The effects of the E3 ubiquitin–protein ligase UBR7 of Frankliniella occidentalis on the ability of insects to acquire and transmit TSWV

Introduction

Tomato spotted wilt orthotospovirus (TSWV), a member of the order Bunyavirales, family Tospoviridae and genus Orthotospovirus, was first discovered by Brittlebank in Australia in 1915 (Brittlebank, 1919). In the last decade, due to climate change, human activities, agricultural production, and arthropod spread, the incidence of TSWV has been increasing (Liang, Yuan & Li, 2020; Mandal et al., 2007; Sivaprasad et al., 2018). TSWV can infect over 1,060 plants in 85 families (Parrella et al., 2003). Because of the significant damage, TSWV is listed as one of the world’s ten most harmful plant viruses (Scholthof et al., 2011). Consequently, the European and Mediterranean Plant Protection Organization (EPPO) classifies it as an A2 quarantine pathogen.

TSWV is only spread by thrips (Ullman et al., 2002), including western flower thrips (Frankliniella occidentalis), flower thrips (F. intonsa), palm thrips (Thrips palmi), and tobacco thrips (T. alliorum). The western flower thrips (F. occidentalis (Pergande)) is the most efficient species for the transmission of TSWV and as such, has attracted much attention. Because of its small size, good concealment, agile action, rapid reproduction, broad host range, and high insecticide resistance, F. occidentalis has brought severe economic losses to many countries and regions (Gupta, Kwon & Kim, 2018). China has included F. occidentalis in the IAS1000 Project (a genome project comprising 1,000 invasive alien species).

Like most Tospoviridae, TSWV replicates in its insect vectors and is transmitted in the vector’s saliva during persistent feeding (Picó, Díez & Nuez, 1996; Wijkamp et al., 1995). F. occidentalis is primarily infected by feeding on plants during the first instar larval stage. During the transmission of TSWV by F. occidentalis, TSWV needs to replicate and break through the vector’s multiple infection barriers to reach the salivary glands of the thrips (Chen et al., 2020; Hogenhout et al., 2008). TSWV first infects the midgut epithelial cells of F. occidentalis (Gupta, Kwon & Kim, 2018; Montero-Astúa, Ullman & Whitfield, 2016). After TSWV infects thrips, it replicates in multiple tissues, similar to the mode of other Bunyavirales virus infections (Whitfield, Ullman & German, 2005). Furthermore, TSWV proliferates in the salivary glands and other tissues of F. occidentalis (Ullman et al., 1993; Wetering, Goldbach & Peters, 1996). The route of virus infection is transferred from the midgut and tubular salivary glands in the larval stage to primary salivary glands in the adult stage (Montero-Astúa, Ullman & Whitfield, 2016). Infected adult insects carry the virus for life and spread it by feeding on healthy plants, thereby infecting them (Wetering, Goldbach & Peters, 1996). Therefore, identifying the key proteins that affect the transmission of TSWV by F. occidentalis is crucial to understanding the relationship between pathogens and their hosts. This interaction is also the basis for the control measures of TSWV and its transmission vector, F. occidentalis.

The transcription, replication and viral particle assembly processes are similar for viruses entering the host cell, both in plant and insect cells (Wang et al., 2019). The study of Bunyavirales viruses in infected animals by reverse genetic system revealed that the viral RNA-dependent RNA polymerase (RdRp) is responsible for the transcription of mRNA in the replication cycle of viruses; it steals the host mRNA5 ‘cap and replicates the RNA genome through the “CAP-snatching” mechanism (Elliott, 2014). The viral nucleocapsid protein N is closely related to the formation of ribonucleoproteins transcription and replication of viral genome (Yu et al., 2015). Thus, viruses are believed to transcribe, replicate and assemble TSWV viral particles in mesophilic cells through the RdRp and N proteins. Numerous studies on virus-host interactions demonstrate that the viral N protein plays a critical role (Liu et al., 2015; Ganaie & Mir, 2014; Zu et al., 2019). However, whether the TSWV N protein interacts with F. occidentalis and is associated with the virus transmitted by thrips needs to be clarified.

