Watery Saliva Secreted by the Grain Aphid Sitobion avenae

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Watery saliva secreted by the grain aphid Sitobion avenae stimulates aphid resistance in wheat Yong Zhang, Jia Fan, Frederic Francis, and Julian Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03141 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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Journal of Agricultural and Food Chemistry

Watery saliva secreted by the grain aphid Sitobion avenae stimulates aphid resistance in wheat *

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Yong Zhang1,2, Jia Fan1, Frédéric Francis2 , Julian Chen1

1 State Key Laboratory of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of

Agricultural Sciences, Beijing, 100193, PR China

2 Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liège, Gembloux, B-5030,

Belgium

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ABSTRACT

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Infestation with Sitobion avenae induces localized defense responses in wheat; in this

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study, the role of S. avenae watery saliva in resistance induction was examined by

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infiltrating aphid saliva into wheat leaves. After feeding S. avenae on an artificial diet

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for 48 h, we first collected watery saliva from them and then separated the salivary

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proteins using one-dimensional gel electrophoresis. Gene expression studies showed

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that infiltration of S. avenae watery saliva in wheat leaves induced strong salicylic

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acid-responsive defense but moderate jasmonic acid-dependent defense. Feeding on

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wheat leaves infiltrated with aphid saliva, compared with untreated leaves,

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significantly decreased the number of nymphs produced per day and the intrinsic rate

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of increase of the population of S. avenae. In a choice test against untreated wheat,

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saliva-infiltrated wheat had repellent effects on aphids. Additionally, electrical

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penetration graph results showed that the feeding behavior of S. avenae on

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saliva-treated wheat was negatively affected compared with untreated wheat. These

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findings provided direct evidence that salivary components of S.avenae are involved

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in the induction of wheat resistance against aphids and further demonstrated the

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important roles of watery saliva in aphid-plant interactions.

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KEYWORDS: Sitobion avenae, saliva infiltration, defense responses, aphid

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performance, choice preference, feeding behavior

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INTRODUCTION

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During the long course of their co-evolution with insects, plants have evolved a

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range of defense mechanisms induced by herbivore attacks, including direct defenses

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and indirect defenses. Direct defenses include herbivore-induced toxic secondary

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metabolites and proteinase inhibitors (PIs) that have negative effects on insect

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development, while indirect defenses consist of volatile emissions that repel

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herbivores or attract their natural enemies and are released by plants in response to

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herbivore feeding 1, 2.

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Jasmonic acid (JA) and salicylic acid (SA) function as two important signaling

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molecules in the induction of plant defense responses 3. Current theory posits that

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plants respond to necrotrophic pathogen infestations and leaf-chewing herbivores by

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activating the JA-mediated defense pathway and that SA-dependent defenses are

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mainly triggered by biotrophic pathogens and phloem feeders 4.

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As one of the largest groups of phloem-feeding insects, aphids (Hemiptera:

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Aphidoidea) are economically important pests that cause heavy losses in agriculture

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and horticulture worldwide 5. The induction of a SA-dependent defense pathway by

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aphid feeding has been demonstrated in many aphid-plant interactions, for example,

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green peach aphid (Myzus persicae) in tomato, tobacco and Arabidopsis

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Russian wheat aphid (Diuraphis noxia) in wheat 9. However, several genes involved

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in the jasmonic acid signaling pathway, such as lipoxygenase (LOX) and PIs, were

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also found to be induced by the feeding of the potato aphid (Macrosiphum euphorbiae)

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on tomato in compatible and incompatible interactions 3

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6-8

and

, the greenbug aphid

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(Schizaphis graminum) feeding on sorghum

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Arabidopsis 12.

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, and M. persicae feeding on

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Plants have the ability to perceive herbivore-derived chemical cues in saliva,

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such as herbivore-associated elicitors or herbivore-associated molecular patterns

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(HAMPs), to activate specific defense responses at a minimal fitness cost

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salivary elicitors or HAMPs have been identified in chewing insects, including fatty

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acid-amino acid conjugates, glucose oxidase, inceptins in lepidopterans, and

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disulfooxy fatty acids in the American grasshopper (Schistocerca americana), all of

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which can induce the activation of SA-, JA-, ethylene (ET)- and reactive oxygen

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species (ROS) defense responses in plants 14-18.

