# Pre-infestation of Tomato Plants by Aphids Modulates Transmission-Acquisition Relationship among Whiteflies, Tomato Yellow Leaf Curl Virus (TYLCV) and Plants

Xiao L. Tan, Ju L. Chen, Giovanni Benelli, Nicolas Desneux, Xue Q. Yang, Tong X. Liu, Feng Ge

► To cite this version:

Xiao L. Tan, Ju L. Chen, Giovanni Benelli, Nicolas Desneux, Xue Q. Yang, et al.. Pre-infestation of Tomato Plants by Aphids Modulates Transmission-Acquisition Relationship among Whiteflies, Tomato Yellow Leaf Curl Virus (TYLCV) and Plants. Frontiers in Plant Science, 2017, 8, 10.3389/fpls.2017.01597. hal-01615933

HAL Id: hal-01615933

https://hal.science/hal-01615933

Submitted on 12 Oct 2017

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Check for
updates

# Pre-infestation of Tomato Plants by Aphids Modulates Transmission-Acquisition Relationship among Whiteflies, Tomato Yellow Leaf Curl Virus (TYLCV) and Plants

Xiao L. Tan<sup>1,2,3†</sup>, Ju L. Chen<sup>2†</sup>, Giovanni Benelli<sup>4</sup>, Nicolas Desneux<sup>5</sup>, Xue Q. Yang<sup>6</sup>, Tong X. Liu<sup>3*</sup> and Feng Ge<sup>1*</sup>

Herbivory defense systems in plants are largely regulated by jasmonate-(JA) and salicylate-(SA) signaling pathways. Such defense mechanisms may impact insect feeding dynamic, may also affect the transmission-acquisition relationship among virus, plants and vectoring insects. In the context of the tomato - whitefly - Tomato Yellow Leaf Curl Virus (TYLCV) biological model, we tested the impact of pre-infesting plants with a non-vector insect (aphid *Myzus persicae*) on feeding dynamics of a vector insect (whitefly *Bemisia tabaci*) as well as virus transmission-acquisition. We showed that an aphid herbivory period of 0-48 h led to a transient systemic increase of virus concentration in the host plant (root, stem, and leaf), with the same pattern observed in whiteflies feeding on aphid-infested plants. We used real-time quantitative PCR to study the expression of key genes of the SA- and JA-signaling pathways, as well as electrical penetration graph (EPG) to characterize the impact of aphid pre-infestation on whitefly feeding during TYLCV transmission (whitefly to tomato) and acquisition (tomato to whitefly). The impact of the duration of aphid pre-infestation (0, 24, or 48 h) on phloem feeding by whitefly (E2) during the transmission phase was similar to that of global whitefly feeding behavior (E1, E2 and probing duration) during the acquisition phase. In addition, we observed that a longer phase of aphid pre-infestation prior to virus transmission by whitefly led to the up-regulation and down-regulation of SA- and JA-signaling pathway genes, respectively. These results demonstrated a significant impact of aphid pre-infestation on the tomato - whitefly - TYLCV system. Transmission and acquisition of TYLCV was positively correlated with feeding activity of *B. tabaci*, and both were mediated by the SA- and JA-pathways. TYLCV concentration

## OPEN ACCESS

**Edited by:**

Gero Benckiser,
Justus-Liebig-Universität Gießen,
Germany

**Reviewed by:**

Yuanyuan Song,
Fujian Agriculture and Forestry
University, China
Yaobin Lu,
ZheJiang Academy of Agricultural
Sciences, China

**\*Correspondence:**

Tong X. Liu
txliu@nwsuaf.edu.cn
Feng Ge
gef@ioz.ac.cn

**Specialty section:**

This article was submitted to
Plant Microbe Interactions,
a section of the journal
Frontiers in Plant Science

Received: 05 December 2016

Accepted: 31 August 2017

Published: 22 September 2017

**Citation:**

Tan XL, Chen JL, Benelli G, Desneux N, Yang XQ, Liu TX and Ge F (2017) Pre-infestation of Tomato Plants by Aphids Modulates Transmission-Acquisition Relationship among Whiteflies, Tomato Yellow Leaf Curl Virus (TYLCV) and Plants. *Front. Plant Sci.* 8:1597. doi: 10.3389/fpls.2017.01597

<footnote n="1">¹ State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, ² State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, ³ State Key Laboratory of Crop Stress Biology for Arid Areas and the Key Laboratory of Crop Pest Management on the Losses Plateau of Ministry of Agriculture, Northwest A&F University, Yangling, China, ⁴ Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy, ⁵ INRA (French National Institute for Agricultural Research), UMR 1355-7254 Institute Sophia Agrobiotech, CNRS, Université Côte d'Azur, Sophia Antipolis, France, ⁶ Key Laboratory of Economical and Applied Entomology of Liaoning Province, College of Plant Protection, Shenyang Agricultural University, Shenyang, China</footnote>

<footnote>† These authors have contributed
equally to this work.</footnote>
during the transmission phases was modulated by up- and down-regulation of SA- and JA-pathways, respectively. The two pathways were inconsistent during the acquisition phase; SA- related genes were up-regulated, whereas those up- and down-stream of the JA pathway showed a more complex relationship. These findings enhance our understanding of plant - herbivore - virus interactions, which are potentially important for development of ecologically sound pest and pathogen management programs.

**Keywords:** TaqMan Real-Time PCR, systemic induced defense, *Bemisia tabaci*, *Myzus persicae*, electrical penetration graph, jasmonic acid, salicylic acid, signaling pathways

INTRODUCTION

In recent decades, there have been many studies on the ecological and physiological mechanisms underlying plant-mediated interactions among herbivore arthropods and plant pathogens, particularly regarding plant defense responses (Al-Bitar and Luisoni, 1995; Beale et al., 2006; Bleeker et al., 2011; Baldin et al., 2013; Mouttet et al., 2013). Although it is well established that infection by pathogenic microorganisms may indirectly influence the behavior of the subsequent herbivore insects of the plant (Beale et al., 2006; Goggin, 2007; Mouttet et al., 2011), there has been less work on the effect of insect herbivore infestation on plant pathogens (Czosnek et al., 1988; Mouttet et al., 2011, 2013). The defense responses induced in plants after incidence of herbivory is central to the longstanding co-evolutionary system of host plant, pathogenic microorganism and herbivore arthropod (including vectors). Furthermore, these responses often impact prevalence of disease and pests, as well as the degree of damage to plants (Mayer et al., 2002; Cory and Hoover, 2006; Huot et al., 2013). However, there are key gaps in understanding of host plant - vector insect - pathogen interaction systems. For example, it is unclear how phytohormone-mediated plant defenses which are induced by insect infestations or virus transmission may influence behaviors of virus-carrying insects. Thus, understanding the mechanisms by which virus transmission occurs in plants showing herbivore-induced defense may be helpful for development of novel pest management strategies.

There have been numerous studies describing various defense systems that are induced in plants in response to damage caused by pathogens and herbivore arthropods (Karban and Baldwin, 1997; Huot et al., 2013). Key signaling pathways identified as regulators of plant defense are Jamonate (JA) for insect infestations and Salicylate (SA) for pathogen infestations, although the relationship between gene expression and function can be complex (Thaler et al., 2010). Further, understanding signal interactions within a plant - insect - pathogen system requires consideration of ecological context and the temporal sequence of interactions, such as the effect that an initial herbivore attack might have on the system during subsequent infestations. These types of interactions are more representative of incidents in natural and agricultural settings, and thus must be studied to develop more effective solutions for pest/pathogen management (Agrawal et al., 2000; Howe and Jander, 2008; Taggar and Gill, 2016).

