Comparative proteomic analysis provides novel insight into the interaction between resistant vs susceptible tomato cultivars and TYLCV infection

Background Tomato yellow leaf curl virus (TYLCV) is a member of the family Geminiviridae, genus Begomovirus. The virus is a widespread plant virus that causes important economic losses in tomatoes. Genetic engineering strategies have increasingly been adopted to improve the resistance of tomatoes to TYLCV. Results In this study, a proteomic approach was used to investigate the molecular mechanisms involved in tomato leaf defense against TYLCV infection. Proteins extracted from leaves of resistant tomato cultivar ‘Zheza-301’ and susceptible cultivar ‘Jinpeng-1’ after TYLCV infection were analyzed using two-dimensional gel electrophoresis. Eighty-six differentially expressed proteins were identified and classified into seven groups based on their functions. For several of the proteins, including CDC48, CHI and HSC70, expression patterns measured using quantitative real-time PCR differed from the results of the proteomic analysis. A putative interaction network between tomato leaves and TYLCV infection provides us with important information about the cellular activities that are involved in the response to TYLCV infection. Conclusions We conducted a comparative proteomic study of TYLCV infection in resistant and susceptible tomato cultivars. The proteins identified in our work show a variety of functions and expression patterns in the process of tomato–TYLCV interaction, and these results contribute to our understanding of the mechanism underlying TYLCV resistance in tomatoes at the protein level. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0819-z) contains supplementary material, which is available to authorized users.


Background
Tomato yellow leaf curl virus (TYLCV), is a member of the genus Begomovirus of the family Geminiviridae, which has three other genera, namely, Mastrevirus, Curtovirus and Topocuvirus, and contains a circular single-stranded DNA (ssDNA) molecule 2.7-2.8 kb in length [1,2]. The TYLCV genome encodes six open reading frames (ORFs): two overlapping ORFs AV1 (encoding capsid protein), AV2 (encoding movement protein MP), and ORFs AC1-AC4 (encoding replication-associated protein Rep, transcriptional activator TrA, replication enhancer protein REn and induction of plant cell division protein, respectively) on the complementary-sense strand [3][4][5][6][7][8]. TYLCV, which is transmitted by the whitefly Bemisia tabaci, threatens up to 20 different plant species, including pumpkin, tobacco and tomato [9,10]. TYLCV infection begins when the viruliferous whitefly B. tabaci feeds by inserting its proboscis into leaves, transmitting the virus to the host cells of the plant [11]. Upon entering the host cell, the ssDNA genome of TYLCV begins replicating through a rolling circle mechanism. TYLCV can spread to adjacent cells via the complex cytoplasm and plasmodesmata, inducing a new infection process.
After about 2 weeks of TYLCV infection, symptoms become increasingly apparent, with yellow and shriveled leaves, dwarfed plants, withered flowers and growth retardation, causing serious economic loss. Increasing numbers of genes involved in the response to TYLCV infection have been identified, such as GroEL [12], a developmentally regulated lipocalin-like gene SlVRSLip [13], and transcription factors, including bHLH [14], AP2/ERF [15], NAC [16]. Comparative transcriptomics and metabolomics have also been conducted in resistant and susceptible tomato cultivars to analyze the mechanisms of resistance to TYLCV infection [16,17].
In recent years, several strategies have demonstrated the advantages of the proteomics approach for studying underlying plant physiological processes, such as plant defense response. A proteomic analysis of root proteins in avocado infected with Phytophthora cinnamomi identified 21 differentially expressed proteins, including homologs to glutathione S-transferase, isoflavone reductase and several abscisic acid stressripening proteins [18]. Moreover, a study of interaction with Lasiodiplodia theobromae infection in cashew plants identified 73 proteins with significantly different expression levels, which were mainly involved in energy metabolism pathways, stress and defense, protein metabolism and cell signaling [19].
Whitefly-transmitted geminiviruses such as the begomovirus TYLCV pose a serious threat to tomato production. Breeding of tomatoes resistant to TYLCV started in the late 1960s [20]. Tomato genotypes with resistance to begomoviruses are derived from different wild species. To date, five loci linked to resistance have been identified: Ty-1/Ty-3 and Ty-4 from S. chilense, Ty-2 from S. habrochaites and Ty-5 from S. peruvianum [17]. And two genes associated with these Ty loci, Ty-1/Ty-3 and Ty-5, have been identified [21,22]. To investigate the tomato-TYLCV interaction, we conducted a proteomic analysis in resistant and susceptible tomato cultivars after TYLCV infection. Leaf proteins of resistant tomato cultivar 'Zheza-301' and susceptible cultivar 'Jinpeng-1' were extracted at 19 days post infection (dpi) and analyzed using two-dimensional gel electrophoresis . Eighty-six proteins were identified and classified into different functional categories. The expression levels of 19 genes encoding these proteins were analyzed at six time points after infection (2, 4, 6, 10, 15 and 19 dpi) by quantitative real-time PCR (qRT-PCR). A putative TYLCV infection response network in tomato leaves was constructed.
This work mainly (1) identified proteins significantly expressed in response to TYLCV infection in tomatoes; (2) analyzed the proteins that were differentially expressed between resistant and susceptible tomato cultivars; (3) revealed the defense mechanism between tomato and TYLCV in proteins and biochemical process level; and (4) analyzed the interaction network in the cells of tomato leaves after TYLCV infection. The overall results contribute significantly to our understanding of protein response and alteration and provide insights into the molecular mechanisms involved in response to TYLCV infection in tomatoes.

