
discharged until ingestion ceases (Dixon 1998, Miles
1999). Russian wheat aphid feeding in susceptible
plants typically results in longitudinal white and yel-
low streaking, chlorosis, leaf rolling, subsequent leaf
and head trapping, stunted growth, and even death in
heavily infested plants (Quick et al. 1991, Burd and
Burton 1992, Puterka et al. 1992, Heng-Moss et al.
2003). At least 11 genes (Dn1, Dn2, Dn3, Dn4, Dn5,
Dn6, Dn7, Dn8, Dn9, Dnx, and Dny) conferring re-
sistance to Russian wheat aphid have been found in
wheat and its relatives (Du Toit 1989; Nkongolo et
al. 1991; Saidi and Quick 1996; Liu et al. 2001, 2002;
Anderson et al. 2003).
Plant resistance and aphid virulence undergo an
adaptation and counteradaptation evolutionary battle
(Walling 2000). Aphid adaptation to an existing resis-
tant host results in novel biotypes that are morpho-
logically similar to the original biotypes, but different
in their behavioral performance, such as their prefer-
ence for different host genotypes (Dreyer and Camp-
bell 1987). Several North American biotypes of the
Russian wheat aphid were recently discovered. From
2003 to 2005, at least six new biotypes were reported
in Colorado and neighboring states (Haley et al. 2004;
Burd et al. 2006; Weiland and Peairs, unpublished
data). Russian wheat aphid biotype 1 was the Þrst
biotype found in the United States. Biotype 2 was
discovered in Colorado in 2003, and it was virulent to
most known resistance genes (Dn1, Dn2, Dn4, Dn5,
Dn6, Dn8, Dn9, Dnx, and Dny) except Dn7 (Haley et
al.2004).VirulentD.noxiapopulationsalsohavebeen
identiÞed in Asia, Africa, and Europe (Puterka et al.
1992, Basky 2003, Smith et al. 2004).
1DepartmentofSoilandCropSciences,ColoradoStateUniversity,
Fort Collins, CO 80523.
2Corresponding author, e-mail: nlapitan@lamar.colostate.edu.
3DepartmentofGenetics,ForestryandAgriculturalBiotechnology
Institute, University of Pretoria, Pretoria, ZA0002, South Africa.
0022-0493/07/0990Ð0999$04.00/0 ? 2007 Entomological Society of America

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The Dn7 gene is a single dominant gene from rye
chromosomearm1RSpresentina1R/1Bchromosome
translocationinthewheatlineÔ94M370?(Andersonet
al. 2003, Marais et al. 1994). Dn7 confers resistance to
biotypes 1 and 2 (Lapitan et al. 2007). Resistance is
based on antixenosis (referring to nonpreference by
aphids for a host; Kogan and Ortman 1978) and likely
antibiosis as well (J.P. and N.L.V.L., unpublished
data).Dn7providesahigherlevelofresistanceagainst
Russianwheataphidbiotype1thanDn4(Andersonet
al. 2003). The molecular mechanism of resistance in
Dn7geneandtheinteractionofthisgenewithRussian
wheat aphid biotypes 1 and 2 are not understood.
In the last decade, our understanding of the mo-
lecular interaction between pathogens and plants has
advanced signiÞcantly. A gene-for-gene model was
proposed (Flor 1971) where genes determining viru-
lence in the pathogen (known as Avr genes) are par-
alleled by genes conferring resistance in the host
(Staskawiczetal.1995).Avrgenesinmanypathogens
andcorrespondingresistance(R)genesinplantshave
been isolated (Pedley and Martin 2003).
Plants can distinguish damage done mechanically
fromthatcausedbyinsectfeeding(Zhu-Salzmanetal.
2005).Discriminationbetweenmechanicalwounding
and chewing insect herbivory is thought to be due to
recognition of insect-derived elicitors by plant cells
(Alborn et al. 1997, Korth and Dixon 1997, Halitschke
et al. 2001). Probably, this is also true for phloem-
feeding insects. Insect saliva contains various hydro-
lytic enzymes that may function as elicitors (Miles
1999, Felton et al. 2001).