Tomato Yellow Leaf Curl Virus is a cosmopolitan geminivirus which can infect dicotyledons, including tomato (Goggin, 2007; Heil, 2008), and is usually transmitted by the globally invasive whitefly *Bemisia tabaci* (Green and Ryan, 1972; Rubinstein and Czosnek, 1997). The transmission of TYLCV by *B. tabaci* causes huge losses in tomato yield in many regions, including China (Pan et al., 2012). The acquisition of TYLCV by *B. tabaci* occurs when a non-infected whitefly feeds on the phloem of a TYLCV-infected plant, and transmission occurs when a virus infected *B. tabaci* feeds on a previously healthy plant (Czosnek et al., 2002). It has been shown that within this defense system, a prior attack by aphid *Myzus persicae* can influence the feeding strategy and population dynamics of subsequent herbivores (Tan et al., 2014). It is unclear whether TYLCV transmission and acquisition are also affected by the feeding behavior of whiteflies, or whether these are influenced by aphid pre-infestation.

Plants infected by TYLCV may attract more whiteflies, and generally create conditions suiting colonization and suppression of the virus (Stout et al., 2006; Luan et al., 2013). Previous research has shown that plant defense responses induced by aphids are similar to those induced by pathogens (Thompson and Goggin, 2006). Aphid attacks can activate the SA-signaling pathway and suppress the JA-signaling pathway of the plant, and consequentially attract more whiteflies (via JA) and suppress TYLCV (SA) (Zarate et al., 2007; Verhage et al., 2010). Increasing this attraction can lead to aggregation of whiteflies and increase their feeding, both of which may increase TYLCV transmission and acquisition. Therefore, evaluation of the SA- and JA-pathway under aphid pre-feeding may help us to understand the mechanism by which induced defense is regulated.

In order to better understand the interactions in the plant- herbivore-virus system under conditions of sequential herbivory, we tested the hypothesis that pre-infestation by aphid *M. persicae* affects the expression of hormone-related genes and the SA- and JA-signaling pathways, consequentially impacting feeding behavior of subsequent whiteflies and thus the transmission and acquisition of TYLCV. TaqMan Real-Time PCR has been used for rapid and efficient quantitative detection of organismal DNA. It has proven highly sensitive for detecting DNA viruses in insect vectors and plants (Lei et al., 1987; Kunkel and Brooks, 2002), and there have been reports on using this technology for detection of TYLCV in tomato (Liu et al., 2007). We use Real-Time PCR to quantify TYLCV in infected tissues of tomato plant as well as in *B. tabaci*. We tested if pre-infestation with *M. persicae* (i) affect the feeding behavior of *B. tabaci*, (ii) modulate TYLCV transmission and acquisition between host
plant and *B. tabaci*, and (iii) impact the expression of SA- and JA-signaling pathway genes (and potential link with vector feeding behavior and virus transmission and acquisition).

# MATERIALS AND METHODS

## Virus, Insects, and Plants

Tomato plants, *Solanum lycopersicum* L. (var. Baofen-F1, 2008, Changfeng Institute of Vegetable, Lintong, Xi'an, Shaanxi, China) were cultivated in plastic trays (50.0 × 25.0 × 15.0 cm), with eight plants per tray. Seedlings, about 4–5 cm in height, were transplanted into plastic pots (20 cm in depth and 15 cm in diameter) and placed in clean cages (60 × 60 × 60 cm, plastic frame, screened with 120-mesh nylon yarn net). Plants used in all experiments were approximately 30 cm in height with 4–6 true, fully expanded leaves. The experiments were conducted in an environmental chamber at 25 ± 2°C, 65 ± 5% relative humidity and photoperiod of 16/8 h of light/dark with artificial lighting of 3500 lux. About 15,000 *M. persicae* individuals were collected from pepper plants (*Capsicum annuum* L. var. Jingyuan New Prince, provided by Qing County Modern Agricultural Technology Promotion Center, Hebei Province) in a greenhouse on the campus of Northwest A&F University, Yangling, Shaanxi (116°22′42″E and 39°59′58″N) in March 2011. The aphids were maintained on tomato plants under the laboratory conditions described above. All tested aphids were offspring (> five generations) of the original collected specimens.

*Bemisia tabaci* whiteflies (507 males and 631 females) of Middle East-Asia Minor 1 (MEAM 1) species were collected from tomato plants in a greenhouse and their identity confirmed by mitochondrial DNA marker analysis as described by Chu *et al.* (2007). They were reared on cotton plants under the same conditions described for green peach aphids during March and April 2011. Newly emerged adult whiteflies were used in all experiments after they had occupied the tomato plants for more than five generations. TYLCV-infected tomato plants were provided by the Natural Enemy Application and Research Laboratory, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China. The plants were cultivated in the State Key Laboratory of Crop Stress Biology of Northwest A&F University using ventilated culturing cages (60 × 60 × 60 cm, plastic frame, screened with 120-mesh nylon yarn net) containing 100 pairs of whitefly adults. The whiteflies were moved to the cages containing 15 tomato plants with 4–6 leaves each. TYLCV-infected plants were tested after 20 days. *Bemisia tabaci* specimens were obtained from cotton leaves from which pseudo pupae had been cut off and the petiole had been inserted in a glass bottle filled with water and placed in a plastic cage (13 × 13 × 30 cm; 100-mesh nylon yarn net door on one side). Non-viruliferous whiteflies were maintained on cotton plants (*Gossypium arboreum* cv. SN407) that had been identified as non-infected hosts for TYLCV. Newly emerged Non-viruliferous adult whiteflies were used in experiments after they were moved from cotton plants and had occupied the tomato plants for more than five generations. Newly emerged whiteflies were

collected and released onto the TYLCV-infected tomato plants in ventilated culturing cages for a duration of 48 h, after which the whiteflies were used in the experiments; previous studies indicate that the viral concentration in whitefly peaks after 12–48 h of feeding on tomato plants infected with TYLCV (Mason *et al.*, 2008; Luo *et al.*, 2010; Lucas-Barbosa *et al.*, 2011).

## DNA Extraction and Virus Detection

Leaves of five TYLCV-infected tomato plants were ground into powder in a pre-chilled motor in liquid nitrogen. Genomic DNA was extracted from 40 mg powder using the Wizard™ Genomic DNA purification kit (Promega Corporation, Madison, WI, United States) according to manufacturer's instructions. Genomic DNA from healthy leaves of non-infected tomato plants were used as control. The extracted DNA was used as a template for PCR reaction or kept at -20°C for later use.

Based on the complete genomic sequence of TYLCV (GenBank accession number: NC_004005.1), two primer pairs: F1 (5'-CCAATAAGGCGTAAGCGTGTAGAC-3') and R1 (5'-A CGCATGCCTCTAATCCAGTGTA-3'), F2 (5'-TCCCCTCTAT TTCAAGATAACAGAACA-3') and R2 (5'-TCTGAGGCTGTAA TGTCGTCCA-3'), and TaqMan probe (5'-FAM- CCCAATGCC TTCCTG-MGB-3') were designed by GeneCore Bio Technologies Co. Ltd. (Shanghai, China) to amplify 543 and 161 bp regions of the TYLCV gene. Actin (ACT) gene of *B. tabaci* (GenBank accession number: AF071908) and S. lycopersicum (GenBank accession number: AB199316) were used as internal controls, and 174 and 191 bp regions of BtACT and SlACT genes were amplified using F3 (5'-TCCTTCCAGCCATCCTTCTTG-3') and R3 (5'-CGGTGATTTCCTTCTGCATT-3') (Li *et al.*, 2013) and F4 (5'-TGGTCGGAATGGGACAGAAG-3') and R4 (5'-CTCAGTCAGGAGAACAGGGT-3') (Chen *et al.*, 2017) as primer pairs, respectively.

