Comparative proteomic analysis provides new insight into differential transmission of two begomoviruses by a whitefly

A culture of MED whitefly was reared on un-infected tomato plants (S. lycopersicum cv. Hezuo 903) in insect proof cages. For the experiments, un-infected tomato plants of both Hezuo 903 and Zheza 502 were cultivated to 7–8 true leaf stage when used. For preparation of TYLCV-infected and PaLCuCNV-infected plants, tomato of Hezuo 903 were first cultivated to 3–4 true leaf stage when virus inoculation was conducted, and then the virus-inoculated plants were further cultivated to 7–8 true leaf stage when used. The status of virus-infection of these plants with typical symptoms was verified by PCR detection, and the primers used here are listed in Additional file 1: Table S1.

To obtain whiteflies feeding on un-infected, TYLCV-infected and PaLCuCNV-infected plants, whitefly adults from the MED culture were collected 5–7 d post emergence, and then were placed on un-infected, TYLCV-infected and PaLCuCNV-infected tomato plants of Hezuo 903 respectively to feed for 2 d. The whitefly adults of each of the three treatments were then transferred to feed on un-infected tomato plants of a begomovirus-resistant cultivar (S. lycopersicum cv. Zheza 502) [35] for a further 2 d to reduce/eliminate effects of host plant differences on whiteflies during the 2d treatments (Fig. 1a). The virus-infection status of viruliferous whiteflies or no-viruliferous whiteflies was verified using PCR detection, and the primers used are listed in Additional file 1: Table S1. At this time, adults were collected for proteomics analysis (Fig. 1b). For each of the three treatments, two biological replicates in two separate cages were conducted. All whitefly cohorts of the three treatments were reared in cages at 25–27 °C, 60 ± 10% relative humidity and 14 h light/10 h darkness.

For iTRAQ analysis, whitefly adults of the three treatments as described above, i.e. whiteflies feeding on un-infected, TYLCV-infected and PaLCuCNV-infected plants, were arranged into three combinations for comparison: (i) un-infected vs. TYLCV-infected, (ii) un-infected vs. PaLCuCNV-infected, and (iii) TYLCV-infected vs. PaLCuCNV-infected. For each of the two treatments in a combination for comparison, 0 .1g sample was taken for protein extraction. The methods and procedures of quantitative proteomics analyses followed those of Zhong et al [33]. Briefly, (i) protein extraction, digestion and iTRAQ labeling, (ii) LC-MS/MS analysis, and (iii) proteomic data analysis (Fig. 1b). The raw MS/MS data was converted into MGF format using ProteoWizard tool msConvert (version 3.0.1), and then peptides were identified by searching the MED transcriptomes. We used a MS/MS data interpretation algorithm within Mascot (version 2.3.02). At least one unique peptide was necessary for an identified protein. Based on the data of protein extraction, the bands and repeatability were qualified, and the total content of protein in each of the treatments was greater than 400 μg.

The identified proteins were categorized according to their Gene Ontology (GO) annotation (http://​www.​geneontology.​org/​). The metabolic pathway analysis of the proteins was conducted according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database (http://​www.​genome.​jp/​kegg). The cluster analysis was conducted using the software Genesis (version 1.8.1). The cuticle protein family analysis was conducted using CutProtFam (aias.biol.uoa.gr/CutProtFam-Pred/home.php) [36].

To validate results from iTRAQ analysis, genes encoding DEPs among the three treatments were subjected to the RT-qPCR analysis. Twenty adult whiteflies were collected as a group for analyzing the gene expression of DEPs, and three replicates were set for each treatment. For gene expression analysis, total RNA of whitefly was isolated by TRIzol (Ambion, USA) and reverse transcribed using PrimeScript RT reagent Kit (TaKaRa, Japan) following the manufacturer’s protocol. Quantitative PCR (qPCR) was performed on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA) with SYBR Premix ExTaq II (Takara, Japan). β-actin was used as internal reference, relative abundance of begomovirus or transcripts was calculated by 2^-ΔCt. Primers used for real-time PCR are listed in Additional file 1: Table S1.

Among the 296,454 spectra generated, 46,699 spectra were identified with 42,153 being unique, 13,042 peptides were identified with 11,954 being unique as judged using 1% PSM (Peptide-spectrum matches) FDR (false discovery rate) (spectrum level), and 3555 proteins were identified with the 1% protein FDR protein levels. In the iTRAQ data, the values of CV exhibit centralized distributions within 0–10% (Additional file 2: Figure S1), indicating a fine reproducibility.

