A peptidoglycan recognition protein acts in whitefly (Bemisia tabaci) immunity and involves in Begomovirus acquisition

Peptidoglycan recognition proteins (PGRPs) are multifunctional pattern recognition proteins. Here, we report that a PGRP gene, BtPGRP, encodes a PGRP from the whitefly Bemisia tabaci (MEAM1) that binds and kills bacteria in vitro. We analyzed BtPGRP transcriptional profiling, and the distribution of the cognate protein within the midgut. Fungal infection and wasp parasitization induced expression of BtPGRP. Silencing BtPGRP with artificial media amended with dsRNA led to reduced expression of a gene encoding an antimicrobial peptide, B. tabaci c-type lysozyme. Begomovirus infection also led to increased expression of BtPGRP. We propose that BtPGRP has a potential Tomato yellow leaf curl virus (TYLCV) binding site because we detected in vitro interaction between BtPGRP and TYLCV by immunocapture PCR, and recorded the co-localization of TYLCV and BtPGRP in midguts. This work addresses a visible gap in understanding whitefly immunity and provides insight into how the whitefly immunity acts in complex mechanisms of Begomovirus transmission among plants.

Scientific RepoRts | 6:37806 | DOI: 10.1038/srep37806 (imd) pathway and the Toll pathway 9,10 . Others, for example, the Drosophila PGRP-LF interacts with PGRP-LC to down-regulate the imd pathway 11 . Still other PGRPs have amidase activity. These proteins, including PGRP-SC and PGRP-LB, also act as negative modulators of the imd pathway, which protects flies from lethal excessive immune reaction to transient infection 12 . PGRPs also act in homeostasis of the Drosophila alimentary canal 2,13 , enabling the tolerance of indigenous microbes and elimination of pathogens. Taken with related findings in vertebrates, the PGRP family has a principal role in innate immune responses.
The whitefly Bemisia tabaci (Gennadius) is a species complex composed of at least 35 morphologically indistinguishable species 14,15 . Some members of the B. tabaci species-complex, especially the cryptic species Middle East-Asia Minor 1(MEAN 1) and Mediterranean, make up a serious pest complex in agricultural ecosystems. Aside from direct feeding damage, B. tabaci is an effective vector of plant pathogenic viruses 16 . Of the whitefly-transmitted virus species, approximately 90% belong to the genus Begomovirus, such as the Tomato yellow leaf curl virus (TYLCV), the Tomato yellow leaf curl China virus (TYLCCNV), and the Tobacco curly shoot virus (TbCSV) 17 . Begomoviruses are transmitted in a persistent circulative manner in which viruses move from gut into hemolymph and on to other tissues within their hosts 16,18 . Some Begomovirus species, TYLCV, for example, may replicate within the whitefly vector, while others, such as the tomato mottle virus, probably do not 19,20 . In general, Geminiviruses, including Begomovirus, replicate in their host plants 21 . Several factors operate in the transmission of Begomovirus. For example, a 63-kDa GroEL homolog produced by endosymbiotic bacteria has high binding affinity for TYLCV and may protect the virus from destruction during its passage through the hemolymph 16 . Two members of the heat-shock protein (HSP) family (HSP70 and BtHSP16) and a midgut protein (MGP) also bind begomoviruses in vitro; HSP70 may act in protecting the vector against begomoviruses while translocating within the whitefly [22][23][24] .
Several B. tabaci transcriptome analyses were designed to identify whitefly genes involved in Begomovirus transmission [25][26][27][28] . Despite considerable progress, however, the molecular and biochemical mechanisms underlying Begomovirus transmission, particularly mechanisms related to maintaining whitefly fitness, are not yet thoroughly elucidated. The situation is probably due to the diversity and complexity whitefly/virus relationships 16 .
We addressed a visible gap in understanding whitefly immunity by posing the hypothesis that a PGRP gene, BtPGRP, encodes a PGRP that binds and kills bacteria and that Begomovirus infection induces BtPGRP expression. Here, we report on the outcomes of experiments designed to test our hypothesis.

