Watery Saliva Secreted by the Grain Aphid - American Chemical Society

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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 *

*

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

1

ACS Paragon Plus Environment


1

ABSTRACT

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

13

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

19

performance, choice preference, feeding behavior

20

21 22

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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

26

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

27

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

28

development, while indirect defenses consist of volatile emissions that repel

29

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

30

herbivore feeding 1, 2.

31

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

32

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

33

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

34

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:

37

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

38

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

41

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

42

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)

44

on tomato in compatible and incompatible interactions 3


10

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.

13

. 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

19

. Additionally, aphids inject watery saliva, a more complex

8, 21

4


. Although, to date, no

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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

29

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

5



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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

31

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

165

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


35

.

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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

228

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

237

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

238

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

239

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

240

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

241

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

242

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

245

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

246

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

247

kDa.

248

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

251

genes showed no significant difference between treatment and control conditions after

252

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

253

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

254

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

255

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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



265

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

266

Aphid performance

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

Watery Saliva Secreted by the Grain Aphid - American Chemical Society

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