For TYLCV-infected vs. un-infected whiteflies, 259 DEPs were identified with 182 being up-regulated and 77 down-regulated (Fig. 2a and b; Additional file 1: Table S2). For PaLCuCNV-infected vs. un-infected, 395 DEPs were identified with 265 being up-regulated and 130 down-regulated (Fig. 2c and d; Additional file 1: Table S3). For TYLCV-infected vs. PaLCuCNV-infected, 74 DEPs were identified with 36 being up-regulated and 38 down-regulated (Fig. 2e and f; Additional file 1: Table S4). Among the 259 DEPs in TYLCV-infected vs. un-infected, 93 were subcategorized into GO classes. These DEPs were found related to receptor group, protein modification process and other processes. Among the 395 DEPs evaluated in PaLCuCNV-infected vs. un-infected, 155 DEPs were subcategorized into GO classes. Most of them were involved in cell cycle, immune response and catabolic process. Among the 74 DEPs evaluated in TYLCV-infected vs. PaLCuCNV-infected, 29 DEPs were subcategorized into GO classes. Groups related to metabolic process exhibited significant enrichment. GO classes with P < 0.05 in each comparison are shown in Fig. 3. In addition, DEPs in the three comparisons were assigned to the reference pathways in KEGG. As a result, 189, 242 and 99 DEPs in the comparison of TYLCV-infected vs. un-infected, PaLCuCNV vs. un-infected, and TYLCV-infected vs. PaLCuCNV-infected were assigned to the reference pathways in KEGG respectively. Finally, 45, 27, 15 pathways were significantly enriched for whiteflies of TYLCV-infected vs. un-infected, PaLCuCNV-infected vs. un-infected, and TYLCV-infected vs. PaLCuCNV-infected, respectively (P < 0.05) (Fig. 4).

A cluster analysis of DEPs was conducted for the three comparisons (Fig. 5), including DEPs related to viral transport, defense response, cell cycle and other processes. Whiteflies infected with different begomoviruses showed some similar responses, including the pathway of ECM-receptor interactions (Table 1), and the up-regulations of some cuticle proteins (Table 2) and proteins related to defense responses (Table 3). In addition, some responses, such as 20S proteasome subunits, were up-regulated only in the comparison of PaLCuCNV-infected vs. un-infected whiteflies (Table 4).

To validate the iTRAQ data, we tested the expression levels of some selected candidate genes in the three treatments. Figure 6 shows the expression patterns of 7 genes, including ras-like protein 3, dystroglycan, integrin alpha-PS2, laminin subunit alpha, cuticle protein 67, PITH domain-containing protein CG6153, and proteasome subunit beta type-6. Consistent with the iTRAQ data, ras-like protein 3, dystroglycan, integrin alpha-PS2, laminin subunit alpha and PITH domain-containing protein were significantly up-regulated after TYLCV/PaLCuCNV infection compared with un-infected whiteflies. Proteasome subunit beta type-6, a kind of 20S proteasome, was significantly up-regulated in the comparison of PaLCuCNV vs. un-infected whiteflies, a result in agreement with the iTRAQ data. However, no significant change was observed in the mRNA expression level of cuticle protein 67.

Binding of a virus to cellular receptors is the key determinant of the physiological outcome of infection [37]. To infect vector cells, viruses need to gain access to the cell surface and then bind their receptor (s). Some attachment factors are required for allowing viral concentration at the cell surface, and following this primary attachment, the viral interaction with specific receptors permits its internalization. This process often requires more than one receptor [38, 39]. A previous study demonstrated that clathrin-mediated endocytosis was involved in TYLCV transport across the vector midgut wall [10], but the receptors that mediated this process remain unknown. According to our iTRAQ data, the expressions of laminin subunit alpha, dystroglycan and integrin alpha-PS2, three proteins in the pathway of extracellular matrix (ECM)-receptor interactions, were significantly up-regulated by 1.36, 1.35 and 1.45 fold respectively in the comparison of TYLCV-infected vs. un-infected whiteflies. Similarly, in the comparison of PaLCuCNV-infected vs. un-infected whiteflies, these three proteins were significantly up-regulated by 1.41, 1.5, 1.36 fold respectively. Laminin has been stated as a cellular attachment receptor for some animal viruses [40, 41]. Alpha-dystroglycan is a type of important cellular viral receptor [37, 42‐44]. Many integrin subunits have been reported to be usurped by a number of viral and bacterial pathogens in order to gain entrance into host cells [45‐48]. Then, laminin subunit alpha, dystroglycan and integrin alpha-PS2, and three enriched cell surface receptors identified through our iTRAQ analysis after begomovirus infection, may synergistically regulate the endocytosis of begomovirus infection. The fact that clathrin-mediated endocytosis can affect virus penetration of the vector midgut epidermal cells implies that the receptor-mediated endocytosis was essential for viral transmission.