The PCR reactions were conducted in a C1000 Thermal Cycler (BioRad, Hercules, CA, United States) using F1 and R1 as primer pair in 30 µL of mixture containing 1 µL of template DNA, 1 µL of each primer (10 µM), 3 µL of 10 × PCR buffer (150 mM Tris-HCl, 500 mM Tris-HCl, pH 8.0 with 25 mM MgCl₂), 1 µL of dNTP Mix (2.5 mM), 0.5 µL of Premix Ex Taq DNA polymerase (5 U/µL, Takara Biotechnology Co. Ltd., Dalian, China) and 22.5 µL of double distilled H₂O. The PCR conditions were 10 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 57°C and 2 min at 72°C and a final extension at 72°C for 10 min. The PCR products were separated on a 2% agarose gel and purified using the Biospin Gel Extraction Kit (Bioer Technology Co. Ltd., Hangzhou, China) as described by the manufacturer. The recycled PCR products were ligated into the pMD-19 T vector (Takara) and transformed into DH5α cells. After blue-white selection, positive clones were sequenced by the Shanghai Sunny Biotech Co. Ltd., China. The sequence-verified clones were cultivated and plasmids were extracted. The concentration of extracted plasmid DNA was measured using a UV-Vis spectrophotometer (NanoDrop 2000 c). Copy number was determined according to the equation: copy number/mL = 6.02 × 10²³ (cn/mol) × plasmid concentration (g/mL)/MW (g/mol). The standard curve of TYLCV coat protein gene was obtained by using serial 10-fold diluted plasmids
(9.16 × 10¹⁰ to 9.16 × 10 copies) as templates, and by using F2 and R2 as primer pairs and TaqMan fluorescence probe (Figure 1). Amplifications were performed in 25 µl volumes containing 12.5 µL of Premix Ex Taq (Probe qPCR), 2 µL of template, 0.5 µL of each 10 µM F2 and R2 primers using thermocycler conditions as follows: 95°C for 10 s, followed by 40 cycles of 10 s at 95°C and 30 s at 58°C. Then, the linear equation of log concentration versus Ct curve was generated, where Ct values were plotted from 10-fold serial dilutions of the plasmid DNA. The estimation of copy number in samples was performed by computing the estimates of linear regression coefficients. The quantifications of DNA samples were calculated based on the fluorescence (ΔRn) values. All samples were run in duplicate by TaqMan Real-Time PCR assay for accuracy of results.

High specificity was determined for primers (Figure 1). PCR amplification showed that the product of TYLCV DNA corresponded to a 161 bp band, and lacking primer dimers. The data for the estimation of a standard curve for absolute quantitative and TaqMan Real-Time PCR assays were obtained and analyzed by an iQTM5 Multicolor Real-Time PCR Detection System. Thus, sample Ct values were obtained automatically. Ct values and the logarithm of TYLCV DNA copy number

(log) had a linear relationship. The logarithmic average of TYLCV DNA copy number (log) was calculated through a standard curve, giving y = -3.4015x + 42.465, slope -3.4015, R² = 0.9973 from each sample of TYLCV, an amplification efficiency (E) of 96.8%, and showed a robust linear relationship (Figure 1B).

## Impact of Aphid Pre-infestation Duration on TYLCV Transmission and Acquisition by Whiteflies on Tomato Plants

We assessed pre-infestation effects on viruses, separately for the processes of transmission and acquisition, and each by varying the duration of pre-infestation. We sequentially added aphids then whiteflies, then measured virus concentration. For transmission, Each tomato plant with 4-5 expanded leaves was infested separately with 60 fourth-instar aphid nymphs, then the aphids were removed using a soft brush after 12, 24, or 48 h. Aphids were randomly assigned to each plant. Sixty viruliferous adult whiteflies were introduced throughout the tomato plant (with sufficient space for flight in the mini cage) and fed for 24 h after removal of aphids. The whiteflies were removed using an aspirator. A group of five tomato plants (including roots) was collected, the roots washed with sterile distilled water, and stored at -80°C. Muniyappa et al. (2000) showed that the virus can be transmitted from whiteflies that have fed on TYLCV-infected plants for 24 h, to uninfected plants after exposure of only 20 min. Jiu et al. (2006) reported that the shortest time of transmission of TYLCV to plants by whiteflies was 15-30 min. Thus, in the present study, a 24 h feeding time was thought sufficient to transmit TYLCV to tomato plants. The experiment was repeated five times, each time with five tomato plants. Control tomato plants were not infested with aphids. Leaves of five TYLCV-infected tomato plants were ground into powder in a pre-chilled motor in liquid nitrogen. Virus concentration was assessed by DNA extraction and PCR; the TaqMan PCR amplification system followed the methods described above. For virus acquisition, we also measured TYLCV for *B. tabaci* via feeding on virus-infested tomato plants. Viruliferous whitefly were first used to transmit virus to the tomato plant, followed by aphid preinfestation and subsequent whitefly exposure. Tomato plants treated as above were individually placed in a square plastic cage (13 × 13 × 30 cm), and 60 viruliferous adult whitefly introduced using an aspirator. The whitefly adults were removed after feeding for 24 h, and then the plants were pre-infested with aphids as above. Fifty newly emerged non-viruliferous whitefly adult females were introduced into the cage to acquire virus from the infected tomato plant after removing the aphids. After feeding for 24 h, 40 whiteflies per plant from each group of five tomato plants were collected and stored at -80°C. Control tomato plants were not infested with aphids. The experiment was replicated five times, each time with five different tomato plants. For virus detection in whitefly, DNA was extracted using the Wizard™ Genomic DNA purification kit (Promega, Madison, WI, United States) from 200 whiteflies of a group of five tomato plants, which were ground into homogenate in 600 µL of nuclear pyrolysis liquid. The extracted DNA was diluted 10 times for

<figure><img src="image_3.png" /></figure>
TaqMan PCR assay or saved at -28°C for later use. PCR amplification system and response procedures were as reported above.

## Effects of Whitefly Feeding Behavior on Virus Transmission and Acquisition

### Tomato Plants Were Treated as Outlined in Section "Impact of Aphid Pre-infestation Duration on TYLCV Transmission and Acquisition by Whiteflies on Tomato Plants"

Each tomato plant with 4-5 expanded leaves was infested separately with 60 fourth-instar aphid nymphs, then the aphids were removed using a soft brush after 24 or 48 h. Aphids were randomly assigned to each plant. Because the concentration of TYLCV was similar for aphid feeding of 12 and 24 h, this test used tomato plants infested with aphids for 24 or 48 h. The experiment was repeated three times, each time with eight whiteflies recorded.

For transmission, whitefly feeding during the virus transmission phase was recorded. Feeding behavior was assessed for newly emerged viruliferous whiteflies from TYCLV infected tomato plants. For acquisition, whitefly probing behavior during virus acquisition (from tomato) was recorded for newly emerged whiteflies from non-infected tomato.

Whitefly feeding was monitored using the electrical penetration graph (EPG) method (Liu et al., 2013). In EPG, the insect and its host plant creates an electrical circuit that is completed when the mouthparts of the phloem-feeding insect penetrate the plant. The resulting electrical signals are amplified and digitized. Fluctuations in voltage and electrical resistance are recorded on computer, and can be matched to specific feeding events. Prior to recording, whiteflies were immobilized by placing them in a freezer for several minutes, then waiting 1 h without feeding. The recording electrode of the EPG system (a thin gold wire of length 2 cm and diameter 10 µm), was connected to the whitefly's dorsum with silver paint. Electrode adhesion was conducted under a microscope, and then the whitefly was put on the abaxial surface of the leaf and allowed to crawl freely. The wired whiteflies were then connected to the Giga-8 probe input, and another electrode was inserted into the soil at the base of the tomato plant. The whole system was placed into an electrically grounded Faraday cage to guard against external electrical noise. The EPG signals were digitized with a DI710-UL analog-to-digital converter, and the output was acquired and stored with PROBE3.4 software. The data were analyzed subsequently with STYLE7 2.0 software. Phases of feeding behavior are described by 14 EPG parameters related to the pathway phase (C waveform, F waveform and potential drops), phloem phase (E waveform) and xylem phase (G waveform). Phloem subphase 1 (E1: salivary secretion into sieve elements) and phloem subphase 2 (E2: phloem ingestion) were extracted from each recording and compared among treatments. 12 h of EPGs were continuously recorded for each replicate. All experiments were carried out under artificial light (1,500 lx) with a 16 h light/8 h dark regime and at 25°C ± 2°C, at 70% relative humidity (RH).