Symptoms of resistant and susceptible tomato cultivars in response to TYLCV infection
Two leaf-stage tomato cultivars (resistant variety: 'Zheza-301' , susceptible variety: 'Jinpeng-1') were exposed to viruliferous whiteflies. The two tomato cultivars showed no symptoms at 10 dpi. At 17 dpi, there was a little yellowing in the leaves of 'Jinpeng-1' (Additional file 1: Figure S1). Compared with the control plants, 'Jinpeng-1' samples presented a typical TYLCV phenotype, with curly yellow leaves at 19 dpi. In contrast, 'Zheza-301' exhibited no symptoms (Fig. 1). For better comparative understanding of the development of TYLCV infection, the leaves of the two tomato cultivars at 19 dpi and control plants were collected and frozen in liquid nitrogen to extract protein and total genomic DNA. To detect TYLCV accumulation, semi-quantitative PCR was conducted using the primers TYLCV-01 (Additional file 2: Table S1). As shown in Fig. 2, in control plants, there was no TYLCV accumulation in either 'Zheza-301' or 'Jinpeng-1' at different PCR cycles. In TYLCV infected plants, after 23 PCR cycles, there was high expression of TYLCV DNA in 'Jinpeng-1' , while there was weak or no expression in 'Zheza-301'. TYLCV DNA was clearly present in 'Zheza-301' after about 25 cycles, but at lower levels than in 'Jinpeng-1'.

2-DE analysis of tomato leaf proteins after TYLCV infection
As described above, samples for protein extraction were collected at 19 dpi, when the leaves of susceptible cultivar 'Jinpeng-1'were yellow and curly. Total proteins of the two tomato cultivars ('Zheza-301': control and TYLCV infection; 'Jinpeng-1': control and TYLCV infection) were separated using 2-DE. Representative maps of three biological replicates are shown in Additional file 3: Figure S2.
Over 500 protein spots were detected in each gel with different expression abundance. To detect differentially expressed proteins, the protein abundance ratios Z T /Z C (treatment /control plants of 'Zheza-301') and J T /J C (treatment/control plants of 'Jinpeng-1') were calculated for each spot. Z C /J C and Z T /J T were also calculated to analyze differences between the two tomato cultivars. A total of 86 spots with apparent molecular mass between 26.08 and 114.02 kDa and isoelectric point (pI) between 4.69 and 8.96 showed significantly differential expression with more than 2.0-fold or less than 0.5-fold differences in abundance ratios (Fig. 3). Identified proteins are shown in Table 1. Among the 86 protein spots, four spots (spot 34, spot 35, spot 36 and spot 53) showed no significant difference in expression with infection but significant differences in expression between the two tomato cultivars. These proteins did not respond significantly to TYLCV infection at 19 dpi, but were related to differences between the two tomato cultivars. The significantly differentially expressed proteins in TYLCV infection in the two tomato cultivars are shown in Fig. 4.