Results
Cloning and sequence analysis of BtPGRP. The full-length BtPGRP cDNA (GenBank accession number KJ868812) is 1228 bp, containing an open reading frame of 708 bp encoding a 235 amino acid protein (Fig. 1a). The predicted molecular weight (MW) of the mature protein is 22.67 kDa. The sequence was followed by a 3′ untranslated stretch of 256 nucleotides containing a possible polyadenylation signal (AATAAA). By confirming the full length of BtPGRP sequence using both DNA and RNA of B. tabaci (MEAN1, MED and ZHJ1), we found that BtPGRP from the three cryptic species shared 99.9% identity. Multiple sequence alignment of the deduced amino acid sequence shows high similarity to other members of PGRP superfamily. The amino acids H 57 , T 182 , and S 184 are the predicted amidase catalytic sites and H 57 is a Zn 2+ binding site motif. Phylogenetic analysis showed that BtPGRP from B. tabaci forms an orthologous group with other insect PGRPs (Fig. 1b). Microbial binding and bactericidal activity assays. Our western blot analysis shows rBtPGRP bound E. coli and S. aureus with high affinity (Fig. 2a, lanes P for both bacterial species) and fluorescence microscopy shows the rBtPGRP localized to the cell walls (seen as the green staining) of both bacterial species (Fig. 2b). We recoded aggregation of S. aureus after rBtPGRP treatment. The protein did not bind to the C. albican cell walls ( Supplementary Fig. S2). Compared to the positive control enzyme, lysozyme, the refolded BtPGRP lacked amidase activity against insoluble PGN from E. coli and S. aureus ( Supplementary Fig. S3), although it was strongly bactericidal to both tested bacteria (Fig. 2c). Compared with controls, 50% of E. coli was killed after 1 h incubation, which increased to 73% during the second hour. Similarly, 33% of S. aureus was killed after 1 h incubation and 60% after 2 h. In negative control experiments, the N-terminal tag peptide (His• tag (6 aa) -Thrombin-T7• tag) did not bind to bacteria and had no antibacterial activity (Fig. S4).
BtPGRP gene expression. BtPGRP was constitutively expressed in all examined tissues, midgut, ovary, fat body and egg, and all developmental stages. There was relatively low expression in egg and 2 nd instar stages ( Supplementary Fig. S5a). We found the expression profiles of BtPGRP were not influenced after B. tabaci fed on selected host plants ( Supplementary Fig. S5b). After we applied B. bassiana to infect the white flies, BtPGRP expression was up-regulated after 24 h post-inoculation (Fig. 3a). With respect to the influence of parasitoids, BtPGRP expression increased 50-fold at the penetrated stage after Eretmocerus hayati parasitization (Fig. 3b).
Expression of BtPGRP gene following dsRNA ingestion. The abundance of BtPGRP transcripts was substantially decreased (up to 50% at 3 days) after dietary exposure to dsRNA for 1, 3, and 5 days (Fig. 4a). At day 5, experimental whiteflies were analyzed for the expression of the selected AMP genes, lysozyme (Btlys-C, Btlys-i1 and Btlys-i2) and defensin (Btdef). Knockdown of BtPGRP led to significantly decreased abundances of mRNA encoding Btlys-C, but not the other AMP genes (Fig. 4b).
Begomovirus acquisition influenced BtPGRP expression. Newly emerged whiteflies were released onto the leaves of healthy tomato plants in one cage and, separately, onto virus-infected (TYLCV-, TYLCCNV-, or TbCSV-infected) tomato plants in other cages. Two cages of nonviruliferous and viruliferous whiteflies were randomly collected at the end of 5 acquisition periods (6 h, 12 h, 24 h, 48 h and 72 h). Figure 5 shows the BtPGRP expression was significantly higher from 12 h to 72 h after acquisition of TYLCV and, separately, TYLCCNV. Compared with control, nonviruliferous whiteflies, the expression level was increased by 3-fold at 72 h after acquisition. The highest BtPGRP expression after acquisition of TbCSV, up by 2-fold occurred at 48 h. Although it appears otherwise, these multi-fold increases were significant, except for the influence of TbCSV.
BtPGRP interacts with TYLCV. We used a IC-PCR assay to validate the interaction between TYLCV and BtPGRP. TYLCV-specific PCR products were obtained after viruliferous whiteflies were applied to PCR tubes coated with an anti-BtPGRP antibody (Fig. 6, lane 7-9) as also seen with tubes coated with an anti-TYLCV CP antibody (Fig. 6, lane 1-3). Negative control experiments with tubes not coated with the antibody yielded no TYLCV PCR products after analysis of viruliferous whiteflies (Fig. 6, lane [4][5][6] or in analysis of extracts from nonviruliferous whiteflies (Fig. 6, lane 10-18).