## Aphid Pre-infestation Effects on Tomato Defense-Related Genes before Transmission and Acquisition

Each treatment combination was replicated three times with eight plants for biological replicates, and each biological replicate contained three technical replicates. The expression of target genes involved in the JA-signaling [lipoxygenase D (LOXD), proteinase inhibitor II (PI-II)] and SA-signaling [phenylalanine ammonia lyase (PAL), and β-1,3-glucanase (PR2b)] pathways were determined.

The RNeasy Mini Kit (Qiagen, Valencia, CA, United States) was used to isolate total RNAs from tomato leaves (100-mg samples) before viruliferous whitefly and non-infected whitefly feeding, and 1 µg of RNAs were used to generate the cDNAs. The mRNA amounts of seven target genes were measured by real-time quantitative PCR.

Specific primers for each gene were designed from the tomato plant expressed sequence tag sequences using PRIMER5 software (Table 1). The reaction mixture contained the following reagents: 1 µL of first-strand cDNA, 2 µL of reaction buffer, 1 µL of MgCl₂ (50 mM), 0.3 µL of SYBR Green, 0.2 µL of PLATINUM Taq polymerase, 0.5 µL of dNTPs (10 mM) and 10 µM of each primer in a total volume of 20 µL. Reactions were carried out on the Mx 3500P detection system (Stratagene), and PCR was performed under the following conditions: 2 min at 95°C; followed by 40 cycles of 30 s at 95°C and 30 s at 60°C. Following the real-time quantitative PCR, a melting curve was generated by gradually increasing the temperature to 95°C to test the homogeneity of PCR products. Relative standard curves for the transcripts of every gene were prepared with linear gradient (five-fold) cDNA as template and were included within each real-time quantitative PCR. The relative level of each target gene was standardized by comparing the copy numbers of target mRNA with *SLACT* and *SITUB* CT values, which remained constant under different treatment conditions. The fold-changes of target genes were calculated using the 2^ΔΔCT method. All PCR runs were performed with three technical replicates. The three

<table><caption>**TABLE 1** | Overview of the target genes used in this study, showing their GenBank accession numbers and the primer pair used for qRT-PCR.</caption><thead><tr><td>Target gene</td><td>GenBank no.</td><td>Sequence (5'-3')</td><td>Length (bp)</td></tr></thead><tbody><tr><td>SITUB-F</td><td>DQ205342</td><td>AACCTCCATTCAGGAGATGTTTT</td><td>180</td></tr><tr><td>SITUB-R</td><td></td><td>TCTGCTGTAGCATCCTGGTATT</td><td></td></tr><tr><td>SIActin-F</td><td>AB199316</td><td>5'-TGGTCGGAATGGGACAGAAG-3'</td><td>191</td></tr><tr><td>SIActin-R</td><td></td><td>5'-CTCAGTCAGGAGAACAGGGT-3'</td><td></td></tr><tr><td>PAL-F</td><td>M83314.1</td><td>5'-ACAGAATTGTTGACGGGTGA-3'</td><td>122</td></tr><tr><td>PAL-R</td><td></td><td>5'-CCATTCAGCTCTTCAGACA-3'</td><td></td></tr><tr><td>PR-2b-F</td><td>M80608.1</td><td>5'-CCCATTTCAAGTTCCTGCTT-3'</td><td>112</td></tr><tr><td>PR-2b-R</td><td></td><td>5'-AGAATTGCCAATCAACGTCA-3'</td><td></td></tr><tr><td>LOXD-F</td><td>U37840.1</td><td>5'-GGCTTGCTTTACTCCTGGTC-3'</td><td>72</td></tr><tr><td>LOXD-R</td><td></td><td>5'-AAATCAAAGCGCCAGTTCTT-3'</td><td></td></tr><tr><td>PI-II-F</td><td>AY129402.1</td><td>5'-TGATGAACCCAAGGCAAATA-3'</td><td>154</td></tr></tbody></table>

<footnote>*SITUB and SIActin are reference genes.</footnote>
independent biological replicates, each containing eight plants, were analyzed.

## Data Analysis

One-way analysis of variance (ANOVA) was used to analyze the data of log copy number of TYLCV DNA for the effects of duration of infestation by *M. persicae* on the transmission and acquisition of TYLCV, whitefly probing profiles (EPG) and defense gene expressions. ANOVA was performed with IBM SPSS statistics version 20.0 (SPSS, Chicago, IL, United States). An independent *t*-test was used to test the effects of aphid feeding duration on defense gene expression during transmission and acquisition. All data were checked for normality and equality of residual error variances, and were transformed (log or square root) where appropriate to satisfy assumptions of ANOVA. General Linear Mixed Model (GLMM) was used to analyze the effects of the duration of aphid pre-infestation, plant position and their interaction on TYLCV. In both cases, when a significant effect was found, Duncan's test was used as *post hoc* test to compare the log copy number of TYLCV DNA (*P* < 0.05).

<figure><img src="image_4.png" /></figure>

**FIGURE 2** | TYLCV DNA concentrations in different tomato plant parts after feeding by TYLCV-infested *Bemisia tabaci* according to various *Myzus persicae* pre-infestation duration treatments. The error bars are standard errors of the mean. Colored letters indicate significant differences of DNA concentration among different aphid pre-infestation durations in each tomato plant part, and ‘*’ indicates significant differences among three plant parts in the same pre-infestation duration according to Duncan's test at *P* = 0.05.

## RESULTS

### Impact of Aphid Pre-infestation Duration on TYLCV Transmission and Acquisition by Whiteflies

The GLMM tests highlighted significant differences in TYLCV DNA concentrations for different durations of aphid pre-infestation (*F* = 12.622, *df* = 3176, *P* < 0.05), and among different tomato plant tissues (*F* = 4.107, *df* = 2177, *P* < 0.05). Further, there was a significant interaction between duration of aphid pre-infestation, tomato plant tissues, and concentration of TYLCV DNA (*F* = 7.208, *df* = 6, *P* < 0.05) (Figure 2). The concentration of TYLCV DNA in *B. tabaci* after feeding on virus-infected tomato was influenced by duration of aphid pre-infestation (*F* = 3.769, *df* = 3176, *P* < 0.05; Figure 3).

### Aphid Pre-infestation Duration Effects on Feeding Behavior of Whiteflies during Transmission and Acquisition Phase Transmission

Twenty-four hour aphid infestation significantly increased the time that viruliferous whiteflies spent in the salivation phase [E1] (*F* = 3.586, *P* = 0.033, *df* = 2, 69) and duration of phloem feeding [E2] (*F* = 4.296, *P* = 0.017, *df* = 2, 69; **Figure 4A**). By contrast there was no significant effect on the total duration of probing by whiteflies (*F* = 2.658, *P* = 0.077, *df* = 2, 69; **Figure 4A**). For 48-h aphid infestation, the duration of E2 was similar to plants not infested with aphids; whereas significant differences are observed at 48 h for the duration of E1 and total duration of probing, compared to 0 h.

### Acquisition

Pre-infestation with *M. persicae* significantly impacted several aspects of whitefly feeding on virus-infected leaves (E1: *F* = 4.836, *P* = 0.011; E2: *F* = 6.173, *P* = 0.003; and total duration of probing: *F* = 3.669, *P* = 0.031; all *df* = 2, 69; **Figure 4B**). 24 h of aphid infestation significantly increased E1, E2 and the total duration of probing by whiteflies. For 48-h aphid infestation, the durations of E1, E2 and of probing were similar to plants not infested with aphids. This trend was similar to the virus concentration in whiteflies with increasing aphid feeding duration on viruliferous tomato plants (**Figure 4B**).