Differentially expressed proteins identified in resistant and susceptible tomato cultivars after TYLCV infection
Expression patterns in terms of the protein abundance ratios of 86 identified proteins were different between the two tomato cultivars. A heat map of differentially expressed protein spots provides an overview of protein expression modification in the two TYLCV-infected tomato genotypes (Fig. 6). In general, most proteins in 'Zheza-301' showed down-regulated expression profiles. In 'Jinpeng-1' , the proportions of up-and downregulated proteins were almost the same. In 'Zheza-301' , 71 proteins were modulated between the TYLCVinfected and control leaf samples, with the expression levels of 28 proteins increased and 43 proteins decreased. In 'Jinpeng-1' , 59 proteins appeared to be altered, and of these 28 and 31 were up-regulated and down-regulated, respectively (Fig. 4). Among the 86 protein spots, expression levels of 17 proteins were upregulated in both 'Zheza-301' and 'Jinpeng-1' , including OEE1 (spot 5), HSP83 (spot 69) and PPO (spot 85). A total of 24 protein spots were identified with down-regulated expression levels in both cultivars, including 14- showed up-regulation in 'Zheza-301' , but down-regulation in 'Jinpeng-1' ( Table 2). The differences in expression patterns of proteins indicate that several proteins play Two genes, namely, Ty-1 and Ty-5, associated with the Ty locus, have been identified in tomatoes. The expression patterns of Ty-1 and Ty-5 were analyzed using qRT-PCR in 'Zheza-301' and 'Jinpeng-1' tomato cultivars (Fig. 7). In 'Zheza-301' , the expression pattern of Ty-5 was up-regulated at six time points after infection (2, 4, 6, 10, 15 and 19 dpi), and a peak was reached at 15 dpi with a 5-fold increase. The expression level of Ty-1 was not noticeably increased until 19 dpi, when it increased by approximately 3-fold. The expression patterns of Ty-1 and Ty-5 were both up-regulated in 'Jinpeng-1'. The expression level peaks of Ty-1 and Ty-5 in 'Jinpeng-1' were observed at 2 dpi with a 30-fold increase and at 10 dpi with a 70-fold increase, respectively.   The expression levels of CHI, AH, HSC70 and MAT in early stages (2, 4 and 6 dpi) were different from those at 19 dpi. At 2, 4 and 6 dpi, CHI, AH and HSC70 were negatively regulated in 'Zheza-301'. Expression of CHI, AH and HSC70 was up-regulated at 19 dpi (Figs. 8 and  9). The expression patterns of the genes that function in detoxification and antioxidation varied among the six infection periods in the two tomato cultivars. Glo I can convert 2-oxoaldehydes into less reactive 2-hydroxyacids by cooperation with Glo II. In 'Zheza-301' , the transcription levels of Glo I and APX were decreased at 2 and 4 dpi (Fig. 8). At 19 dpi, the transcription levels of Glo I and APX in 'Zheza-301' were up-regulated (Fig. 9). The expression patterns of PPO and PRO were negatively and positively regulated, respectively, in both tomato cultivars at all time points after infection. Previous studies have revealed that CDC48 can impair tobacco mosaic virus (TMV) movement by removing virus-encoded MP from the endoplasmic reticulum transport pathway and by promoting the interference of MP with microtubule dynamics. The expression levels of CDC48 in 'Jinpeng-1' were higher than those in 'Zheza-301' at all time points. At 2, 4 and 6 dpi, the expression patterns of LFNR, AH, CHI, CDC48, CYS, HSC70 and PPO were downregulated in 'Zheza-301'. By contrast, the expression levels of OEE, CAB, ID, AH, CAR, APX, Glo I, CHI, CDC48, GLDC, THD, HSC70 and PRO were upregulated in 'Zheza-301' at 19 dpi. Therefore, these genes may respond to TYLCV infection via a positive regulatory mechanism (Figs. 8 and 9).

Comparative analysis of expression patterns detected using qRT-PCR and 2-DE
The expression patterns at 19 dpi observed using qRT-PCR and 2-DE were compared. In 'Zheza-301' , five proteins, namely, OEE, CAR, GLDC, THD and PRO were positively regulated in both qRT-PCR and 2-DE results. In 'Jinpeng-1' , the protein abundance of OEE, AH, CHI, CDC48, THD, HSC70 and Glo I increased. The expression level of LFNR was down-regulated in both analyses. The transcription levels of the genes detected using qRT-PCR were not well correlated with the protein abundance of proteins detected using 2-DE. CAB, which functions in photosynthesis, showed opposite expression patterns in the two analyses. The protein abundance of   Glo I in 'Zheza-301' was decreased but transcription level measured by qRT-PCR was increased approximately 3-fold at 19 dpi. In 2-DE, the protein abundance of PPO in the two tomato cultivars increased by approximately 10-fold; in contrast, its transcription levels decreased, as measured using qRT-PCR (Fig. 9, Table 1).

Discussion
Plants use compensatory strategies to protect themselves from various aggressions by pathogens such as viruses and insects. Infestation with viruliferous whiteflies can cause extensive damage in tomatoes. The proteins identified in control and TYLCV-infected tomato cultivars were classified into different groups, such as defenserelated proteins, chaperones and signal transduction proteins. These groups played different roles in response to TYLCV infection. Several of the proteins identified produced multiple spots with different pI and molecular masses, which indicates the presence of isoforms and posttranslational modification [28]. The ability to resist TYLCV infection varies between different tomato cultivars. Previous studies have shown that 'Zheza-301' and 'Jinpeng-1' exhibit different resistance to TYLCV infection [15,29]. After TYLCV infection for two months, 'Zheza-301' had resistance ability with 45.0 % disease incidence and with 9.0 disease indexes. 'Jinpeng-1' showed susceptible to TYLCV infection with 90.0 % disease incidence and 60.9 disease indexes [29]. The two tomato cultivars 'Zheza-301'and 'Jinpeng-1' were thus chosen to further analyze the mechanism of the response to TYLCV infection.