Co-localization of BtPGRP and TYLCV in B. tabaci midgut.
We investigated the distribution of BtPGRP in B. tabaci midguts using a polyclonal anti-BtPGRP antibody. Immunofluorescent microscopy confirmed that BtPGRP is present throughout the midgut (Fig. 7a,b), extracellular to the midgut cells (Fig. 7b). We performed localization studies using an immunological detection of BtPGRP and TYLCV viral DNA in midguts dissected from viruliferous whiteflies. The presence of BtPGRP is indicated by red fluorescence and can be seen in each part of the midgut (Fig. 7c). The presence of TYLCV is shown by green fluorescence, observed in the whole midgut. Co-localization of BtPGRP and TYLCV is seen as yellow patches in the ascending midgut.

Discussion
The data reported in this paper strongly support our hypothesis that a PGRP gene, BtPGRP, encodes a PGRP that binds and kills bacteria and that Begomovirus infection, except for TbCSV, induces BtPGRP expression. Our results form a sound argument. First, the nucleotide and calculated amino acid sequences of BtPGRP are similar to known PGRPs. Second, our phylogenetic analysis shows the BtPGRP fits into the insect PGRPs, with closest connection to another hemipteran, the brown planthopper. Third, we cloned the BtPGRP and expressed it in competent E. coli cells. The rBtPGRP protein bound to E. coli (Gram negative) and S. aureus (Gram positive) cells, with potent bactericidal activity. Fourth, BtPGRP is expressed in several tissues and all life stages from egg to adult, and is up-regulated after fungal infection and wasp parasitization. Fifth, a dietary dsRNA construct effectively reduced BtPGRP expression from days 3 to 5. This indicates BtPGRP acts in whitefly immunity. Sixth, exposure to separate plant pathogenic viruses (TYLCV, TYLCCNV and TbCSV) showed that TYLCCNV infection led to a time-dependent increase in BtPGRP expression up to 72 h post exposure. Finally, immunohistochemical protocols revealed the co-localization of BtPGRP protein and TYLCV within the midguts of viruliferous whiteflies. Taken together, each element of our results amounts to a convincing demonstration at least one BtPGRP operates in whitefly immunity and in Begomovirus vector capacity.
BtPGRP encodes a signal peptide, with no transmembrane domain, and is likely a secreted protein, which would place it in the short PGRP family (a PGRP-S). Multiple sequence alignment of the deduced amino acid shows that BtPGRP is similar to PGRPs in other insect species and contains a conserved PGRP domain at the C-terminus. Aside from assorting PGRPs into short-and long-transcript categories, some, but not all, of these proteins express amidase activity. With respect to phylogenetics, BtPGRP is an ortholog of PGRP -SB/ -SC in D. melanogaster. Although Drosophila PGRP-SB1/-SB2 and PGRP-SC1/-SC2 are thought to have conserved the amidase function 29,30 , BtPGRP lacks amidase activity, owing to the substitution of critical amino acids in the amidase-related motifs. The refolded BtPGRP did not have amidase activity against insoluble E. coli and S. aureus, confirming our inference. This is similar to Drosophila non-catalytic PGRPs (PGRP-SA, -SD, -LE, -LD or -LC), which function as pattern recognition receptors for PGN [31][32][33][34] . We identified only one S-type PGRP in whitefly, which stands in contrast to D. melanogaster, in which many S-type PGRPs, with varying roles, have been identified 35,36 . We infer from the orthology between BtPGRP and Drosophila PGRP -SB/-SC that BtPGRP functions in multiple roles in whitefly immunity as a combination of multiple S-type PGRPs, a concept we will test in future research. Figure 6. BtPGRP-TYLCV CP interaction in viruliferous whiteflies. TYLCV-specific PCR products were obtained after viruliferous whiteflies were applied to PCR tubes coated with BtPGRP antibody (lanes 7-9 ), as seen in tubes coated with TYLCV CP antibody (lanes 1-3). Negative controls, applying viruliferous whiteflies to uncoated tubes are shown in lanes 4-6, and applying extracts from nonviruliferous whiteflies to coated tubes are shown in lanes 10-18. Unlike Drosophila PGRP-SB/-SC, the BtPGRP contains an Arg 106 , which is associated with recognizing DAP-type PGN, as seen in the Drosophila PGRP-LE and PGRP-LC 37 . The Arg 106 does not restrict BtPGRP binding exclusively to DAP-type PGN because it binds both Gram-negative and -positive bacteria. Previous studies have also implied that in addition to PGN, other envelope components such as beta-glucan, LPS and LTA, may be recognized by PGRPs 38,39 . BtPGRP reduced populations of E. coli and S. aureus, from which we infer that BtPGRP has antibacterial activity against both Gram-negative and Gram-positive bacteria.