### Effects of Aphid Pre-infestation on Defense Genes in Tomato Prior to Transmission and Acquisition

The relative mRNA expressions of *PAL* (before transmission: *F* = 9.213, *P* = 0.0001, *df* = 2, 69; and before acquisition: *F* = 4.727, *P* = 0.012, *df* = 2, 69) and *PR2b* (before transmission: *F* = 19.881, *P* = 0.0001, *df* = 2, 69; and before acquisition: *F* = 2.553, *P* = 0.085, *df* = 2, 69) positively correlated with aphid feeding time prior to transmission by whiteflies (**Figure 5**). Similarly, prior to acquisition by whiteflies, expressions of *PAL* and *PR2b* in plants with 24 h of aphid feeding were higher than for those without aphids, but similar to those with 48 h of aphid feeding. Moreover, expressions of both genes were higher prior to acquisition by whiteflies than prior to transmission by whiteflies: *PAL* for 0 h, *t* = -2.924, *df* = 33.022, *P* = 0.006; for 24 h, *t* = -4.709, *df* = 35.63, *P* = 0.0001; and for 48 h, *t* = -2.132, *df* = 35.117, *P* = 0.04; and *PR2b* for 0 h, *t* = -6.654, *df* = 23.494, *P* = 0.0001; for 24 h, *t* = -4.547, *df* = 26.518, *P* = 0.0001; and for 48 h, *t* = -3.111, *df* = 35.117, *P* = 0.003 (**Figure 5**).
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** | TYLCV DNA concentrations in *Bemisia tabaci* adults after feeding on TYLCV-infested tomato plants for 24 h according to various *Myzus persicae* pre-infestation. The error bars are standard errors of the mean. Letters indicate significant differences of DNA concentration among different aphid pre-infestation durations according to Duncan's test at *P* = 0.05.</figcaption></figure>

In contrast, the relative mRNA expressions of JA-signaling genes *LOXD* (before transmission: *F* = 12.418, *P* = 0.00001, *df* = 2, 69; and before acquisition: *F* = 12.566, *P* = 0.00001, *df* = 2, 69) and PI-II (before transmission: *F* = 20.617, *P* = 0.00001, *df* = 2, 69; and before acquisition: *F* = 2.611, *P* = 0.081, *df* = 2, 69) were significantly reduced with increase in aphid feeding time, except for *PI-II* in plant prior to acquisition by whiteflies. The expression of *LOXD* in plants was lower prior to acquisition than prior to transmission by whiteflies, except for during an aphid infestation period of 24 h (*LOXD* for 0 h, *t* = 2.497, *df* = 46, *P* = 0.016; for 24 h, *t* = 1.666, *df* = 45.831, *P* = 0.103; and for 48 h, *t* = -2.741, *df* = 28.652, *P* = 0.01). However, expression of *PI-II* was lower in the non-infested plant prior to acquisition but not prior to transmission (*PI-II*, 0 h: *t* = 2.304, *df* = 46, *P* = 0.026; 24 h: *t* = -0.532, *df* = 30.834, *P* = 0.598; 48 h: *t* = -4.889, *df* = 27.284, *P* = 0.00001) (Figure 5).

DISCUSSION

This study is the first investigation on impact of herbivore pre-
infestation on virus transmission and acquisition by subsequent
herbivores, in terms of key interactions such as feeding
behavior and plant defenses. We demonstrated that virus
transmission and acquisition between tomatoes and vector
whiteflies under aphid pre-infestation follow two different
mechanisms. The SA- and JA-signaling pathways had different
roles and the feeding behavior of whiteflies dominated the
acquisition process. We believe that this overlooked issue
will be of wide ecological interest, since knowledge of the
effects of herbivorous feeding activity on the acquisition and
transmission of viruses can shed light on complex plant-
mediated relationships among herbivores, arthropod vectors
and pathogens (Mehta et al., 1994; Messina et al., 2002;
Beale et al., 2006; Mithöfer and Boland, 2012). Feeding

behavior (salivation/phloem ingestion/duration of feeding) of
viruliferous and non-infected *B. tabaci* was also influenced
by different aphid pre-infestation times, in contexts of both
transmission and acquisition. Furthermore, we found that the
expression of two SA-signaling pathway genes may increase
when aphid pre-infestation period is prolonged, and expression
of the related JA-signaling pathway is reduced, especially
during the transmission phase. Previous studies have shown
that attack by aphids can inhibit the JA-signaling pathways
and consequentially reduce negative impacts of herbivory
(Papayiannis et al., 2009, 2010; Giordanengo et al., 2010).
Plant defense responses induced by sap-sucking herbivores or
pathogens are often similar, since both responses can act to
resist both insects and pathogens (Thaler et al., 2002; Murugan
and Dhandapani, 2007). The defense signaling pathways relying
on SA and JA may vary with injury time (Preston et al.,
1999; Nombela et al., 2009). Tobacco plants infected with TMV
developed SAR, while damage response via JA was not normally
activated, possibly because infection by TMV increased SA
levels, inhibiting the JA pathway (Ohgushi, 2005). However, this
might reduce JA/ET-associated insect resistance (Salzman et al.,
2005).

TYLCV Transmission/Acquisition

The pattern in transmission of virus from viruliferous *B. tabaci* to non-infected tomato was consistent among different plant organs tested (attacked leaves, stems, and roots), as well as under different durations of aphid pre-infestation. When measured directly on the aphid-attacked leaf, the amount of TYLCV initially increased, and was followed by a decline, with extension of period of pre-infestation. The stem and the root showed similar tendencies; the pre-infestation was found to stimulate systemic resistance, resulting in a more effective response to subsequent attacks. Generally, herbivore feeding action leads to a diverse and synergistic systemic defense, performed across different tissues of the plant (Pico et al., 1999; Schilmiller and Howe, 2005; Jung et al., 2009). The defense pathways may be induced in tissues away from the position of attack, although this reaction is somewhat delayed (Kessmann et al., 1994). In this study, TYLCV DNA concentration in roots showed a belated increase, which demonstrated that a period of time is required for systemic transmission of TYLCV within tomato plants. Thus, a threshold period is needed for transmission of injury messages within the plant and for producing the defense compounds responsible for defense (Sauge et al., 2002). The acquisition of TYLCV by whitefly from TYLCV-infected tomato plants also displayed a tendency to rise and then decline. When increasing duration of aphid infestation beyond the 12 h threshold, the TYLCV concentration in whiteflies began to decrease. Aphids feeding on tomato plants for sufficient time can induce a plant defense response which inhibits whiteflies from acquiring TYLCV. Our result indicated that extension of the pre-infestation phase may increase virus transmission and acquisition by whitefly in the short term. This result might be explained by feeding efficacy, as influenced by the plant induced defense response.
Feeding Behavior of *Bemisia tabaci*
According to Aphid Pre-infestation

As our results show, the feeding behavior strategies of either TYLCV infected *B. tabaci* on non-infected tomato (virus transmission) or non-infected whitefly on viruliferous plants (virus acquisition) were both influenced by duration of pre-infestation by *M. persicae*. We found that the duration of phloem ingestion (E2) of *B. tabaci* may increase at 24 h and decrease at 48 h, with increasing of duration of pre-infestation. However, the total probing duration for whitefly was inconsistent between transmission/acquisition treatments. The transmission and acquisition of virus between whitefly and tomato plant may be due to different feeding strategies; for transmission of virus from whitefly to plant, net virus infestation is associated with salivation (E1). Thus, as indicated by the amount of TYLCV