It has been proposed that the interaction between
phloem-feeding insects, such as the Russian wheat
aphid,andplantsinvolvesagene-for-geneinteraction
(Dixon 1998; Stotz et al. 1999; Botha et al. 2005, 2006;
Boyko et al. 2006). It has been predicted that aphid
salivacontainssomeelicitingagent(s)orAvr-encoded
elicitor(s) that is recognized by the corresponding
plant R gene-encoded receptor (Miles 1999). This
recognition is a key step in determining plant resis-
tance or susceptibility to a speciÞc aphid attack, be-
causesuchsuccessfulrecognitioncanquicklyactivate
defense-relatedpathways,e.g.,thesalicylicacid(SA)-
and jasmonic acid (JA)/ethylene-dependent signal-
ingpathways(vanderWesthuizenetal.1998a,1998b;
Fidantsef et al. 1999; Moran and Thompson 2001; Mo-
hase and van der Westhuizen 2002a; Botha et al. 2006;
Thompson and Goggin 2006). Recent transcript pro-
Þling of gene expression during Russian wheat aphid
feeding in wheat showed differential regulation of
nucleotide binding site leucine-rich repeat proteins,
which make up the conserved domains of genes for
resistancetopathogens(Bothaetal.2006).Transcrip-
tome analysis also indicates that plant responses to
phloem-feeding insects seem to be quantitatively and
qualitatively different from responses to other insects
or pathogens (Thompson and Goggin 2006).
Several defense-related products have been shown
to rapidly accumulate in Russian wheat aphid-resis-
tantwheatcultivars,suchasproline,phenolics,patho-
genesis related proteins, including ?-1,3-glucanases,
chitinase, peroxidase, and catalase (van der Westhui-
zen and Pretorius 1995, 1996; van der Westhuizen et
al. 1998a, 1998b; Ni et al. 2001). Although Russian
wheat aphid-susceptible plants also could accumulate
defense-related products as part of a general defense
mechanism, the accumulation is generally slower and
at a lower level than in resistant plants. This delayed
reaction may diminish the level of defense against
Russian wheat aphid feeding (van der Westhuizen et
al. 1998a, 1998b).
If plantÐaphid interaction follows a gene-for-gene
model,itshouldbepossibletoidentifyanelicitorfrom
the aphid. Previous studies using in vitro and in vivo
assays suggest the existence of elicitors from Russian
wheat aphid. When Brigham (1992) introduced total
soluble compounds extracted from ground Russian
wheat aphid into susceptible wheat and barley plants
throughpunctureandbymeansofdimethylsulfoxide,
plants showed the susceptible leaf rolling symptom.
Dong et al. (1994) incubated cut etiolated leaves of
resistant and susceptible wheat genotypes in Russian
wheat aphid extract, and they found that initially
rolled leaves of susceptible genotypes remained
rolled, whereas rolled leaves of resistant genotypes
unfolded during an 8-h treatment. The results suggest
that whole extract from Russian wheat aphid may
contain some eliciting agent(s) that is recognized by
an R gene product in wheat. The biochemical nature
of eliciting agents remains unknown. A more recent
analysis of glycoproteins found to accumulate in the
intercellular spaces in Russian wheat aphid-infested
resistantÔTugelaDn1Õwheatplantsalsofailedtoiden-
tify Russian wheat aphid-speciÞc elicitors (Mohase
and van der Westhuizen 2002b).
In the current study, our objectives were to 1)
detect fractions from Russian wheat aphid biotypes 1
and 2 containing elicitor(s) based on phenotypic and
biochemical responses of resistant and susceptible
wheat plants injected with aphid extracts; and 2) de-
termine the effect of these fractions on activities of
defense-response enzymes.
Materials and Methods
Plant Materials. Two wheat genotypes were used:
94M370containsDn7andisresistanttoRussianwheat
aphid biotypes 1 and 2; and Gamtoos (dn7), which is
susceptible to both biotypes. The 1RS/1BL chromo-
some in 94M370 was from Gamtoos, and Dn7 was
transferred to this chromosome through a recombi-
nation with the 1RS telosome from ÔTurkey 77Õ
(Marais et al. 1994). Seedlings were grown in 20-cm-
diameter pots containing Metro-Mix 350 growing me-
dium (Scotts Miracle-Gro Company, Marysville, OH)
under a photoperiod of 16:8 (L:D) h with daytime
temperatures ?29Ð32?C and nighttime temperatures
?21Ð26?C. Each pot contained two to four seedlings.
Plants were allowed to grow to two- to four-leaf stage
so that leaves were large enough for syringe-driven
injection.
D. noxia Culture. Russian wheat aphid biotypes 1
and 2 were identiÞed and separated according to the
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differential response of wheat plants containing dif-
ferent resistance genes to aphid feeding as described
in Haley et al. (2004). Isofemale colonies from bio-
types 1 and 2 were developed from a single Þrst instar
femalegrowninsusceptibleÔGamtoosÕunderthesame
growing conditions as described above.