The antibacterial activity is not directly correlated with amidase activity. In our study, BtPGRP showed no amidase activity against E. coli or S. aureus but had strong antibacterial activity to both bacterial classes. This is similar to some mammalian PGRPs, such as the human PGLYRP3 and PGLYRP4 40 . These PGRPs are bactericidal, but they are most likely not amidase-type PGRPs, as they do not have all five conserved catalytic residues. Other PGRPs also show bactericidal activity, such as Drosophlia PGRP-SB1 30 , Bombus ignitus PGRP-S 41 , Helicoverpa armigera HaPGRP-B and HaPGRP-C 38 and the amphioxus Branchiostoma japonicum PGRP-S 39 . It is not clear how these PGRPs function but we speculate they constitute a new class of bactericidal proteins with structures and action mechanisms that differ from the currently known AMPs.
Many insect PGRPs are expressed in immune conferring organs and tissues, fat body, gut and hemolymph. For example, the Drosophlia, PGRP-SA, PGRP-SB and PGRP-SD are primarily present in hemolymph and expression is induced in fat body and PGRP-SC is induced in gut 9 . BtPGRP is constitutively expressed in all life stages and in the three tissues we analyzed, fat body, midgut and ovaries. AMPs, a component of insect humoral immunity, are produced in fat body. Insects also express epithelial immunity, recorded in salivary glands, Malpighian tubules and tracheal epithelia 3 . Ovaries represent the next generation and Drosophila ovaries up-regulate expression of the AMP, attacin, when ovarian tumors are detected 42 . The idea that BtPGRP acts in immune functions is bolstered by results of infection and parasitization and by the outcomes of dietary dsRNA experiments. Fungal infection and wasp parasitization led to upregulation of BtPGRP mRNA expression, from which we infer the BtPGRP protein acts in one or more roles in white fly immunity. Dietary dsRNA led to reduced BtPGRP mRNA accumulatoin, which translated into a severe reduction in mRNA encoding an AMP, Btlys-c. BtPGRP may specifically influence Btlys-c, because it did not influence other AMP-encoding genes, Btdef, Btlys-il or Btlys-i2 in immunologically naïve whiteflies. The overall picture indicates that BtPGRP acts in multiple immune-related functions.
Our data document co-localization of BtPGRP with TYLCV in the midgut. The IC-PCR aasay indicates a specific interaction between BtPGRP and TYLCV during TYLCV acqusition. We infer BtPGRP has a TYLCV binding site to form a BtPGRP and TYLCV complex. This is consistent with other studies reporting viral-associated up-regulation of PGRPs. Drosophila C virus infection led to increased PGRP-SA expression and SIGMAV infection resulted in increased expression of PGRP-SB1 and PGRP-SD in Drosophila 43 . Similarly, Gao et al. 2 reported that B. mori cytoplasmic polyhedrosis virus infection led to inceased expression of another PGRP gene, BmPGRP-S3. Overall, we infer that BtPGRP acts in multiple immune-response functions.

Materials and Methods
Insects, host plants and microorganism strains. Our whitefly rearing procedure followed 27 . Adults of B. tabaci MEAM1 were released onto cotton plants and maintained for five generations in the lab at 26 ± 1 °C, 60% ± 10% relative humidity and natural photoperiod. Cotton plants were individually grown in plastic pots in greenhouses and no chemicals were applied during the growth and test periods.
Clones of TYLCCNV isolate Y10 (GenBank accession number AJ319675) and its DNAβ (GenBank accession number AJ421621), TYLCV isolate SH2 (GenBank accession number AM282874) and TbCSV isolate Y35 (GenBank accession number AJ420318) were agroinoculated into 3-4 true-leaf stage of tomato (Solanum lycopersicom L. cv. Hezuo903) as described by 44 . Viral infection of test plants was verified by the typical viral symptoms and confirmed by PCR using the procedure described by 45 . All plants were grown in a greenhouse under natural lighting at 25 °C.