Meanwhile, little is known about thrips’ response to TSWV during infection from larval acquisition to adult inoculation of plants. RNA sequencing (RNA-seq) is a powerful tool for identifying differentially expressed transcripts during host–pathogen interactions. Therefore, several RNA-seq-based transcriptome analyses have been performed to determine the differentially expressed genes in TSWV-infected F. occidentalis (Schneweis, Whitfield & Rotenberg, 2017; Shrestha et al., 2017; Zhang et al., 2013). De novo transcriptome sequencing identified 278 unigenes genes involved in plant virus transmission and insecticide resistance (Zhang et al., 2013). Schneweis, Whitfield & Rotenberg (2017) used RNA-seq to determine the overall transcriptome response of first instar larvae, pupae, and adults of F. occidentalis to viral infection. The differentially expressed putative innate immunity-related transcript genes included zinc finger protein, hexamerin 2 B, and thyroid peroxidase precursor (Schneweis, Whitfield & Rotenberg, 2017). The transcripts that responded to TSWV differed during various developmental stages, reflecting the relationship between thrips development and virus transmission via insect vectors and the coordination between development and the virus dissemination route (Schneweis, Whitfield & Rotenberg, 2017; Shrestha et al., 2017). Transcriptomic and network integration analysis of the larval gut of F. occidentalis found that zinc finger proteins were associated with TSWV infection (Han & Rotenberg, 2021).

Additionally, we referred to the primary sequence of a draft genome (https://doi.org/10.15482/USDA.ADC/1503960) of F. occidentalis and three developmental-stage transcriptomes to identify TSWV responsive genes (Schneweis, Whitfield & Rotenberg, 2017). As inferred from the Gene Ontology (GO), TSWV infection interferes with host defenses, insect cuticle structure, development, metabolism, and transport processes and functions. Moreover, most of these studies are at the level of sequencing and prediction, and there are few basic biological studies on the interaction between TSWV and F. occidentalis. Badillo-Vargas et al. (2019) identified six interacting proteins of the glycoprotein G_N by blot overlay assays, among which cyclophilin and endoCP-G_N could directly interact with G_N. Wan et al. (2019) successfully constructed a yeast two-hybrid library and TSWV membrane protein G_N and G_C bait vector. Subsequently, they screened proteins interacting with TSWV G_N, including ubiquitin-related proteins (Zheng et al., 2020). These studies suggest a possible interaction between TSWV and F. occidentalis at the protein level.

Ubiquitin, one of the most common posttranslational protein modifiers, is widely and highly conserved in all eukaryotes. The ubiquitination process, consisting of a tertiary enzyme-linked reaction consisting of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin–protein ligase, is an essential posttranslational modification (PTM) protein degradation process and a crucial aspect of protein interaction between host and pathogen (Alejandro et al., 2019; Schinz & Littlefield, 1985). E3 ubiquitin–protein ligase is responsible for protein-specific ubiquitination and promotes ubiquitin transfer by producing E2 ubiquitin–protein conjugating enzyme. E3 is the critical factor in the last step of the ubiquitination cascade (Alejandro et al., 2019; Schinz & Littlefield, 1985). Through the interaction with specific substrates, this enzyme determines spatiotemporal properties and the pathway specificity (Spratt et al., 2012). Many E3 ubiquitin–protein ligase members have been reported to degrade human/animal virus proteins, thus negatively affecting virus accumulation in host cells (Wang et al., 2021; Yang et al., 2019). Viruses use host cells to synthesize proteins that influence and control the host, and thus protein expression in the host is conducive to viral proliferation (Snippe, Goldbach & Kormelink, 2005). The host’s E3 ubiquitin–protein ligase system can selectively degrade viral proteins, restrict viral growth, and bring the virus and host to homeostasis. (Tang et al., 2018). To counter the antiviral mechanism of the host ubiquitin–proteasome system (UPS), the viruses have evolved strategies to use or destroy the UPS to inactivate or degrade cellular proteins and thereby promote viral reproduction (Tang et al., 2018).

E3 ubiquitin–protein ligases are divided into three subfamilies according to the characteristic domain and the mechanism of ubiquitin transfer: the RING (including the U-box protein family), HECT (homologous to the E6AP C-terminus), and RING-in-related-RING (RBR) family (Hatakeyama, 2017). Although E3 ubiquitin–protein ligases from different families can catalyze the covalent connection between ubiquitin and lysine residues in target proteins, the mechanisms are different (Berndsen & Wolberger, 2014; Mattiroli & Sixma, 2014; Zheng & Shabek, 2017). RING-type E3s are the most common E3 ubiquitin–protein ligases (Deshaies & Joazeiro, 2009), related to DNA repair and immune signaling pathways (Hatakeyama, 2017).