Preparation of D. noxia Whole Extract. Apterous
late instars and adult Russian wheat aphids collected
from the greenhouse were frozen in liquid nitrogen,
and then ground to powder by using a prechilled
mortar and pestle. Potassium phosphate buffer (0.025
M KH2PO4, pH 6.8) was added with an aphid/buffer
ratio of 1 g/10 ml. The bufferÐaphid homogenate was
centrifuged at 10,000 ? g for 15 min at 4?C. The
supernatant was transferred to a ßask and further
diluted to 50 ml/g fresh aphids. The approximate pro-
tein concentrations of the whole Russian wheat aphid
extracts and protein extracts were estimated based on
UV absorbance at A280by assuming that 1 mg/ml
protein has an UV absorbance of 1.3 (Coligan et al.
2003). The average protein concentrations after 1:50
dilution were 2.23 and 2.37 mg/ml for Russian wheat
aphidbiotypes1and2,respectively,fortheÞrstsetof
experiments. A second set of experiments involving
only biotype 2 had protein concentrations ranging
from 1.16 to 18.30 mg/ml. Phosphate buffer was used
as blank control against whole extract.
Preparation of Extracts of Russian Wheat Aphid
Proteins and Metabolites. Fresh aphids (1.25 g) were
frozen in liquid nitrogen, ground, and then homoge-
nized in buffer (0.025 M KH2PO4, pH 6.8). After 10-
min incubation on ice, the homogenate was centri-
fuged at 10,000 ? g for 15 min. The supernatant was
transferredintoaßaskandproteinswereprecipitated
byadditionof56.8gammoniumsulfate/100mlprotein
solutionfollowingtheprotocolprovidedbyColiganet
al. (2003). The solution was centrifuged at 10,000 ? g
for 10 min. The supernatant, including nonprotein
water-solublecompounds,wasdialyzedovernightin4
liters of phosphate buffer to remove all salt residues
(“metabolite” fraction). Before injection, metabolites
were incubated at 65?C for 10 min to denature poten-
tial leftover proteins. The protein-containing pellet
was resuspended in 1Ð2 volumes of the 0.025 M
KH2PO4buffer, pH 6.8. Any insoluble materials were
removed by centrifugation at 10,000 ? g for 5 min,
whereasammoniumsulfatewasremovedusingPD-10
Columns (GE Healthcare, Little Chalfont, Bucking-
hamshire, United Kingdom). The Þltered protein so-
lution was diluted by adding the 0.025 M KH2PO4to
a Þnal volume of 50 ml. All steps were performed at
4?C. The average protein concentrations for the Þrst
setofexperimentsafter1.25:50dilutionwere1.644and
1.768 mg/ml for Russian wheat aphid biotypes 1 and
2, respectively. In the second set of experiments, pro-
tein concentration ranged from 0.42 to 6.77 mg/ml
isolatedfrom1.25to4.7gofaphids.Blankcontrolsfor
Russian wheat aphid proteins, and metabolites were
the phosphate buffer treated as described for protein
and metabolite preparations, respectively.
Preparation of Glycol Chitin. Chitin, a polysaccha-
ride composed of ?-134-linked N-acetyl-D-glu-
cosamine, is a structural element found in insect ex-
oskeletons(Wanetal.2004,Ramonelletal.2005),and
it has been shown to be an important oligosaccharide
elicitorofplantdefense(Ramonelletal.2005).Torule
out the effect of aphid chitin (Ramonell et al. 2005),
glycol chitin was prepared and included as a treat-
ment. Glycol chitin was obtained by acetylation of
glycol chitosan by a modiÞcation of the method used
by Molano et al. (1979). Glycol chitosan (5 g) was
dissolved in 100 ml of 10% (vol:vol) acetic acid by
grindinginamortar.Theviscoussolutionwasallowed
tostandovernightatroomtemperature.Methanolwas
added, and the solution was vacuum Þltrated through
a 50-ml disposable vacuum Þltration system with
0.22-?m Express PLUS Membrane (Millipore, Bil-
lerica,MA).TheÞltratewastransferredintoabeaker,
and7.5mlofaceticanhydridewasadded.Thegelwas
covered with methanol and homogenized. The sus-
pensionwascentrifugedat27,000?gfor15minat4?C.
The gelatinous pellet was resuspended in 1 volume of
methanol,homogenized,andcentrifugedasdescribed
in the preceding step. The pellet was resuspended in
500 ml distilled water containing 0.02% (m/v) sodium
azide, kept overnight, and then methanol-saturated
glycol chitin was Þltered. In total, ?92.5 ml methanol
was added. The Þnal volume was ?700 ml, and the
approximate chitin concentration was 7.14 mg/ml. A
mixture of 7.5 ml acetic acid, 100 ml 10% (vol:vol)
acetic acid, 500 ml water plus 0.02% (wt:vol) sodium
azideand92.5mlmethanolwereusedasblankcontrol
for chitin treatment. The 7.14 mg/ml chitin solution
and its blank control were used as stock solutions for
further dilutions of chitin and its blank control, re-
spectively. The chitin solution was diluted to 4.0, 2.0,
1.0,0.5,and0.1mg/ml.Similardilutionsweremadefor
theblankcontrol.Thedilutedsolutionswereinjected
into Gamtoos and 94M370 plants, as described below.