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. Many

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During the process of probing and feeding, aphids initially secrete gelling saliva

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that can solidify into a tube-like sheath to protect the stylets from mechanical damage

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and chemical attacks

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mixture of enzymes and other defense-eliciting components, into the plant cells and

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apoplasts 20. It has been proposed that aphid interactions with plant immunity involve

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a gene-for-gene model in which some eliciting components or elicitors can be

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recognized by nucleotide binding site-leucine rich repeat (NBS-LRR) resistance (R)

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protein in plants leading to resistance against aphids

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specific elicitors have been identified from aphids, the eliciting activity of watery

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saliva in plant defense has been well demonstrated in M.persicae. Infiltration of M.

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persicae salivary components in the range of 3-10 kDa into Arabidopsis plants

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activated resistance against aphids resulting in reduced fecundity, and 52 genes such

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. Additionally, aphids inject watery saliva, a more complex

8, 21

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as senescence associated protein 1 and cytochrome P450, involved in stress responses

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were also induced as being activated after aphid feeding

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oxidative enzymes, such as pectinases and polyphenol oxidase (PPO), were also

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detected in aphid saliva, and have been shown to trigger plant defense responses as

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eliciting agents

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salivary elicitors of M. persicae, reduced aphid fecundity, while Mp10 induced

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chlorosis and activated the SA and JA signaling pathways in Nicotiana benthamiana,

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all of which indicates their important roles in plant defense induction 26, 27.

22, 23

. Several hydrolytic and

20, 24, 25

. The overexpression of Mp10 and Mp42, two candidate

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The grain aphid, Sitobion avenae, is one of the most dominant and destructive

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pests of wheat in the world in that it both feeds directly on phloem sap and transmits

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barley yellow dwarf viruses 28. The feeding of S. avenae increased the enzyme activity

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of LOX, PPO, phenylalanine ammonia lyase (PAL) and β-1,3-glucanase (BGL2)

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related to both the JA and SA pathways in wheat, as well as the mRNA levels of

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allene oxide synthase (AOS) and PAL, which are involved in JA and SA synthesis,

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respectively

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S.avenae 30, little is known about the roles of saliva in aphid-wheat interactions. In our

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study, the watery saliva of S. avenae was collected and then infiltrated into wheat

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leaves to investigate the roles of aphid saliva in the induction of wheat defense and

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the attendant effects on aphid performance by reverse transcription quantitative

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real-time PCR (RT-qPCR), bioassays and electrical penetration graph (EPG)

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recording.

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MATERIALS AND METHODS

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. Although some proteins have been identified in the watery saliva of

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Insects and plants

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Winter wheat seeds, Triticum aestivum var. Beijing 837, were immersed in 0.5 %

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sodium hypochlorite (Amresco, OH, USA) for 30 min to sterilize the surface, then

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washed 3 times in distilled water. These seeds were transferred into sterilized petri

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dishes and germinated in distilled water for 3-4 days at a temperature of 25±1 °C; the

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water was changed every day. Healthy seedlings of similar size were carefully

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transferred into plastic pots with organic soil (peat: vermiculite=3:1) and continued to

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be reared in the climate chamber until the two-leaf stage for use in further study (L:

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D=16 h: 8 h; 20±1 °C).

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A clone of S. avenae was initially established from a single aphid collected from

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a wheat field in Langfang City, Hebei Province, North China, and has been reared on

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wheat plants (Beijing 837 variety) for 6 yrs (25-30 generations every year) in an

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indoor environment with a temperature of 20±1 °C, relative humidity of 40-60 % and

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a photoperiod of L: D = 16 h: 8 h.