Responses to TYLCV infection in resistant and susceptible tomato cultivars at different stages
Responses to TYLCV infection in tomatoes involve a complicated defense mechanism that is related to transcription factors and gene regulatory networks involving mitogen-activated protein kinases [15,16]. Such responses are correlated with anatomical structure [30], tomato genotypes [31][32][33] and different infection stages [17,33]. Glick and his colleagues reported that symptoms and responses differ between the early (before 2 weeks) and later (after 4 weeks) stages of TYLCV infection in tomatoes [34]. Tomato plants appear normal after TYLCV infection for 2 weeks, then the leaves of susceptible tomato cultivars become yellow and curly over time, and newly formed leaves are smaller and shriveled. In later stages, whole plants stop growing and this leads to severe yield loss [15,34]. The response of tomatoes to TYLCV infection should be analyzed at different stages of infection. To determine the genes encoding resistance to TYLCV infection, some research has been conducted at early and later stages of TYLCV infection [16,17,33,35,36]. A comparative transcriptomic analysis at 3, 5 and 7 dpi has been carried out for resistant and susceptible tomato cultivars [16]. Comparative metabolomics and transcriptomics in response to TYLCV infection at 1, 3, 7 and 14 dpi have also been carried out in two tomato lines [17,36]. Adi et al. analyzed stress responses to TYLCV infection in tomato after 4 weeks, which was sufficient to allow the development of differential coat protein (CP) aggregation [33]. Miozzi et al. investigated the transcriptional changes induced by TYLCSV infection after 6 weeks post-infection with typical systemic symptoms [35]. However, responses of tomatoes to TYLCV infection in the middle stage (~3 weeks) have not been described before now. The responses of 'Zheza-301' (resistant tomato cultivar) and 'Jinpeng-1' (susceptible tomato cultivar) to TYLCV infection are complex and long term. A proteomics approach was used to investigate resistant and susceptible tomato cultivars and to understand the response to TYLCV infection in tomato cultivars in the middle stage (~3 weeks). At 19 dpi, a typical TYLCV phenotype was found in 'Jinpeng-1' , but no symptoms were observed in 'Zheza-301'. When stress conditions are encountered, plants can trigger a network of events linked to energy metabolism [37]. Most proteins in this network are involved in photosynthesis, carbohydrate metabolism and energy metabolism. This finding indicates the importance of energy metabolism in response to TYLCV infection. Changes in carbohydrate metabolism are related to energy allocation and long-term defense processes [38]. Upon TYLCV infection, changes in energy metabolism and wound response in tomato plants happen over the whole infection period [2,[7][8][9][10]. The expression patterns of 19 selected genes that encoded proteins with different functions were analyzed through qRT-PCR after 2, 4, 6, 10, 15 and 19 dpi. Changes in metabolic pathways and different expression levels were revealed through qRT-PCR and 2-DE. The symptoms in the two tomato cultivars were the combined response results caused by TYLCV infection at 19 dpi.

Defense-related proteins
Recent studies in certain processes of virus-host interactions have reported that some oxidative stress is produced, and base damage is caused by reactive oxygen species (ROS) such as H 2 O 2 [39][40][41][42]. Superoxide dismutase and the ascorbate-glutathione cycle are involved in the breakdown of H 2 O 2 into H 2 O and O 2 [43,44]. Three isoforms of APX (spots 80-82) were identified as having different expression patterns. Spot 81 showed upregulated expression in terms of protein abundance ratio, which was higher in 'Zheza-301' ( Table 1). The expression level of APX as measured using qRT-PCR was up-regulated at 19 dpi, which was consistent with the result of 2-DE. PPOs are ubiquitous in plants and play important roles in plant defense against pests and pathogens [45][46][47]. PPOs can catalyze the oxygendependent oxidation of phenols to quinones, which may have direct antibiotic and cytotoxic activities to pathogens [45]. In our study, one PPO protein was highly expressed in both cultivars as measured using 2-DE, especially in 'Zheza-301' , in which it increased 15-fold in protein abundance ratio. By contrast, the expression pattern of PPO gene measured using qRT-PCR exhibited a down-regulated expression profile both in 'Zheza-310' and 'Jinpeng-1'.
In addition to PPO, APX, a lactoylglutathione lyase protein (spot 83), known as glyoxalase I (Glo I) was induced after TYLCV infection in both tomato cultivars. This enzyme catalyzes the formation of S-D-lactoylglutathione from methylglyoxal and glutathione to reduce damage [48]. Methylglyoxal is a cytotoxic and mutagenic αketoaldehyde that is significantly increased (2-6 fold) under abiotic stress in plants [49]. This compound can form adducts with proteins and nucleic acids, and damages cellular functions [49][50][51][52]. Glo I, encoding the Glo I protein, showed up-regulated transcription levels of about 3.0-fold and 2.5-fold in 'Zheza-301' and 'Jinpeng-1' at 19 dpi, respectively, while the protein abundance ratio was down-regulated in 'Zheza-301' (Fig. 9, Table 1). CHI is a kind of pathogenesis-related protein that can be dramatically induced by fungal, bacterial or viral attacks, and which can hydrolyze chitin in the fungal cell wall to protect the plant from biotic and abiotic stresses [52][53][54]. After TYLCV infection, the activity of CHI protein is increased more than in uninfected tomatoes sprayed with eugenol. In the study, CHI protein was induced in both 'Zheza-301' and 'Jinpeng-1'. The protein abundance ratio in 'Jinpeng-1' was more than 4.2-fold that in 'Zheza-301'. Quantitative PCR showed significantly increased expression of the CHI gene in 'Zheza-301' and 'Jinpeng-1' from 10 dpi and 12fold and 35-fold increases, respectively, at 19 dpi (Figs. 8 and 9). The similar expression patterns of CHI protein in the two cultivars indicates that CHI protein plays an important role in the resistance of plants to TYLCV.