Experimental Design. Two sets of experiments
wereperformed.TheÞrstset,conductedinNovember
2005, consisted of three extracts from Russian wheat
biotypes 1 and 2: whole extracts, protein extracts, and
metabolite. A buffer control, chitin solution, and
chitin control also were included. The six treatments
wereseparatelyinjectedintoseedlingsof94M370and
Gamtoos. Three replications, with six plants per rep-
lication, were conducted in a randomized complete
block design. Forty-eight hours after injection, three
seedlings from each replication were harvested sep-
arately, frozen in liquid nitrogen, and stored at ?80?C
for enzyme analysis. The remaining three plants per
replication were grown for further observation of
symptoms.
A second set of experiments was conducted in De-
cember 2006 to test whether the observed effect of
Russianwheatextractinjectionsonphenotypeduring
the Þrst set of experiments can be quantiÞed. Only
Russian wheat aphid biotype 2 was used based on
availability. The treatments included 1) no injection
(control), injection with 2) buffer, 3) whole extract,
4) protein, and 5) metabolite. Treatments were ap-
plied on 94M370 and Gamtoos. There were three
replications, with two plants per treatment per rep-
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lication, in a randomized complete block design.
Measurements of plant height were taken 16 d after
the initial injection.
Injection Procedure. Wheat seedlings at the two-
to four-leaf stage were used for injections. For the
Þrstsetofexperiments,eachplantwasinjectedonly
once. In the second set of experiments, each plant
was injected three times at 4-d intervals. Approxi-
mately 200 ?l of each treatment was injected
through leaf veins in the abaxial side of the leaves of
each seedling by using a 1-ml B-D Latex Free Sy-
ringe and B-D 30G 1/2 Precision Guide needle (BD
Biosciences, Bedford, MA).
Enzyme Activity Assay. Frozen injected seedlings
weregroundandproteinswereextractedusingT-PER
TissueProteinExtractionReagentwithadditionofthe
HaltProteaseInhibitorCocktailkit(PierceChemical,
Philadelphia, PA). Protein concentrations were de-
termined according to absorbance at 595-nm wave-
length by using Quickstart Bradford Dye Reagent
(Bio-Rad,Hercules,CA)andBovineGammaGlobulin
(Bio-Rad) as a standard. Commercial enzymes, in-
cluding catalase (EC 1.11.1.6), peroxidase (EC
1.11.1.7), and ?-glucanase (EC 3.2.1.6), were used for
building standard curves for each enzyme activity
assay.
Peroxidase activity was measured using a method
modiÞed by van der Westhuizen et al. (1998b) from
Zieslin and Ben-Zaken (1991). The assay solution (1
ml) contained 0.1 M phosphate buffer, pH 5, 3 mM
H2O2, 3 mM guaiacol, and an aliquot (20 ?l) of the
wheat protein extract. The formation of tetraguaiacol
was monitored at 470-nm wavelength. A standard
curve related A470to guaiacol concentration was built
to calculate peroxidase activity (micromoles of tetra-
guaiacol per hour per milligram of protein).
Catalase activity was measured according to
Chance and Maehly (1955) and Hildebrand et al.
(1986),andactivitywasdeterminedbymeasuringthe
decrease in absorbance that reßects the decomposi-
tionofhydrogenperoxidecatalyzedbycatalase.After
scanning in a wide range of wavelengths (210Ð700
nm), the highest absorbance peaks were found at 220
nm. Catalase activity was monitored at 220 nm 1 min
after initiation of the reaction at room temperature.
Enzymatic activity was initiated by adding 50 ml of
wheat protein extract into the reaction mixture con-
taining500mlof0.1Mpotassiumphosphatebuffer,pH
6.5, 250 ml of distilled water, and 200 ml of 75 mM
H2O2. Enzyme activity was measured against a blank
reactionmixturecontainingnoproteinextract.Astan-
dard curve related A220to H2O2concentration was
used to calculate catalase activity (micromoles of
H2O2per minute per milligram of protein).
?-Glucanase activity was measured using a method
modiÞed by van der Westhuizen et al. (1998a) from
Fink et al. (1988). An aliquot (20 ml) of the wheat
protein extract was incubated with 0.5 ml of substrate
laminarin at 1 mg/ml and 50 mM sodium acetate, pH
4.5,at37?Cfor10min.Subsequently,0.5mlofreagent
of Somogyi (1952) was added, and the mixture was
heated at 100?C for 10 min. After cooling and addition
of 0.5 ml of Arsenomolybdate reagent (Nelson 1944),
absorbance of the colored product was measured at
540 nm. A standard curve relating A540to glucose
concentrationwasusedtocalculateenzymeactivity
(milligrams of glucose per hour per milligram of
protein).