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Aphid infestation treatments

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At the two-leaf stage, 20 wingless adults of S. avenae were transferred to the first

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leaf (the older leaf) of wheat and the movement of aphids was restricted in a plastic

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ecological cage (2.7×2.7×2.7 cm) to avoid the escape of aphids. The edge of the

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ecological cage was covered with sponge to avoid causing mechanical wounds to the

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leaf. This aphid feeding site was designated the “local leaf” group. The other leaf of

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the same plant was also caged without aphids as the “systemic leaf”. In the control

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plant, both leaves were caged at corresponding sites with ecological cages containing 6

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no aphids. Each pot contained one wheat plant and was kept in a climate incubator

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with a temperature of 20 ± 1°C and a photoperiod of 16 h: 8 h (L: D). After 30 min,

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all aphids had begun settling and feeding; this time was recorded as 0 h. After 48 h of

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feeding, all aphids were removed, and leaf samples were then collected. Three

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experimental replicates were conducted for each treatment.

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Aphid saliva collection and 1D gel electrophoresis Chemically defined diets for S. avenae were formulated as previously described

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118

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and sterilized with 0.22 µm Millipore membrane filters (Merck Millipore,

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Germany). Then, 1 mL of artificial diet was sandwiched between two layers of

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Parafilm membrane (Bemis, WI, USA) stretched across a PVC tube, 27 mm in

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diameter and 40 mm high, under sterile conditions. The Parafilm was sterilized and

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exposed to UV light for a minimum of 1 h before use. Approximately 200 S. avenae

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of different instars were carefully collected from wheat plants and starved for 2 h, then

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transferred to the PVC tubes to feed on the artificial diet for 48 h in an environmental

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chamber (20±1°C, L: D=16 h: 8 h); tubes with the same volume of artificial diet but

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without aphids feeding were used as a control. The secreted saliva was collected from

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a total of 100 mL of diet (approximately 20,000 aphids) and stored at -70°C until use.

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The salivary sample was concentrated to a volume of 2 mL using a Vivaspin 20

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centrifuge concentrator (Sartorius, Gottingen, Germany) with a 3000 Da molecular

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weight cut-off PES membrane at 4 °C, 15000 g for at least 1 h. Ten microliters of

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concentrated saliva sample or 5 µL of protein ladder (PageRulerTM Unstained Protein

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Ladder, Thermo Scientific, USA) mixed with an equal volume of loading buffer was 7

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heated in boiling water for 5-10 minutes then loaded into the wells of the gel. Proteins

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were separated by one-dimensional polyacrylamide gel electrophoresis with 5%

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stacking and 12 % separating gel. Silver nitrate staining was conducted as previously

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described to detect aphid salivary protein bands 32.

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Saliva infiltration

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Twenty microliters of concentrated saliva or control sample was diluted to a

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volume of 200 µL with distilled water and then infiltrated into the first leaf of a wheat

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plant at the two-leaf stage using a 1 mL syringe without the needle. Leaves infiltrated

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with same volume of control sample were used as control groups. The plants were

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then reared in the same environment as described above for further study. The

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infiltrated leaves of the plants were collected after 6 h and 24 h of infiltration.

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Total RNA isolation and cDNA synthesis

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Leaf samples were collected using sterilized scissors and transferred into liquid

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nitrogen immediately, then stored at -70 °C until use. Total RNA was extracted from

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leaves using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) following the

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protocols provided by the manufacturer. The quality and quantity of RNA were

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assessed with NanoDrop™ 2000 Spectrophotometers (Thermo Scientific, CA, USA).

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A total of 1 µg of RNA was reverse transcribed into cDNA with a Transcript One-Step

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gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing,

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China) following the manufacturer’s instructions, and cDNA templates were stored at

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-20°C until they were used for RT-qPCR.