DNA in the host plant (Figure 2), virus infection in tomato
may have a corresponding increase with salivation by viruliferous
*B. tabaci*, although unaffected by total duration of probing. In
contrast, the acquisition of virus by whitefly from infected plants
was mainly related to phloem ingestion (E2). Clearly, our results
showed similar responses to variation in aphid pre-infestation
between the amount of TYLCV in whitefly and the duration
of their phloem ingestion (Figures 3, 4). It seems that virus
transmission/acquisition is influenced by the whitefly's different
feeding strategies. The feeding of whiteflies may be inhibited by
the plant defense response triggered by aphid pre-infestation.
Empirical reports indicate that defensive materials (like JA, SA
and its derivants) may suppress phloem ingestion (Kehr, 2006).
Thus, the non-viruliferous whitefly showed a reduced ability to
combat this response, and struggle to maintain normal phloem

<figure><img src="image_6.png" /><figcaption>**FIGURE 4** | Aphid pre-infestation duration effects on feeding behavior (EPG) of transmission (A) of whiteflies on virus non-infected tomato plants and acquisition (B) of whiteflies on virus infected tomato plants. Letters indicate significant differences of feeding behavior among different aphid pre-infestation durations.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**FIGURE 5** | Aphid pre-infestation duration effects on related defense genes in the SA- (A) and JA- (B) signaling pathways in tomato plants. The relative mRNA expression was calculated according to the 2⁻^(ΔΔCt) method, and the y axis represents the level of change. The expression level of each target gene was first normalized to the quantity of *SlActin* and *SItub*. The error bar represents the standard deviation calculated from three replicates. Letters indicate significant differences in relative expression among different aphid pre-infestation durations, and '+' indicates significant differences in the same aphid pre-infestation duration according to Duncan's test at *P* = 0.05.</figcaption></figure>
ingestion feeding time. Interestingly, the virus infection may be
quite specific in the way it regulates feeding behavior, with the
whitefly enhancing only key processes, particularly salivation,
which is vital for virus transmission.

Gene Expression Related to SA- and
JA-Signaling Pathways

Defense response to herbivore or plant pathogens occurs mostly in the Salicylic Acid (SA) and Jasmonic Acid (JA) signaling pathways (Lamb et al., 1989). Feeding by aphids induce plant responses similar to those induced by pathogens (Czosnek et al., 2002; Thompson and Goggin, 2006). Aphid infestation may activate the SA-signaling pathway and suppress the JA-signaling pathway, which will attract whiteflies and suppress TYLCV (Verhage et al., 2010). SA activation and JA suppression both inhibit viruses and improve feeding behavior of herbivore insects, respectively (Zarate et al., 2007). Our results showed that extending the period of aphid pre-infestation may reduce expression of the genes involved in the JA-signaling pathway, and increase expression of the genes involved in the SA-signaling pathway. According to our results, it seems that upregulating the SA pathway and suppressing the JA pathway with aphid pre-infestation may directly impact the feeding strategies of the whitefly, and consequentially, the transmission and acquisition of TYLCV between whitefly and tomato plant. Similarly, there have been several reports showing that induced plant defense responses are stronger against pathogens compared to insect herbivores (Ghanim et al., 2001; Tan and Liu, 2014). Ohgushi (2005) reported that infestation by tobacco mosaic virus can induce systemic acquired resistance, while not activating the JA signaling pathway. Generally, the SA pathway is activated by pathogens, and the JA pathway is induced by insects (Salzman et al., 2005). Previous studies have shown that attack by sap-sucking herbivores can induce plant responses similar to those induced by pathogen infestations, inhibiting the JA-signaling pathways and consequentially reducing negative impacts on herbivores (Papayiannis et al., 2009, 2010; Giordanengo et al., 2010). Plant defense responses induced by herbivores or pathogens are often similar, since both responses can act to resist both insects and pathogens, or to minimize interference between herbivores and pathogen defenses (Thaler et al., 2002; Murugan and Dhandapani, 2007). Tomato plants infested by phloem-feeding insects (e.g., aphids) evoked JA- and SA-signaling pathways to co-regulate the expression of plant defense genes (Muniyappa et al., 2000). The antagonism between SA and JA impacts feeding behavior of whiteflies and viral propagation. In the transmission test, the SA and JA was dominant during the whole process. However, during the acquisition test, the JA-signaling pathway was the key factor affecting virus acquisition, and the feeding behavior of whiteflies dominated the whole process. SA activity may deter the transmission of pathogens, but inhibition of JA might attract herbivorous insects (Chu et al., 2007). The results showed that a decrease in JA combined with an increase in SA led to a net positive effect of aphid infestation on

increased feeding of whiteflies, outweighing inhibition of TYLCV
transmission.

Furthermore, the defense responses of these two pathways
may vary with duration of injury (Preston et al., 1999; Nombela
et al., 2009). Our results not only showed a temporal component
to variation in associated gene expression, but also showed that
indirect systemic responses (variation in amount of TYLCV
across plant organs) also vary according to duration of pre-
infestation. Generally, herbivore feeding leads to systemic defense
in the plant, enacted in a concerted manner across tissues and
organs (Polston et al., 1990; Pico et al., 1999). Thus, further
evaluation of defense-related gene expression induced by insect
feeding on different plant parts is required, and how this is
impacted by pre-infestation.

Generally, we have confirmed the hypothesis that aphid pre-
infestation impacts transmission and acquisition of TYLCV
between tomato and *B. tabaci*. Pre-infestation showed different
respective influences to the feeding strategies during the
transmission or acquisition processes, while associated with
opposing trends in expression of the SA- and JA- pathways.
Based on current understanding of the interactions between
the SA- and JA- pathways, particularly cross-inhibition, we
believe that the mechanism delineating the complex and precise
interaction among these organisms is worthy of further research.
Notwithstanding, due to the economic importance of tomato
crop, our study might have implications in the development of
novel strategies for the regulation of plant pathogens and insect
vectors.

AUTHOR CONTRIBUTIONS

XT, JC, TL, and FG designed the study. All authors drafted the
manuscript. XT, GB, ND, and XY implemented the analyses. All
authors gave final approval for publication.

ACKNOWLEDGMENTS

We thank Dr. T. Giordani (University of Pisa, Italy) for reading and commenting on an earlier version of the manuscript, and thanks Dr S. Wang and Dr. H. X. Xu for providing part of the material and methods. This work was supported by the National Basic Research Program of China (973 Program) (grant No. 2013CB127605), the Special Fund for Agroscientific Research in the Public Interest (grant Nos. 201303024 and 201303108), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11050400), the National Nature Science Fund of China (grant No. 31370438), the R&D Special Fund for Public Welfare Industry (Agriculture 201303019) and the State Key Laboratory of Integrated Management of Pest Insects and Rodents (Grant No. ChineseIPM1611). We are grateful for the assistance of all staff and students in the Key Laboratory of Applied Entomology, Northwest A&F University at Yangling, Shaanxi, China.
REFERENCES

Agrawal, A. A., Karban, R., and Colfer, R. G. (2000). How leaf domatia and induced plant resistance affect herbivores: natural enemies and plant performance. *Oikos* 89, 70–80. doi: 10.1034/j.1600-0706.2000.890108.x

Al-Bitar, L., and Luiisoni, E. (1995). Tomato yellow leaf curl geminivirus: serological evaluation of an improved purification method 1. *EPPO Bull.* 25, 269–276. doi: 10.1111/j.1365-2338.1995.tb01467.x

Baldin, E. L. L., Crotti, A. E. M., Wakabayashi, K. A. L., Silva, J. P. G. F., Aguiar, G. P., Souza, E. S., et al. (2013). Plant-derived essential oils affecting settlement and oviposition of *Bemisia tabaci* (Genn.) biotype B on tomato. *J. Pest Sci.* 86, 301–308. doi: 10.1007/s13744-015-0356-8

Beale, M. H., Birkett, M. A., Bruce, T. J., Chamberlain, K., Field, L. M., Huttly, A. K., et al. (2006). Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior. *Proc. Natl. Acad. Sci. U.S.A.* 103, 10509–10513. doi: 10.1073/pnas.0603998103