Statistical Analysis. Enzyme activity measurements
were analyzed by analysis of variance (ANOVA) by
using a split-plot model with genotype as the main
plot.Thismodelwasusedtoachievegreaterprecision
incomparingtreatmentsandÞndingdifferentialtreat-
ment effects relative to the genotype (interaction).
Comparisons between two treatment means in the
same genotype were made using the least signiÞcant
difference test (P ? 0.01).
ANOVA also was conducted for plant height mea-
surementsofthetwogenotypes,byusingmeanoftwo
plants per replication as values. An F-test was used
to test the signiÞcance of sources of variation. The
StudentÐNewmanÐKeulstest(Newman1939)wasap-
plied to conduct multiple comparisons of the Þve
treatments. Statistical analyses were conducted using
SAS (SAS Institute 1988).
Results
Susceptible Symptoms Are Induced by the Russian
Wheat Aphid Protein Extract. In the Þrst set of ex-
periments, we observed susceptible symptoms in
Gamtoos 3 to 7 d after injection of extracts from
biotypes 1 and 2. Leaf rolling, a typical susceptible
symptom, was observed in Gamtoos plants injected
withwholeextractandproteinextract(Fig.1A).Leaf
rolling or other susceptible symptoms were not ob-
served in Gamtoos plants injected with metabolite,
buffer,chitin,orchitincontrol.Theresistantgenotype
94M370 had ßat leaves and normal appearance in all
the treatments, including whole extract and protein
extract.
A second set of experiments using extracts from
biotype 2 was conducted to verify these observations
and to test whether the effect of the different treat-
ments can be quantiÞed. In contrast to the Þrst set of
experiments, multiple injections of the treatments
wereappliedoneachplant.AftertheÞrstinjectionof
extracts, no differences in plant response were ob-
served between the resistant and susceptible geno-
types. This could be due to the lower protein con-
centration used compared with the Þrst set of
experiments (0.42 versus ?1.8 mg/ml, respectively).
The second and third injections, however, contained
higher protein concentrations (3.04 and 6.77 mg/ml,
respectively). Three days after the second injection,
phenotypic differences between the two genotypes
began to manifest. Gamtoos plants treated with pro-
teinextractfrombiotype2showedleafrolling(Fig.1B
and C), similar to observations in the Þrst set of ex-
periments. In addition, chlorosis, head trapping, and
stunting also were observed (Fig. 1CÐE). 94M370
plants injected with protein extract did not show any
of these symptoms. Furthermore, none of the other
treatments showed symptoms in Gamtoos or 94M370.
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After the third injection, Gamtoos plants treated
with proteins were weaker and showed visibly
stunted growth compared with plants in the other
treatments. Leaf rolling also occurred in Gamtoos
plants treated with whole extracts. Gamtoos in-
jected with metabolite (Fig. 1F) seemed the same
as those injected with buffer (Fig. 1G). 94M370
plants did not show susceptible symptoms with any
ofthetreatmentsafterthethirdinjectionofRussian
wheat aphid extracts (Fig. 1H).
Injection of chitin at a concentration of 2.0Ð0.5
mg/ml caused leaf burning and death (data not
shown). Chitin injection at 0.1 mg/ml concentration
did not cause leaf burning and death, but it led to
necrosis in the leaves of both 94M370 (Fig. 1I) and
Gamtoos (Fig. 1J) around the injection sites. Unlike
injectionwithwholeorproteinextracts,therewereno
differencesintheresponseof94M370andGamtoosto
chitin or the blank control. Furthermore, injection of
chitin did not induce leaf rolling.
Plant Height Measurements Can Be Used to Quan-
tifytheEffectofRussianWheatAphidExtracts.Stunt-
ing is one of symptoms of susceptible plants after
Russianwheataphidinfestation.Totestwhetherplant
height can be used to quantify the effects of Russian
wheataphidextractinjectionsinwheat,wemeasured
seedling height in injected wheat plants after the ma-
jor symptoms (i.e., leaf rolling and chlorosis) have
fullydeveloped.ResultsofANOVAindicatedthatthe
treatment effect in Russian wheat aphid-susceptible
Gamtoos is highly signiÞcant (F ? 36.00, P ?
0.000037).Plantstreatedwithproteinshowedthelow-
estmeanheight,followedbythosetreatedwithwhole
extract, metabolite, buffer, and no treatment (Table
1). These results agree with phenotypic observations
and indicate that plant height measurement can be
used to quantify the effect of Russian wheat aphid
extract injections in the susceptible plant.