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RT-qPCR analysis 8

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The relative expression of genes involved in the JA and SA defense signaling

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pathways of wheat after aphid infestation and saliva infiltration were detected using

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RT-qPCR. Target genes for the JA-responsive pathway included LOX, AOS and Ω-3

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fatty acid desaturase (FAD) , which are involved in JA biosynthesis

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tested for the SA-responsive pathway were the SA synthesis enzymes PAL and

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isochorismate

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pathogenesis-related protein 1 (PR-1) 34. Actin was used as an internal control and

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was synthesized according to Liu et al. 33. Primers for RT-qPCR were designed using

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Primer Premier 5.0. All primer sequences are shown in Table 1.

synthase

(ICS)

and

the

induced

SA

33

. The genes

marker

protein

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RT-qPCR was performed on an ABI 7500 Real-Time PCR System (Applied

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Biosystems, Carlsbad, CA, USA). cDNA was diluted 10-fold and then used as

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templates to detect the relative expression of the target genes in a 20 µL reaction

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system containing 2 µL of cDNA, 0.5 µL each of 10 µmol L-1 forward primer and

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reverse primer, 10 µL of 2× SYBR premix Ex TaqTM (Tli RNaseH Plus, Takara,

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Dalian, China) and 0.4 µL of 50× ROX Reference Dye II (Tli RNaseH Plus, Takara,

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Dalian, China) under the following conditions: 30 s at 95 °C followed by 40 cycles of

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30 s at 95 °C and 40 s at 60 °C. In RT-qPCR, there were 3 biological replicates for

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each treatment, and each replicate consisted of 3 technical replicates.

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Aphid bioassay

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Choice test

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After 24 h of saliva infiltration, wheat plants were placed horizontally on the flat

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table, and the same length of leaves (5 cm) with two different treatments were 9

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carefully inserted into a transparent plastic column (24 cm in width, 5 cm in height)

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from holes in opposite sides (Figure 1). Thirty winged S. avenae were collected in a

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2.0 mL centrifuge tube and then released from the middle of plastic column device.

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The number of aphids on each leaf was recorded at 6, 24 and 48 h. There were 15

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replicates for each test, and all of them were conducted in environmentally controlled

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room with a temperature of 20±1°C, relative humidity of 40-60 % and a photoperiod

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of L:D=16 h:8 h.

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Intrinsic rate of increase of aphid population

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At the two-leaf stage, the first wheat leaves were infiltrated with aphid saliva as

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described above. After 24 h, one newborn aphid was transferred onto the

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saliva-treated wheat leaf and the movement of aphids was restricted on the leaf in a

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plastic ecological cage (2.7×2.7×2.7 cm). The edge of the ecological cage was

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covered with sponge to avoid causing mechanical wounds to the leaf. The instar of the

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aphid was checked every 12 h to record the time when it produced the first nymphs.

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Then, the number of newborn nymphs was recorded every day, and the nymphs were

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removed after each count to avoid crowding. The period from the birth of the aphid to

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its first reproduction was defined as development days (Td). The number of newborn

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nymphs during the Td was expressed as Md. The intrinsic rate of increase (rm) for

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each aphid was calculated by the following equation: rm=0.738 × (lnMd) / Td

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Fifteen replicates were conducted in each group. The wheat seedlings were replaced

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every 3 days with new seedlings receiving the same treatment.

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Mean relative growth rate of aphid 10

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Fifteen newborn aphid nymphs were collected into 0.2 mL microcentrifuge tubes

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and weighed, and then all aphids were fed on saliva-treated or control wheat leaves as

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described above. After 7 days, all 15 aphids were collected and weighed again. The

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wheat seedlings were replaced every 3 days with new seedlings receiving the same

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treatment. Each pot contained one wheat plant and was kept in a climate incubator

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with a temperature of 20±1°C and a photoperiod of 16 h: 8 h (L:D). A total of 18

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replicates were performed for each treatment. The mean relative growth rate (MRGR)

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of S. avenae was calculated as described previously: MRGR=(ln 7-day weight - ln

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birth weight)/7 36.

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Detection of aphid feeding behavior by EPG

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EPG (Giga-8d) was conducted to record the feeding behavior of S. avenae on

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wheat leaves. Wingless adult S. avenae were gently collected from wheat plants using

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a brush and starved for 30 min, then 2-3 cm of 18 mm gold wire was attached to the

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abdomen of each aphid with water-based silver glue. The plant electrode was inserted

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into the soil in the pot in order to obtain successful electrical access. The complete

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insect electrode was carefully pinned into the probe input (BNC connector). EPG was

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performed from 10:00 to 16:00 every day and recorded continuously for 6 h. Each

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aphid and plant was used only once. All experiments were carried out in a Faraday

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cage at 20±1°C. The visualization and manual labeling of the various feeding waves

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were carried out using Stylet+d. Characteristics of the aphid feeding waves were

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identified as described in a previous study 37, 38.