Escherichia coli strain BL21 (DE3) was used as the expression host. E. coli DH5α was used as the host for sub-cloning and plasmid amplification. E. coli DH5α , Staphylococcus aureus and Canidia albicans were used to assay the activity of the purified rBtPGRP as representative of Gram-negative and -positive bacteria and fungi.

RNA isolation and rapid amplification of cDNA ends (RACE). An EST sequence homologous to
PGRP was obtained from the B. tabaci transcriptome databases (MEAN1, MED and ZHJ1) by local blast using known insect PGRP sequences as queries. To get the full length of the PGRP cDNA sequence, total RNA was isolated from about 200 B. tabaci adults using SV Total RNA Isolation System (Promega). RNA yield was determined on a NanoDrop 2000 spectrophotometer (Thermo Scientific). 3′ and 5′ RACE were performed with the SMART RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions. Specific primers (Supplementary Table S1) were designed on the basis of the EST sequence. The resultant PCR products were sub-cloned into pMD-19 vector (Takara) and then sequenced by Sangon Biotech (Shanghai, China).
Expression, enrichment and refolding of BtPGRP recombinant proteins. The cDNA fragment encoding BtPGRP, without a signal peptide, was amplified by PCR primers with BamH I and Hind III restriction sites (Takara) (Supplementary Table S1). Products of the PCR assembly were ligated into the pET-28a vector (Novagen) using T4 DNA ligase (Promega). The resulting vector was transformed into the E. coli BL21 (DE3) for DNA sequence analysis. In our standard protocol, a single colony of E. coli BL21 (DE3) cells was grown to OD600 = 0.6 and induced by isopropyl -β -D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM (Sigma). After 3 h induction, the bacterial cells were harvested by centrifugation and protein expression analyzed on 12% SDS-PAGE. We confirmed identity of the rBtPGRP by western blotting using a His-Tag HRP-conjugated rabbit polyclonal antibody (HuaAn Biotechnology, Hangzhou, China) as the primary antibody. His TALON ™ Gravity Columns (Clontech) were used to highly enrich the rBtPGRP.
Scientific RepoRts | 6:37806 | DOI: 10.1038/srep37806 The proteins were refolded using a Float-A-Lyzer G2 (Spectrumlabs) dialysis device. The rBtPGRP solution was dialyzed (with agitation) for 4 h, followed by dialysis in a succession of buffers (Supplementary Table S2). The refolded protein was subjected to a final dialysis against 25 mM Tris (pH 7.0) containing 10 mM NaCl and 5% glycerol overnight and then ultra-filtered with Amicon Ultra-4 (Millipore). The refolded rBtPGRP was analyzed on a 12% SDS-PAGE gel followed by western blotting with the His-tag antibody (HuaAn Biotechnology). The concentration of rBtPGRP was measured by absorbance at 280 nm and then stored at − 80 °C until use.
A polyclonal antibody was prepared by injecting rBtPGRP into rabbits by BGI. A rabbit Immunoglobulin G ELISA Kit (BGI, China) was used to evaluate the reactivity and specificity of the polyclonal antibodies, which were stored at − 80 °C until use.
In vitro assays of rBtPGRP activity. To check the binding specificity, E. coli and S. aureus were grown in MH broth. C. albicans was grown in a modified Martin Broth. When grown to logarithmic phase (OD600 = 0.6), 4 ml of E. coli, S. aureus and C. albicans were harvested by centrifugation at 3500 × g for 10 min, washed three times with PBS and resuspended in 40 μ l of rBtPGRP (2 μ g) in PBS. After 10 min incubation at room temperature, the suspensions were centrifuged and the pellets were washed and resuspended in 40 μ l of PBS. Samples of pellets and supernatants were subjected to 12% SDS-PAGE and western blot analysis using the antiserum against BtPGRP (diluted 1:500 (v/v) in PBS) as just described.
Amidase activity was measured as described by 46 with slight modification. rBtPGRP was incubated with insoluble PGN from E. coli and S. aureus (InvivoGen), and OD540 nm was recorded every five minutes over a 120 min period. Egg white lysozyme [EC 3.2.1.17] (Sangon, China) was used as positive control. Three independent biological replicates were performed.