Chaperones
Plant cells can be injured by protein misfolding or unfolding when they are confronted with adverse conditions [55,56]. Many studies have shown that heat shock proteins (HSPs) can refold proteins to maintain cellular homeostasis and reestablish normal protein conformation under adverse conditions [24]. HSC70s are a major family of chaperones that play essential roles in a range of protein-folding processes, including protein import and translocation, and that facilitate degradation of unstable proteins [57,58]. Another study indicated that HSP83 proteins are involved in a general regulation mechanism and control a variety of cellular functions [59,60]. In avocados, a small 17.3 kDa heat-shock protein is induced after infection with the oomycete Phytophthora cinnamomi [18]. HSP70 is overrepresented in inoculated cashew plants after Lasiodiplodia theobromae infection [19]. In this study, HSC70 and two isoforms of HSP83 were identified in both 'Zheza-301' and 'Jinpeng-1' with up-regulated protein abundance ratios after TYLCV infection. HSP83 and HSC70 showed higher protein abundance ratios, which increased 9.5-fold and 9.2-fold, respectively, in'Jinpeng-1'. As shown in Fig. 9, the transcription levels of HSC70 were up-regulated in both tomato cultivars with 12-fold (in 'Zheza-301') and 8-fold (in 'Jinpeng-1') higher expression. All these results indicate that HSPs play a crucial role in recognizing and correcting misfolded proteins under TYLCV infection in tomato.

Signal transduction proteins
14-3-3 proteins, as important regulators of cellular signal transduction and primary metabolism in plants, are ubiquitous in eukaryotes; these proteins regulate plant development and protect plants from adverse conditions [61,62]. Previous studies have shown that 14-3-3 protein can respond to various abiotic stresses, such as environmental stress [63], metabolism/nutrient stress [64], herbivory and wound stress [65], and biotic stress, such as Magnaporthe grisea and Xanthomonas oryzae in rice [61]. Moreover, 14-3-3 protein may control the activities of kinases and phosphatases by taking part in various signal transduction processes [66]. After TYLCV infection, the protein abundance ratios of 14-3-3 protein in 'Zheza-301' and 'Jinpeng-1' showed a significant decrease. This decrease indicates that 14-3-3 is down-regulated in response to TYLCV infection.

Other important proteins involved in TYLCV infection
Serine carboxypeptidases play important roles in plant growth and development, being involved in protein hydrolyzation, signal transduction and response to trauma and adverse conditions [67][68][69][70][71]. Two isoforms of CAR (spots 41, 42) were identified as having positive protein abundance ratios in the two tomato cultivars (Table 1). CDC48 was up-regulated in both 'Jinpeng-1' and 'Zheza-301' as measured by both qRT-PCR and 2-DE (Fig. 9, Table 1). As shown in Fig. 9, the expression patterns of three genes (OEE1, CAB and LFNR), that encode photosynthesis-related proteins, and four energy genes (GAPDH, EA, AH and ID), related to carbon metabolism, were identified by qRT-PCR at 19 dpi. The transcription levels of OEE1, CAB, ID and AH were higher both in 'Zheza-301' and 'Jinpeng-1'. LFNR and EA, encoding ferredoxin-NADP reductase and enolase, were down-regulated. GLDC, CYS, THD and MAT, which are involved in amino acid metabolism, showed different transcription profiles in the two tomato cultivars. THD  (Fig. 9, Table 1). A complex process from the gene to the corresponding protein involves complicated translation and modification, which may cause the difference in expression levels observed between the gene and protein.