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Statistical analysis 11

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All data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, USA).

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The percentages of S. avenae that settled on plant leaves in the choice test were

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arcsine-square-root transformed before analysis, and the differences between groups

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were examined using Student’s t-test. EPG data were analyzed by a Mann-Whitney U

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test. For RT-qPCR, each treatment was performed in triplicate, and the differential

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expression was calculated using 2–∆∆CT method 39. The fold change of the expression

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of genes involved in the JA and SA signaling defense pathways between the control

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and treatment conditions was calculated and then analyzed using Student’s t-test. P

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values less than 0.05 were considered statistically significant.

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Results

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Aphid feeding induced local defense responses

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To determine whether aphid infestation could induce resistance in wheat, two

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genes involved in JA- and SA-mediated defense responses were identified as

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differentially expressed after S. avenae feeding (Figure 2A and 2B). The relative

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expression of the JA-responsive gene FAD had a significant increase in local leaves

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after infestation by aphids (1.98±0.092-fold, t4=5.745, P=0.005), but in systemic

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leaves of aphid-infested plants, the mRNA levels of FAD were not significantly

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different from those in uninfested plants. Similarly, the relative expression of the

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SA-responsive gene PR-1 was significantly up-regulated in local leaves that had been

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fed upon by aphids previously (4.04±0.88-fold, t4=4.865, P=0.008), whereas no

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significant differences were observed in the systemic leaves of the same plants. These

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results indicated that aphid feeding induced a local defense response in wheat. 12

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Collection of S. avenae saliva

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Aphid salivary protein bands were detected on 12 % separated gel using silver

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staining. The results in Figure 3 show that the protein bands were obviously stained

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and were mainly distributed at approximately 15 kDa and between 40 kDa and 85

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kDa.

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Expression of defense-related genes after S. avenae saliva infiltration

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Some key genes involved in the JA and SA defense pathways were found to be

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differentially expressed in wheat leaves (Figure 4A and 4B). The three JA-responsive

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genes showed no significant difference between treatment and control conditions after

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6 h of infiltration, but the SA synthesis enzyme PAL and the SA downstream signaling

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protein PR-1 were significantly up-regulated, with 2.94±0.76-fold (t2.009=4.441,

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P=0.047) and 4.17±0.73-fold (t4=4.477, P=0.011) increases, respectively.

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After 24 h of saliva treatment, the relative expression of the JA defense-related

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gene AOS increased significantly (1.94±0.42-fold; t4=3.791, P=0.02) compared with

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the level found in the control, but there was no significant difference in the expression

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of FAD (t4=1.216, P=0.291). The expression level of LOX was also up-regulated

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1.57±0.15-fold, showing a significant increase (t4=3.076, P=0.037) between the saliva

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and control treatments. The mRNA levels of both PAL (5.39±1.59-fold; t4=4.36,

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P=0.012) and ICS (3.07±0.52-fold; t4=6.251, P=0.003), enzymes involved in the SA

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synthesis pathway, showed significant up-regulation with saliva treatment, and the

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expression of the SA signaling marker protein PR-1 was up-regulated 14.17±2.71-fold

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after saliva treatment and showed significant increases compared with the control 13

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(t2.016=-4.74, P=0.041).

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Aphid performance

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The results in Table 2 show that there was no significant difference in the

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development time or mean relative growth rate of S. avenae between saliva treatment

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and control. However, the number of nymphs per day produced by S. avenae was

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reduced significantly when they were fed on wheat leaves with saliva infiltration.

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Furthermore, the intrinsic rate of increase of the population of S. avenae was also

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significantly decreased (t28=2.360, P=0.025) after the insects fed on wheat leaves that

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were infiltrated with aphid saliva compared to the control groups. In the choice test,

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the percentage of winged aphids that landed on leaves treated with aphid saliva was

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significantly less than the percentage landing on control groups (t28=6.545, P