Bleeker, P. M., Spyropoulou, E. A., Diergaarde, P. J., Volpin, H., De Both, M. T., Zerbe, P., et al. (2011). RNA-seq discovery, functional characterization, and comparison of sesquiterpene synthases from *Solanum lycopersicum* and *Solanum habrochaites* trichomes. *Plant Mol. Biol.* 77, 323–336. doi: 10.1007/s11103-011-9813-x

Chen, S., Zhao, H., Wang, M., Li, J., Wang, Z., Wang, F., et al. (2017). Overexpression of E3 ubiquitin ligase gene AdBiL contributes to resistance against chilling stress and leaf mold disease in Tomato. *Front. Plant Sci.* 8:1109. doi: 10.3389/fpls.2017.01109

Chu, D., Jiang, T., Liu, G., Jiang, D., Tao, Y., Fan, Z., et al. (2007). Biotype status and distribution of *Bemisia tabaci* (Hemiptera: Aleyrodidae) in Shandong province of China based on Mitochondrial DNA markers. *Environ. Entomol.* 36, 1290–1295. doi: 10.1093/ee/36.5.1290

Cory, J. S., and Hoover, K. (2006). Plant-mediated effects in insect-pathogen interactions. *Trends Ecol. Evol.* 21, 278–286. doi: 10.1016/j.tree.2006.02.005

Czosnek, H., Ber, R., Navot, N., Zamir, D., Antignus, Y., and Cohen, S. (1988). Detection of tomato yellow leaf curl virus in lysates of plants and insects by hybridization with a viral DNA probe. *Plant Dis.* 72, 949–951. doi: 10.1094/PD-72-0949

Czosnek, H., Ghanim, M., and Ghanim, M. (2002). The circulative pathway of begomoviruses in the whitefly vector *Bemisia tabaci*—insights from studies with Tomato yellow leaf curl virus. *Annu. Appl. Biol.* 140, 215–231. doi: 10.1111/j.1744-7348.2002.tb00175.x

Ghanim, M., Morin, S., and Czosnek, H. (2001). Rate of Tomato yellow leaf curl virus translocation in the circulative transmission pathway of its vector, the whitefly *Bemisia tabaci*. *Phytopathology* 91, 188–196. doi: 10.1094/PHYTO. 2001.91.2.188

Giordanengo, P., Brunissen, L., Rusterucci, C., Vincent, C., Van Bel, A., Dinant, S., et al. (2010). Compatible plant-aphid interactions: how aphids manipulate plant responses. *C R Biol.* 333, 516–523. doi: 10.1016/j.crvi.2010.03.007

Goggin, F. L. (2007). Plant-aphid interactions: molecular and ecological perspectives. *Curr. Opin. Plant Biol.* 10, 399–408. doi: 10.1016/j.pbi.2007.06.004

Green, T., and Ryan, C. (1972). Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. *Science* 175, 776–777. doi: 10.1126/science.175.4023.776

Heil, M. (2008). Indirect defence via tritrophic interactions. *New Phytol.* 178, 41–61. doi: 10.1111/j.1469-8137.2007.02330.x

Howe, G. A., and Jander, G. (2008). Plant immunity to insect herbivores. *Annu. Rev. Plant Biol.* 59, 41–66. doi: 10.1146/annurev.arplant.59.032607.092825

Huot, O. B., Nachappa, P., and Tamborindeguy, C. (2013). The evolutionary strategies of plant defenses have a dynamic impact on the adaptations and interactions of vectors and pathogens. *Insect Sci.* 20, 297–306. doi: 10.1111/ 1744-7917.2010

Jiu, M., Zhou, X. P., and Liu, S. S. (2006). Acquisition and transmission of two Begomoviruses by the B and a non-B biotype of *Bemisia tabaci* from Zhejiang, China. *J. Phytopathol.* 154, 587–591. doi: 10.1111/j.1439-0434.2006. 01151.x

Jung, H. W., Tschaplinski, T. J., Glazebrook, J., and Greenberg, J. T. (2009). Priming in systemic plant immunity. *Science* 324, 89–91. doi: 10.1126/science.1170025

Karban, R., and Baldwin, I. T. (1997). *Induced Responses to Herbivory*. Chicago: University of Chicago Press. doi: 10.7208/chicago/9780226424972.001.0001

Kehr, J. (2006). Phloem sap proteins: their identities and potential roles in the interaction between plants and phloem-feeding insects. *J. Exp. Bot.* 57, 767–774. doi: 10.1093/jxb/erj087

Kessmann, H., Staub, T., Hofman, C., Maetzke, T., and Herzog, J. (1994). Induction of systemic acquired disease resistance in plants by chemicals. *Annu. Rev. Phytopathol.* 32, 439–459. doi: 10.1146/annurev.py.32.090194.002255

Kunkel, B. N., and Brooks, D. M. (2002). Cross talk between signaling pathways in pathogen defense. *Curr. Opin. Plant Biol.* 5, 325–331. doi: 10.1016/S1369- 5266(02)00275-3

Lamb, C. J., Lawton, M. A., Dron, M., and Dixon, R. A. (1989). Signals and transduction mechanisms for activation of plant defenses against microbial attack. *Cell* 56, 215–224. doi: 10.1016/0092-8674(89)90894-5

Lei, X. Y., Li, H. F., and Chiu, W. F. (1987). Effect of induced resistance on virus infection of some plants and the mechanisms of the resistance inducer NS-83. *Sci. Agric.* 20, 1–6.

Li, R., Xie, W., Wang, S., Wu, Q., Yang, N., Yang, X., et al. (2013). Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, *Bemisia tabaci* (Hemiptera: Aleyrodidae). *PLOS ONE* 8:e53006. doi: 10.1371/journal.pone. 0053006

Liu, B. M., Preisser, E. L., Chu, D., Pan, H., Xie, W., Wang, S., et al. (2013). Multiple forms of vector manipulation by a plant-infecting virus: *Bemisia tabaci* and tomato yellow leaf curl virus. *J. Virol.* 87, 4929–4937. doi: 10.1128/JVI.J03571-12

Liu, S. S., De Barro, P., Xu, J., Luan, J.-B., Zang, L. S., Ruan, Y. M., et al. (2007). Asymmetric mating interactions drive widespread invasion and displacement in a whitefly. *Science* 318, 1769–1772. doi: 10.1126/science.1149887

Luan, J. B., Yao, D. M., Zhang, T., Walling, L. L., Yang, M., Wang, Y. J., et al. (2013). Suppression of terpenoid synthesis in plants by a virus promotes its mutualism with vectors. *Ecol. Lett.* 16, 390–398. doi: 10.1111/ele.12055

Lucas-Barbosa, D., van Loon, J. J., and Dicke, M. (2011). The effects of herbivore-induced plant volatiles on interactions between plants and flower-visiting insects. *Phytochemistry* 72, 1647–1654. doi: 10.1016/j.phytochem.2011.03.013

Luo, C., Jones, C., Devine, G., Zhang, F., Denholm, I., and Gorman, K. (2010). Insecticide resistance in *Bemisia tabaci* biotype Q (Hemiptera: Aleyrodidae) from China. *Crop Prot.* 29, 429–434. doi: 10.1016/j.cropro.2009.10.001

Mason, G., Caciagli, P., Accotto, G. P., and Noris, E. (2008). Real-time PCR for the quantitation of Tomato yellow leaf curl Sardinia virus in tomato plants and in *Bemisia tabaci*. *J. Virol. Methods* 147, 282–289. doi: 10.1016/j.jviromet.2007. 09.015

Mayer, R. T., Inbar, M., McKenzie, C., Shatters, R., Borowicz, V., Albrecht, U., et al. (2002). Multitrophic interactions of the silverleaf whitefly, host plants, competing herbivores, and phytopathogens. *Arch. Insect Biochem. Physiol.* 51, 151–169. doi: 10.1002/arch.10065

Mehta, P., Wyman, J. A., Nakhla, M. K., and Maxwell, D. P. (1994). Polymerase chain reaction detection of viruliferous *Bemisia tabaci* (Homoptera: Aleyrodidae) two tomato-infecting geminiviruses. *J. Econ. Entomol.* 87, 1285–1290.