For the bactericidal assay, bacterial cultures were grown in MH medium at 37 °C to an OD600 = 0.6. Four ml of E. coli and S. aureus were pelleted by centrifugation at 3500 × g for 5 min, washed twice with PBS and resuspended in PBS. Bacteria were diluted to 10 4 and incubated with rBtPGRP (40 μ g/ml) for 1 h or 2 h. The samples were spread on LB agar plates. The plates were incubated at 37 °C for 18 h and the number of colonies was counted. CFUs per ml are shown as the mean of three independent biological replicates. For the microbial binding and bactericidal activity assays, the N-terminal tag peptide (His•tag (6 aa) -Thrombin-T7•tag) was synthesized and used as a control.
Quantitative real time PCR (qPCR). qPCR assays were conducted following 47 . The reactions were conducted on a BioRad CFX96 qPCR System (BioRad, USA) using SYBR Green Real Time PCR Master Mix-Plus (TOYOBO, Japan) following the manufacturer's instructions. The cycling program was 5 min at 95 °C; 40 cycles of 15 s at 95 °C and 35 s at 56 °C. β -actin was used as the reference gene 19 . Plasmid DNA encoding BtPGRP or β -actin were used to generate standard curves. Three independent reactions were performed for each sample. Data shown are the mean of three independent biological replicates. Relative expression values were calculated after normalizing to the maximum expression value. BtPGRP mRNA abundances were analyzed by one-way ANOVA, at P < 0.05. BtPGRP mRNA abundances. Tissue samples, midgut, ovary and fat body and whole insects, including eggs, 1 st -4 th instar larvae and adults, were separately analyzed by qPCR using primers listed in Table S1.
The influence of B. bassiana infection and E. hayati parasitization on the relative accumulation of BtPGRP transcripts was determined. For fungal infection, we followed the protocol of 48 . For whitefly parasitization, whiteflies in the second to third stage of nymph development were used for parasitization and two stages of parasitization were compared with developmental stage-matched non-parasitized nymphs. The samples for the experiments were taken according to 49 . dsRNA synthesis and RNAi. BtPGRP transcriptional templates were produced from total whitefly cDNA using gene-specific primers. The extended sequence of T7 polymerase promoter (5′ -TAATACGACTCA CTATAGG -3′ ) was fused with gene-specific primers at the 5′ -end, and two separate PCR reactions with single T7 promoter were required to generate two separate single promoter temples. PCR products were purified using a PCR DNA Clean-up Kit (Axygen, USA) and used as template for in vitro transcription. dsRNA was synthesized using the T7 RiboMAX Express RNAi System (Promega, USA). Sense and antisense strands were transcribed from the DNA template in the same reaction. dsRNA was suspended in nuclease-free water, analyzed on 1% agarose gels and quantified by NanoDrop 2000 spectrophotometry. The dsRNA was stored at − 80 °C until use. A 700 bp GFP (GenBank accession number U76561) (Clontech, USA) was used to synthesize dsGFP for a negative control.
Newly emerged whiteflies were collected into plastic tubes (5 × 7 cm). The tube openings were covered with two layers of parafilm, and 200 μ l solutions containing 30 μ g dsRNA were placed into the gap between the two layers. Parafilm was pre-treated with 0.1% diethylpyrocarbonate (DEPC) solution to remove any RNases, and then RNase free water was used to clean the DEPC from the parafilm. The dsRNA solution was changed every 24 h. BtPGRP mRNA expression after dietary dsRNA was analyzed by qPCR by using specific primers (Supplementary Table S1). The influence of dietary dsRNA on expression of genes encoding AMPs, defensin and three lysozymes were analyzed, also using gene-specific primers.
Immunocapture-PCR (IC-PCR) assay. Interaction between BtPGRP and TYLCV was detected by IC-PCR using anti-BtPGRP polyclonal antiserum following 50 . The buffers used for IC-PCR are described by 51  from the TYLCV virions bound to the PGRP protein was performed with the TYLCV-specific primers V61 and C473 52 , with three independent biological replicates.
For co-localization analysis, TYLCV and BtPGRP, whitefly adults were allowed 48 h for TYLCV acquisition, then were isolated and treated as just described. Two primary antibodies (BtPGRP antibody, 1:50 dilution; TYLCV CP antibody, 1:100 dilution) and two secondary antibodies were applied. To visualize TYLCV, we used Alexa Fluor 488 nm donkey anti-mouse IgG, dilution 1:50 and to visualize BtPGRP we used Alexa Fluor 546 nm donkey anti-rabbit IgG, dilution 1:100. The specimens were examined on a LSM 780 confocal laser scanning microscope.