The comparison of plant height means between
differenttreatmentsindicatedthatthefourtreatments
Fig. 1.
aphid.(A)GamtoosinjectedwithRussianwheataphidwholeextractfrombiotype2(arrowshowsleafrolling).(B)Gamtoos
injected with protein extract from Russian wheat aphid biotype 2 (arrows show leaf rolling and chlorosis). (C and D) Same
as B, showing close-up view of leaves with leaf rolling and chlorosis (C) and only chlorosis (D). (E) Gamtoos injected with
protein extract from Russian wheat aphid biotype 2 showing a trapped head. (F) Gamtoos injected with metabolite showing
normal leaf morphology. (G) Gamtoos injected with buffer showing normal leaf morphology; yellow lines along the front
leaf areas are sites of injection. (H) 94M370 injected with protein from Russian wheat aphid biotype 2 showing normal leaf
morphology. (I) 94M370 injected with 0.1 mg/ ml chitin showing ßat leaf and cell death around injection sites. (J) Gamtoos
injected with 0.1 mg/ml chitin showing the same symptoms as the resistant plant in I.
Phenotypes of susceptible Gamtoos and resistant 94M370 wheat after injection with extracts from Russian wheat
994JOURNAL OF ECONOMIC ENTOMOLOGY
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(whole extract, protein, metabolite, and buffer) sig-
niÞcantly (P ? 0.01) reduced seedling height in com-
parison with the control, with the highest effect for
protein followed by whole extract, metabolite, and
buffer(Table1).Theeffectsofprotein,wholeextract,
and metabolite are signiÞcantly (P ? 0.05 or 0.01)
higher than that of the buffer control. The effect of
protein is signiÞcantly higher than those of whole
extract and metabolite. However, the whole extract
had no signiÞcant difference in effect on seedling
height from that of metabolite. The results of pheno-
typic observations and plant height measurements in-
dicate that the protein extract of Russian wheat aphid
containsfactorsthatinducesusceptiblesymptomsand
cause damage in plants.
ANOVA analysis of plant height measurements in
the resistant plant indicated that the same treatments
alsocouldcausevariationofseedlingheightin94M370
(F ? 6.01, P ? 0.015552). However, the effect was
lowerthanthatinthesusceptibleplants.Themultiple
comparison test showed that the treatment variation
wasmainlyduetothedifferenceofplantheightmeans
in the control plants from those in the protein and
metabolite treatments (data not shown). All other
pairsofcomparisonswerenotsigniÞcant,thatis,there
wasnodifferenceamongthefourtreatmentinjections
(protein,wholeextract,metabolite,andbuffer),inthe
effectsonplantheight.Itispossiblethatthereduction
of plant height in injected plants compared with the
uninjected control is an effect of wounding.
Effects of Russian Wheat Aphid Extract Injection
on Defense-Related Enzyme Activities. The results of
the ANOVA F-tests indicate that plant genotypes sig-
niÞcantly(P?0.01)affectedtheactivitiesofcatalase,
peroxidase, and ?-glucanase. In most cases, the resis-
tant 94M370 plants showed much higher activities of
these enzymes than the susceptible Gamtoos (Fig. 2).
The treatments also had signiÞcant effects (P ? 0.01)
on catalase, peroxidase, and ?-glucanase activities.
The comparison of means for the genotypes within
eachtreatmentissummarizedinFig.2.Therewereno
signiÞcant differences in the means of the three en-
zymes between 94M370 and Gamtoos in the blank
control (buffer). Similarly, injection with chitin or
with the chitin control did not produce signiÞcant
differences in the activity of the three enzymes be-
tween 94M370 and Gamtoos.
Injection with whole extracts from Russian wheat
aphid biotype 2 resulted in signiÞcantly higher levels
of enzyme activities in 94M370 compared with
Gamtoos. The absolute differences in enzyme activi-
tiesbetweenthegenotypeswereupto5.99(P?0.05),
0.43 (P ? 0.05), and 0.74 (P ? 0.01) units for catalase,
peroxidase, and ?-glucanase, respectively (Fig. 2). In
comparison, injection of Russian wheat aphid biotype
1wholeextractproducednosigniÞcantdifferencesin
the activity of the three enzymes between the two
wheat genotypes (Fig. 2).
Injection with Russian wheat aphid metabolites
produced no signiÞcant differences in the enzyme
activities between the two genotypes (Fig. 2), except
that Russian wheat aphid biotype 2 metabolites pro-
duced a signiÞcantly higher activity of peroxidase in
94M370 than in Gamtoos (P ? 0.05). The results sug-
gest that Russian wheat aphid biotype 2 metabolites
may play some role in eliciting defense response in
94M370.