TYLCV CP aggregation in different tomato cultivars
The CP of TYLCV forms a capsid and exhibits various functions [72]. Gorovits et al. detected TYLCV CP with weak expression levels in susceptible '906-4' tomato plants at 14 dpi but not in resistant '902' tomato plants until 21 dpi [31]. In our study, TYLCV CP was not detected by 2-DE in the resistant cultivar 'Zheza-301' or the susceptible cultivar 'Jinpeng-1' at 19 dpi. A total of 500 protein spots with different protein abundances were identified using PDQuest software with an automation mode (data not shown). Only 86 protein spots with obviously different expression levels were manually selected, excised and analyzed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS). The CP protein spot may not have been excised; consequently, the CP protein was not detected. The CP protein may be among the 414 spots that were not analyzed.
Viral replication, transcription, translation and movement, as well as virus inoculum dose and inoculation methods, influence the aggregation level of TYLCV CP in tomato plants [31]. Different tomato genotypes and resistance to TYLCV infection also affect TYLCV CP expression. After TYLCV infection, host compounds such as chaperones, proteases and stress-induced proteins also contribute to the maintenance of small aggregates in tomato plants [32]. Genetic backgrounds and resistance to TYLCV infection differ between 'Jinpeng-1' and '906-4'. Thus, these parameters influenced the accumulation of TYLCV CP [20,29]. To analyze TYLCV CP Fig. 10 Possible TYLCV infection response network in tomato leaves. Red color represents significantly differentially expressed proteins identified in the study accumulation in tomato, we will extend the time of infection with TYLCV or use isobaric tags for relative and absolute quantification (iTRAQ) in further studies [73].

A putative TYLCV infection response network in tomato leaves
Based on proteomic analysis, 86 proteins were identified in tomato leaves at 19 dpi. A TYLCV infection response network was proposed that included the majority of the 86 TYLCV-responsive proteins, which are involved in several functional processes, such as systemic acquired resistance, redox homeostasis, photosynthesis rate, energy metabolism, proteolysis and amino acid hydrolysis.
As shown in Fig. 10, tomato leaves perceive a stress signal after TYLCV infection, through some receptors located on the membrane and transmit it to cellular mechanisms by signal transduction processes that alter many cellular and metabolic processes. The level of certain hormones such as salicylic acid (SA), jasmonicacid (JA), abscisic acid (ABA) and some pathogenesis-related (PR) proteins including CHI and β-1.3-glucanase were induced in response to TYLCV infection and confer systemic acquired resistance [32,74]. The increase in ROS content after TYLCV infection could cause an imbalance of redox in tomato leaves. To maintain redox homeostasis, the synthesis of antioxidation enzymes such as SOD, APX, Glo I and PPO, increases to decrease the levels of ROS and allow the cell to achieve redox balance. The photosynthetic rate of tomato leaves is impaired by the downregulation of RuBisCO, LFNR and CAR protein (Table 1). Energy metabolism of tomatoes under TYLCV infection is increased by up-regulation of ID and AH, which improves the ability of tomato leaves to respond to TYLCV infection and decrease the damage (Fig. 9). After TYLCV infection, protein biosynthesis of tomato leaves is inhibited through the down-regulation of EF-4A-2, whereas proteolysis increases because of up-regulation of PRO and CAR proteins. Amino acid metabolism of tomato leaves is enhanced by the over-expression of THD and GLDC proteins.
Through the changes of cellular signal transduction and metabolic processes, tomato leaves strive to resist TYLCV infection and adapt to or decrease the damage caused by TYLCV. In the current study, a putative interaction network between tomato leaves and TYLCV infection provides us with important information about the strategy of cellular activities responding to TYLCV infection in tomato leaves.
Comparative analysis with other 'omics' used to study plant-virus interactions 'Omics' technologies, including transcriptomics, proteomics and metabolomics can be applied to monitor and capture complete changes that occur under adverse conditions at transcript, protein and metabolic levels [75,76]. In plants, geminivirus infections, including TYLCV, have rarely been subjected to transcriptomic analysis. A total of 3604 genes and 34,831 transcripts were identified through transcriptomics, as conducted by Miozzi et al. and Chen et al., respectively [16,35]. Only 86 significantly differentially expressed proteins were identified in this study using proteomics. Changes in gene expression at the transcript level are not well correlated with alterations in protein levels because of transcript instability and post-transcriptional modification. Genes encoding different proteins involved in TYLCV infection were revealed through transcriptomic and proteomic analyses; these genes included HSP, CHI and CYS. Gene ontology analysis also demonstrated that genes identified through transcriptomic and proteomic analyses were mainly involved in response to biotic stress, amino acid metabolism and protein degradation. Regulatory factors, such as MYB, NAC and WRKY, could be identified through transcriptomics but not through proteomic analysis. These proteins are often difficult to investigate because of their low abundance [16,35].
In the innate immune response of plants, small molecules can be analyzed in detail and compounds can be identified through metabolomic approaches [77]. The combination of global gene expression and metabolic profiling is a powerful approach that can provide comprehensive insights into cellular dynamics associated with pathogen infections [17,33]. After TYLCV infection occurs, numerous metabolites are involved in several metabolic pathways, such as phenylpropanoids and lignins; these metabolites exhibit different regulatory mechanisms between resistant and susceptible tomato cultivars [17]. Glucosamine is a metabolic compound involved in ROS metabolism; the amounts of glucosamine decreased in the early stages of infection but sharply increased at 25-28 dpi [33]. The protein abundance ratio of APX (spot 81) involved in antioxidation was upregulated in the two tomato cultivars ( Table 1). The findings indicate that changes in primary and secondary metabolites occur in the defense against TYLCV infection in tomato.
Proteomics has been used to investigate the potential functional mechanisms involved in resistance to pathogen infections in tomatoes [78,79]. Proteins, including APX and OEE, have been identified in proteomics data from TYLCV-infected, TMV-infected, and cucumber mosaic virus (CMV)-infected tomato plants [78,80]. CHI and LAP have been identified in TYLCV-infected and TMV-infected tomato plants. After TYLCV and CMV infection occurs, photosynthesis, energy metabolism and carbon metabolism are altered in tomato plants. Proteins involved in defense responses are also induced. HSP, which functions as a chaperone, is induced at 4 weeks post-TYLCV infection and at 19 dpi [33].