Messina, F. J., Durham, S. L., Richards, J. H., and McArthur, D. E. (2002). Trade-off between plant growth and defense? A comparison of sagebrush populations. *Oecologia* 131, 43–51. doi: 10.1007/s00442-001-0859-3

Mithöfer, A., and Boland, W. (2012). Plant defense against herbivores: chemical aspects. *Annu. Rev. Plant Biol.* 63, 431–450. doi: 10.1146/annurev-arplant- 042110-103854

Mouttet, R., Bearez, P., Thomas, C., and Desneux, N. (2011). Phytophagous arthropods and a pathogen sharing a host plant: evidence for indirect plant-mediated interactions. *PLOS ONE* 6:e18840. doi: 10.1371/journal.pone.0018840

Mouttet, R., Kaplan, I., Bearez, P., Amiens-Desneux, E., and Desneux, N. (2013). Spatiotemporal patterns of induced resistance and susceptibility linking diverse plant parasites. *Oecologia* 173, 1379–1386. doi: 10.1007/s00442-013- 2716-6

Muniyappa, V., Venkatesh, H., Ramappa, H., Kulkarni, R., Zeidan, M., Tarba, C. Y., et al. (2000). Tomato leaf curl virus from Bangalore (ToLCV-Ban4): sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships. *Arch. Virol.* 145, 1583–1598. doi: 10.1007/s007050070078

Murugan, M., and Dhandapani, N. (2007). Induced systemic resistance activates defense responses to interspecific insect infestations on tomato. *J. Veg. Sci.* 12, 43–62. doi: 10.1300/J484v12n03_05

Nombela, G., Garzo, E., Duque, M., and Muniz, M. (2009). Preinfestations of tomato plants by whiteflies (*Bemisia tabaci*) or aphids (*Macrosiphum
*euphorbiae*) induce variable resistance or susceptibility responses. *Bull. Entomol. Res.* 99, 183-191. doi: 10.1017/S0007485308006214

Ohgushi, T. (2005). Indirect interaction webs: herbivore-induced effects through trait change in plants. *Annu. Rev. Ecol. Evol. Syst.* 36, 81–105. doi: 10.1146/annurev.ecolsys.36.091704.175523

Pan, H. P., Chu, D., Yan, W. Q., Su, Q., Liu, B. M., Wang, S. L., et al. (2012). Rapid spread of tomato yellow leaf curl virus in china is aided differentially by two invasive whiteflies. *PLOS ONE* 7:e34817. doi: 10.1371/journal.pone.0034817

Papayiannis, L., Brown, J., Seraphides, N., Hadjistylli, M., Ioannou, N., and Katis, N. (2009). A real-time PCR assay to differentiate the B and Q biotypes of the *Bemisia tabaci* complex in Cyprus. *Bull. Entomol. Res.* 99, 573–582. doi: 10.1017/S0007485308006603

Papayiannis, L., Iacovides, T., Katis, N., and Brown, J. (2010). Differentiation of tomato yellow leaf curl virus and tomato yellow leaf curl Sardinia virus using real-time TaqMan® PCR. *J. Virol. Methods* 165, 238–245. doi: 10.1016/j.jviromet.2010.02.003

Pico, B., Diez, M. J., and Nuez, F. (1999). Improved diagnostic techniques for tomato yellow leaf curl virus in tomato breeding programs. *Plant Dis.* 83, 1006–1012. doi: 10.1094/PDIS.1999.83.11.1006

Polston, J., Al-Musa, A., Perring, T. M., and Dodds, J. (1990). Association of the nucleic acid of squash leaf curl geminivirus with the whitefly *Bemisia tabaci*. *Phytopathology* 80, 850–856. doi: 10.1094/Phyto-80-850

Preston, C. A., Lewandowski, C., Enyedi, A. J., and Baldwin, I. T. (1999). Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants. *Planta* 209, 87–95. doi: 10.1007/s004250050609

Rubinstein, G., and Czosnek, H. (1997). Long-term association of tomato yellow leaf curl virus with its whitefly vector *Bemisia tabaci*: effect on the insect transmission capacity, longevity and fecundity. *J. Gen. Virol.* 78, 2683–2689. doi: 10.1099/0022-1317-78-10-2683

Salzman, R. A., Brady, J. A., Finlayson, S. A., Buchanan, C. D., Summer, E. J., Sun, F., et al. (2005). Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. *Plant Physiol.* 138, 352–368. doi: 10.1104/pp.104.058206

Sauge, M. H., Lacroze, J. P., Poëssel, J. L., Pascal, T., and Kervella, J. (2002). Induced resistance by *Myzus persicae* in the peach cultivar 'Rubira'. *Entomol. Exp. Appl.* 102, 29–37. doi: 10.1046/j.1570-7458.2002.00922.x

Schilmiller, A. L., and Howe, G. A. (2005). Systemic signaling in the wound response. *Curr. Opin. Plant Biol.* 8, 369–377. doi: 10.1016/j.pbi.2005.05.008

Stout, M. J., Thaler, J. S., and Thomma, B. P. (2006). Plant-mediated interactions between pathogenic microorganisms and herbivorous arthropods. *Annu. Rev. Entomol.* 51, 663–689. doi: 10.1146/annurev.ento.51.110104.151117

Taggar, G. K., and Gill, R. S. (2016). Host plant resistance in Vigna sp towards whitefly, *Bemisia tabaci* (Gennadius): a review. *Entomol. Gen.* 36, 1–24. doi: 10.1127/entomologia/2016/0184

Tan, X. L., and Liu, T. X. (2014). Aphid-induced plant volatiles affect the attractiveness of tomato plants to *Bemisia tabaci* (Hemiptera: Aleyrodidae) and associated natural enemies. *Entomol. Exp. Appl.* 151, 259–269. doi: 10.1111/eea.12190

Tan, X. L., Wang, S., Ridsdill-Smith, J., and Liu, T. X. (2014). Direct and indirect impacts of infestation of *Myzus persicae* (Hemiptera: Aphididae) on *Bemisia tabaci* (Hemiptera: Aleyrodidae). *PLOS ONE* 9:e94310. doi: 10.1371/journal.pone.0094310

Thaler, J. S., Agrawal, A. A., and Halitschke, R. (2010). Salicylate-mediated interactions between pathogens and herbivores. *Ecology* 91, 1075–1082. doi: 10.1890/08-2347.1

Thaler, J. S., Fidantsef, A. L., and Bostock, R. M. (2002). Antagonism between jasmonate-and salicylate-mediated induced plant resistance: effects of concentration and timing of elicitation on defense-related proteins, herbivore, and pathogen performance in tomato. *J. Chem. Ecol.* 28, 1131–1159. doi: 10.1023/A:1016225515936

Thompson, G. A., and Goggin, F. L. (2006). Transcriptomics and functional genomics of plant defence induction by phloem-feeding insects. *J. Exp. Bot.* 57, 755–766. doi: 10.1093/jxb/erj135

Verhage, A., van Wees, S. C. M., and Pieterse, C. M. J. (2010). Plant immunity: it's the hormones talking, but what do they say? *Plant Physiol.* 154, 536–540. doi: 10.1104/pp.110.161570

Zarate, S. I., Kempema, L. A., and Walling, L. L. (2007). Silverleaf whitefly induces salicylic acid defenses and suppresses effectual jasmonic acid defenses. *Plant Physiol.* 143, 866–875. doi: 10.1104/pp.106.090035

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Tan, Chen, Benelli, Desneux, Yang, Liu and Ge. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.