In contrast to Russian wheat aphid metabolites, the
levels of the three enzyme activities induced by
Russian wheat aphid biotypes 1 and 2 proteins were
all signiÞcantly (P ? 0.01Ð0.05) higher in resistant
94M370 than in susceptible Gamtoos (Fig. 2). Differ-
ences of the three enzyme activities produced by
Russian wheat aphid biotypes 1 and 2 protein treat-
ments between the two genotypes were the largest in
comparison with differences produced by treatments
withmetabolitefractionsandwholeextracts.Biotypes
1 and 2 protein injections produced 1.4Ð1.5 times
greater activity in catalase and ?-glucanase in 94M370
compared with Gamtoos (Fig. 2). These results fur-
ther conÞrm that the protein fractions from Russian
wheat aphid biotypes 1 and 2 contain eliciting agents.
Differential responses between the resistant and sus-
ceptible genotypes indicate that the interaction be-
tween aphid elicitors and the wheat plant is mediated
by the resistance gene Dn7.
Discussion
Russian Wheat Aphid Elicitors Consist of Proteins.
The presence of an eliciting factor from the Russian
wheat aphid was suggested from earlier studies show-
ing that treatment of plants or cut leaves with total
soluble compounds extracted from the Russian wheat
aphid could elicit leaf rolling, which is one of the
susceptiblesymptomsobservedinplantsinfestedwith
the aphid (Brigham 1992, Dong et al. 1994). In the
Brigham (1992) study, only susceptible plants were
used, and differential responses between plant geno-
types was not determined. Dong et al. (1994) used
resistant and susceptible wheat genotypes. However,
theassayinvolvedcutleavesratherthanwholeplants.
Inthisstudy,wedevelopedaninplantaassaytoverify
the previous results and to further narrow down the
extract that is responsible for eliciting susceptible
symptoms. The results of this study demonstrate that
injection of Russian wheat extracts in wheat can in-
duce the development of susceptible symptoms in a
susceptiblegenotype.Consistentwithpreviousresults
(Brigham1992,Dongetal.1994),injectionofextracts
containing whole, ground aphid produced leaf rolling
Table 1.
2 extract injections on Gamtoos-S plant height
Multiple comparisons of Russian wheat aphid biotype
Treatment
Mean
(cm)
Difference between means
Protein Whole MetaboliteBuffer
Protein
Whole extract
Metabolite
Buffer
Control
25.83
28.75
29.08
32.33
35.83
0
2.92*
3.25*
6.50**
10.00**
0
0.33
3.58*
7.08**
0
3.25**
6.75**
0
3.50**
SigniÞcant difference at *P ? 0.05 and **0.01.
June 2007LAPITAN ET AL.: EFFECT OF RUSSIAN WHEAT APHID EXTRACTS IN WHEAT
995

Page 7

in the susceptible genotype. Furthermore, by sepa-
rating protein and nonprotein components, we deter-
mined that the susceptible symptoms are induced by
theproteinfraction.Byrepeatedinjectionsofprotein
extract from biotype 2, we show induction of leaf
rolling, chlorosis, head trapping, and stunting in sus-
ceptible plants. Plant height measurements also
showedsigniÞcantdifferencesintheeffectsofprotein
injectionfromthoseofwholeextract,metabolite,and
buffer. In contrast, none of the treatments produced
susceptible symptoms in the resistant genotype.
Chitin injection produced the same response in the
Fig. 2.
(milligrams of glucose per hour per milligram of protein), and catalase (micromoles of H2O2per minute per milligram of
protein),inresistant94M370andsusceptibleGamtooswheatgenotypesinducedbyinjectionsofRussianwheataphidextracts,
chitin, and controls. *, signiÞcantly different between 94M370 and Gamtoos at P ? 0.05; **, signiÞcantly different at P ? 0.01.
Graph of activities of peroxidase (micromoles of tetraguicol per hour per milligram of protein), ?-glucanase
996JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 100, no. 3

Page 8

resistantandsusceptiblegenotypes,i.e.,necroticspots
aroundtheinjectionsite.However,susceptiblesymp-
toms associated with Russian wheat aphid infestation
were not observed in either genotype when injected
with chitin. These results rule out the possibility that
theobserveddifferentialphenotypicandbiochemical
responses between 94M370 and Gamtoos induced by
Russian wheat aphid extracts was due to chitin re-
maining in the extracts.