Conclusions
A comprehensive proteomics analysis in the leaves of resistant and susceptible tomato cultivars was conducted. Eighty-six protein spots were identified with more than 2-fold or less than 0.5-fold in protein abundance ratio after TYLCV infection in two cultivars. Classified into seven functional groups, proteins identified after TYLCV infection played different roles in the process of tomato-TYLCV interaction. The interaction network between tomato leaves and TYLCV infection provided us the information about the possible activities in tomato leaf cells. The results will help to find the key proteins involved in tomato-TYLCV interaction to enhance the resistance to TYLCV and obtain protection from virus infection.

Plant materials and TYLCV infection
In this study, resistant tomato cultivar 'Zheza-301' , derived from T5678161-1-1-2-2 and T07-018, and susceptible tomato cultivar 'Jinpeng-1' , a hybrid of Holland tomato cultivar 99-13A and 9708B from America, were chosen as the source for the comparative proteomic analysis. 'Zheza-301' was identified as carrying the Ty-2 locus, which because T07-018 was selected from the 4th generation of the 'CLN2498E' , breeding to contain the Ty-2 resistant gene [81]. Seeds of tomato cultivars 'Zheza-301' and 'Jinpeng-1' were obtained from the Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, and Xi'an Jinpeng Seed Co., Ltd., respectively. Tomato plantlets of 'Zheza-301' and 'Jinpeng-1' were grown in a chamber under 25°C/18°C , 12 h/day and a relative humidity of 60-70 % [29]. Viruliferous whiteflies, provided by Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences (Nanjing, China), were allowed to feed on tomato plants in an insect-proof greenhouse. Tomato plantlets at the two-leaf stage were transmitted to the insect-proof greenhouse to expose them to viruliferous whiteflies. Control plants were transmitted to an insect-proof greenhouse without viruliferous whiteflies. The leaves of TYLCVinfected tomato plants were harvested at 2, 4, 6, 10, 15 and 19 dpi. To guarantee a better comparative understanding of the development of infection between resistant and susceptible tomato cultivars, leaves of the two tomato cultivars were processed at 19 dpi; at this point, systemic symptoms, including curly yellow leaves, were apparent in 'Jinpeng-1' but not in 'Zheza-301'. The leaves of control and infected tomato cultivars at 19 dpi were collected and frozen for protein extraction.