According to the proteins of insects that have a complete genome, over 1% of the total proteins are cuticle proteins [49], indicating the importance of cuticle proteins in insect body. In our iTRAQ data, three cuticle proteins, cuticle protein 6 (1.26-fold), cuticle protein 67, isoform A (1.25-fold) and cuticle structural protein PCP16.7 (1.22-fold), were significantly up-regulated in the comparison of TYLCV-infected vs. un-infected whiteflies, and two cuticle proteins, cuticle protein 21 (1.35-fold) and cuticle protein 7 (1.21-fold), were significantly up-regulated in the comparison of PaLCuCNV-infected vs. un-infected whiteflies. No cuticle proteins showed down-regulation in both comparisons. According to the analysis from CutProtFam, cuticle protein 6 and cuticle protein 21 belong to the CPR family and RR-2 subgroup, cuticle protein 67, isoform A belongs to CPF family. However, none of the cuticle protein families or sub-families was identified when referring to cuticle structural protein PCP16.7 and cuticle protein 7. For a persistently transmitted virus, e.g. cereal yellow dwarf virus-Rhopalosiphum padi virus, at least four cuticular proteins are involved in the transmission process by the greenbug aphid, Schizaphis graminum [50]. In addition, rice stripe virus can utilize a hemipteran cuticular protein of the small planthopper, Laodelphax striatellus, to facilitate its survival in the hemolymph [51]. So far, the functions of cuticle proteins in begomoviruses transmission have not been reported. Here, we provide a reference for further studies on, for example, clarification of functions of these cuticle proteins identified from our data in begomovirus transmission.

Although insects lack an adaptive immune system, they possess internal defense mechanisms when facing foreign pathogens [52, 53]. In whiteflies, several components have been reported to play a role in viral response, such as the heat shock protein 70 protein and autophagy pathway [14, 54]. According to our iTRAQ data coupled with GO and pathway analysis, in the comparison of TYLCV-infected vs. un-infected whiteflies, some proteins related to defense response, including putative defense protein 3, PITH domain-containing protein CG6153, heat shock factor-binding protein 1 and multidrug resistance-associated protein 1, were significantly up-regulated by 1.57, 1.32, 1.28, 1.25 fold respectively. Similarly, in the comparison of PaLCuCNV-infected vs. un-infected whiteflies, putative defense protein 3, PITH domain-containing protein CG6153 and apoptosis regulator BAX were significantly up-regulated by 1.66, 1.33, 1.31 fold respectively.

A given whitefly species can often acquire and transmit different begomoviruses with varied efficiencies [9]. The different characteristics of TYLCV and PaLCuCNV transmission by MED indicate: (i) following viral acquisition, TYLCV can accumulate in the whitefly but PaLCuCNV is unable to [54], and (ii) PaLCuCNV penetrates through the midgut wall of MED less efficiently than TYLCV, resulting in a lower efficiency of PaLCuCNV transmission by MED [11, 24]. In our iTRAQ data, four 20S proteasome subunits were significantly up-regulated in the comparison of PaLCuCNV-infected vs. un-infected whiteflies, namely proteasome subunit beta type-6 (1.31-fold), proteasome subunit alpha type-7-1 (1.27-fold), proteasome subunit alpha type-3 (1.25-fold), and proteasome subunit alpha type-4 (1.24-fold). Interestingly, no 20S or related proteasome subunits showed significant changes in the comparison of TYLCV-infected vs. un-infected whiteflies. Host proteasome-mediated protein proteolysis is known as a common strategy used by both plants and animals for virus degradation and accumulation [55‐57]. The proteasomes are large multi-subunit proteinase complexes and exist as particles of 20S and of 26S. The 20S particle of ≈700 kDa is an important component of 26S complex of ≈2000 kDa, which is responsible for the degradation of many cellular proteins as a proteolytic core [58, 59]. Ubiquitin-proteasome system has been reported to limit the quantity of begomovirus in whitefly [60]. Thus, in consideration of the potential antiviral function of 20S proteasome, the up-regulated 20S proteasome subunits in PaLCuCNV-infected vs. un-infected whiteflies may be one important factor that leads to the failure of PaLCuCNV accumulation in whitefly body and in turn the lower level of PaLCuCNV acquisition and transmission by MED whitefly.