The results show that only protein-containing ex-
tracts(i.e.,wholeextractandproteinextract)fromthe
Russianwheataphidinducedsusceptiblesymptomsin
Gamtoos. Because the whole extract also contains the
nonprotein fraction or metabolites, which did not in-
duce susceptible symptoms, it can be concluded that
Russianwheataphidelicitingagentsareintheprotein
fraction. The development of more symptoms with
proteinfractioninjectionisprobablyduetothehigher
activity of proteins in this fraction than in the whole
extract. The differential responses between resistant
and susceptible wheat genotypes to injection with
protein-containing extracts from the Russian wheat
aphid indicate that a protein elicitor from the Russian
wheataphidisrecognizedbyaplantreceptor,andthe
recognition is mediated by the resistance gene prod-
uct. These observations support the hypothesis that
theinteractionbetweenwheatandtheRussianwheat
aphid involves a gene-for-gene interaction, similar to
thatinplantÐmicrobeinteractions(Dixon1998;Botha
et al. 2006, 2007; Stotz et al. 1999; Moloi and van der
Westhuizen 2006).
It is interesting to note that chlorosis was only ob-
served after multiple injections of protein, whereas
leaf rolling was observed even when plants were in-
jected with protein only once. These results support
a previous statement that leaf chlorosis, leaf rolling,
and plant stunting occur as independent damage
symptoms (Burd et al. 1993; Dorry and Assad 2001).
We speculate that separate biochemical pathways are
involved in the development of leaf rolling and chlo-
rosis and that chlorosis develops with continuous ex-
posure to the Russian wheat aphid elicitor.
Protein Extracts from the Russian Wheat Aphid
Induce Increased Activities in Defense–Response En-
zymes. Previous studies demonstrated that Russian
wheataphidinfestationleadstoincreasedactivityand
accumulation of defense-related enzymes in Russian
wheat aphid-resistant wheat cultivars (van der Wes-
thuizenandPretorius1995,1996;VanderWesthuizen
et al. 1998a, 1998b; Ni et al. 2001). The results of this
study are consistent with previous Þndings. The ac-
tivity of peroxidase, ?-glucanase, and catalase were
signiÞcantly higher in 94M370 than in Gamtoos after
injection with protein extracts from both biotypes 1
and 2. SigniÞcant increases in enzyme activities also
were observed for whole extracts from Russian wheat
aphid biotype 2, which is to be expected, because this
fraction contains proteins. Surprisingly, however,
whole extract injection of biotype 1 did not produce
signiÞcant differences in enzyme activities between
94M370 and Gamtoos. This is probably due to lower
protein activity in the whole extract from biotype 1
than that for biotype 2. It is also possible that protein-
degradingenzymesarepresentinthebiotype1whole
extracts.
Theaphidwaterysalivaconstituentsincludeanum-
ber of enzymes such as hydrolases and oxidases (for
review, see Miles 1999). Some hydrolytic enzymes
(e.g.,cellulases,pectinases,andglucoseoxidases)and
structural proteins (e.g., glycoproteins) have been
suggested as eliciting agents (Eichenseer et al. 1999,
Miles 1999). For Russian wheat aphid, a recent study
suggestedthatglycoproteinisageneralelicitorrather
than a speciÞc one (Mohase and Van der Westhuizen
2002b). This study demonstrated that proteinaceous
substances from Russian wheat aphid biotypes 1 and
2 elicited speciÞc responses in susceptible and resis-
tant wheat genotypes. The signiÞcant difference be-
tween 94M370 and Gamtoos in peroxidase activity
with metabolite injection may indicate a possible in-
volvement of nonprotein compounds from Russian
wheataphidbiotype2inelicitingdefenseresponsesin
94M370. Although aphid saliva contains nonenzymic,
reducingcompoundsthat,inthepresenceofoxidases,
can inactivate defensive phytochemicals in plants
(Miles 1999), nonprotein compounds have not been
known to play an eliciting role in plants.
In conclusion, this study provides evidence for the
existence of an eliciting agent(s) in Russian wheat
aphid that is involved in an R-geneÐmediated bio-
chemical pathway. The major eliciting agents consist
ofsolubleproteins.Furtherstudieswillaimtoidentify
the speciÞc proteins responsible for eliciting the dif-
ferential phenotypic and biochemical responses be-
tweenresistantandsusceptiblewheatgenotypes.Pre-
sumably, interaction between the eliciting agent and
a receptor in the resistant plant leads to induction of
defense response genes, which is consistent with re-
cent Þndings based on microarray analysis (Botha et
al. 2006).
Acknowledgments
WethankProf.F.B.Peairsforeditingthismanuscript,J.B.
Rudolph for providing colonies of Russian wheat aphid bio-
types 1 and 2, H Wang for assistance in the plant injections
and preparation of extracts, R. Lapitan for help with the
statistical analysis of enzyme activities, and D. Badillo for
assistanceinpreparationofRussianwheataphidproteinand
metaboliteextracts.ThisworkwaspartiallyfundedbyUSDA
contract 2003-34205-13636, the Colorado Wheat Research
Foundation, and Hatch funds to N.L.
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Received 7 July 2006; accepted 27 February 2007.
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