Protein extraction and 2-DE
Three biological repeats of each sample were collected to improve accuracy. According to the Bio-Rad (Hercules, CA, USA) 2-D manual, acetone/trichloroacetic acid (TCA) precipitation method was used to extract protein with slight modifications. Tomato leaf sample powder was briefly suspended in 10 % w/v TCA/acetone containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and 0.07 % w/v β-mercaptoethanol, and held at −20°C for 1 h. Approximately 800 μL of lysis solution (containing 7 M urea, 2 M thiourea, 4 % (w/v) 3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate, 65 mM dithiothreitol, 1 mM PMSF and 0.5 % v/v biolytes) was used to dissolve the vacuum-dried pellets after centrifugation and rinse. After centrifugation, the insoluble materials were removed. The Bradford method was performed to quantify the protein concentration for each sample [82]. About 1500 μg of protein of each sample was loaded on a 24 cm nonlinear gradient immobilized pH gradient strip (pH 4-7) and subjected to IEF at 20°C: 50 v for 13 h, 100 v for 1 h, 200 v for 1 h, 1000 v for 1 h, 8000 v for 3 h, and 8000 v for a total of 110,000 VH. Afterward, 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for the second electrophretic dimension. Proteins were observed by staining with Coomassie Brilliant Blue G-250. A gel scanner (Powerlook 2100XL, UMAX) and PDQuest software package (ver 7.2.0; Bio-Rad), with automation mode, were used to digitalize and analyze the gel images, respectively [83]. Based on the total density of gels with the parameter of percentage volume, spots were detected, matched and normalized. Subsequently, the mean relative volume of each spot by three biological repeats was computed and spots with more than 2.0-fold and less than 0.5-fold of protein abundance ration was considered differentially expressed protein. The experimental design was showed in Additional file 4: Figure S3.

In-gel digestion and MALDI-TOF-TOF MS analysis
Differentially expressed protein spots were manually excised, washed three times with Millipore pure water and destained twice with 50 mM NH 4 HCO 3 in 50 % acetonitrile (ACN). Afterward, 10 mM dithiothreitol in 50 mM NH 4 HCO 3 was used to reduce alkylation with 40 mM iodoacetamide in 50 mM NH 4 HCO 3 . ACN (100 %) was employed to dry twice and was digested overnight at 37°C with sequencing grade modified trypsin (Promega, Madison, WI, USA) in 50 mM NH 4 HCO 3 . The peptides were extracted twice with 0.1 % trifluoroacetic acid (TFA) in 50 % ACN. After being pooled and lyophilized, the resulting lyophilized tryptic peptides were dissolved in 5 mg/mL CHCA containing 0.1 % TFA and 50 % acetonitrile. A 4800 plus MALDI-TOF-TOF TM analyzer (Applied Biosystems, Foster City, CA, USA) was used for analyses.
MASCOT program online was employed to search the database of all protein spectra (http://www.matrixscience. com), against NCBInr databases. The search parameters were as follows: 0.15 Da mass tolerance for peptides and 0.25 Da mass tolerance of TOF-TOF fragments, one allowed trypsin miscleavage, carbamidomethyl of cystine (Cys) as fixed modification, oxidation of methionine (Met), and pyro-glutamicacid (Glu) formation of N-terminal glutarnine (Gln) and Glu as variable modification. To select the significant hits, the protein abundance ratio of each spot (treatment/control) with more than 2.0-fold or less than 0.5-fold was formulated as the selection criteria. Only significant hits, which were identified by the MASCOT probability analysis, were accepted.

Bioinformatic analysis
The protein abundance ratio of each spot shown in Table 1 was analyzed and normalized using a log2 transform, and then a heat map with hierarchical clustering was produced using HemI 1.0 software [84].

Semi-quantitative PCR and qRT-PCR analysis
The total RNA of tomato leaves was extracted using an RNA kit (RNA simple total RNA kit, Tiangen, Beijing, China) and then transcribed into cDNA using a Primer Script RT reagent kit (TaKaRa, Dalian, China), respectively. qRT-PCR with SYBR Premix Ex Taq was conducted using an ABI7500 (Applied Biosystems, Foster, CA, USA) according to the following procedure: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s, and melting curve analysis (61 cycles) at 65°C for 10 s. Primer Premier 5.0 software was used to design the primer of each selected protein and A-Tubulin (Solyc04g077020.2) was used to regulate the expression level [85]. The expression patterns of two genes (Ty-1 and Ty-5) associated with the Ty locus were assessed after TYLCV infection in the two tomato cultivars, 18S ribosomal RNA was used as reference with the following primers 5′-GCGACGCATCATTCAAATTTC-3′ and 5′-TCCGGA ATCGAACCCTAATTC-3′ [22].
To detect whether there was accumulation of TYLCV DNA after TYLCV infection, semi-quantitative PCR was conducted using the primers TYLCV-01 F/R. A-Tubulin was used as internal control for semi-quantitative PCR and qRT-PCR. Total DNA was extracted from leaves of two tomato cultivars (control and treatment) using a DNA kit (DNAsecure Plant Kit, Tiangen, Beijing, China). The condition and parameters of PCR were 95°C for 5 m, followed by different cycles (23, 25, 27 and 30) at 95°C for 30 s, 54°C for 1 m, 72°C 1 m, then 72°C 10 m. Primers used in the study were shown in Additional file 2: Table S1.