The UBR7 protein of the E3 ubiquitin–protein ligase family, belonging to the RBR subfamily, encodes a UBR box-containing protein (zinc finger in N-recognin) and contains a plant homeodomain (PHD) in the C-terminus (Dasgupta et al., 2022). UBR7 plays a role in the N-terminal rule proteolytic pathway that is highly conserved in yeast, animals, and plants (Lee et al., 2008; Zimmerman et al., 2014). A study based on TurboID used proximity labeling demonstrated that the UBR7 protein in the E3 ubiquitin–protein ligase family directly interacts with the N Toll-interleukin-1-receptor domain (TIR) of the nucleotide-binding leucine-rich repeat (NLR) immune receptors (Zhang et al., 2019). NLR can recognize viral proteins upon infection and trigger RNA silencing-based antiviral immunity, one type of plant immunity. Hence, UBR7 downregulation led to increased N protein and enhanced TMV resistance (Zhang et al., 2019). So we wondered if insects also have a protein similar to plant UBR7 involved in autoimmunity. There have been many studies on the function of E3 ubiquitin ligase in plant autoimmune regulation, Still, the process function of UBR7 in insects is not straightforward, and no relevant studies have been conducted in thrips.

Here, we verified the UBR7 gene, a protein closely related to TSWV, from F. occidentalis using an existing data (Schneweis, Whitfield & Rotenberg, 2017). We detected the expression of the UBR7 gene in different F. occidentalis instars and explored the effect of UBR7 on the acquisition and transmission of TSWV. Surface plasmon resonance (SPR), GST pull-down, and Co-IP assays were used to demonstrate the direct interaction between the UBR7 protein of F. occidentalis and the TSWV N protein. We aimed to provide insights for the research and development of novel molecules for the simultaneous targeted control of TSWV and thrips.

Materials and Methods

UBR7 gene interference

dsRNA synthesis

RNA interference (RNAi) primers were designed using Harvard’s SnapDragon program based on the UBR7 gene sequence. The T7 promoter sequence (-TAATACGACTCACTATAGGG-) was added to the 5′ ends of the forward and reverse primers. After pretest screening, we selected the dsRNA primer with the highest interference efficiency (Table 1). The PCR was then carried out using ds-UBR7 primers and the negative control EGFP (ds-EGFP) primers by referring to previous studies (Table 1) (Xiang et al., 2011). The DNA template of the experimental group was from F. occidentalis, and the negative control group was EGFP plasmid (Beyotime, Shanghai, China). The T7 RiboMAX™ Express RNAi System (Promega, Madison, WI, USA) synthesized dsRNA. After measuring the concentration, the samples were stored at −80 °C.

dsRNA feed preparation

Sucrose was weighed and dissolved in DEPC water to prepare a solution with a 30% mass fraction as a solute of artificial feed. Then, the sucrose solution was used to dilute the dsRNA to the concentration of 500 ng/μL. Three diets groups were subsequently prepared (blank control, CK: 30% sucrose + ddH₂O; negative control group, ds-EGFP: 30% sucrose + dsRNA-EGFP; and experimental group, ds-UBR7: 30% sucrose + dsRNA-UBR7).

Interference efficiency and survival rate detection

The entire RNAi method and apparatus were referenced from previous reports (Wu et al., 2018). Adult thrips 3 days after eclosion without TSWV were randomly selected regardless of sex. The thrips were divided into three groups, CK, ds-EGFP, and ds-UBR7, and placed in separate plastic cups, and the experiment was repeated three times. The thrips were starved for 4 h, then fed an artificial diet. Twenty-four hours later (Wu et al., 2018; Yuan, 2019), the interference efficiency of the UBR7 gene in F. occidentalis was examined by RT-qPCR, and the survival rate of F. occidentalis was determined.

Gene expression and viral abundance detection

A total of 3 days after eclosion, adult thrips carrying TSWV (V⁺) were randomly selected irrespective of sex and divided into three groups (CK, ds-EGFP, and ds-UBR7). The thrips were fed artificial feed after 4 h of starvation treatment. The samples were collected after 0, 6, 12, 24, 48, 72, and 96 h, with three replicates of 50 thrips in each samples. The expression of the UBR7 gene and the TSWV abundance in thrips were then detected via RT-qPCR. No additional artificial feed was supplemented throughout the experimental process. The entire process is illustrated in Fig. 1A.

Experiments concerning TSWV acquisition

Collected L1 thrips were divided into three groups (CK, ds-EGFP, and ds-UBR7) of 50 thrips each and were fed an artificial diet for 24 h. Subsequently, thrips were transferred into glass pots and were fed with the leaves of D. stramonium plants carrying TSWV. After 48 h, the leaves were replaced with healthy beans, and the thrips continued feeding until they reached L2. The TSWV abundance of L2 thrips was detected using RT-qPCR. F. occidentalis can only acquire poison in larva, the younger the instar, the greater the ability to acquire poison (Wetering, Goldbach & Peters, 1996). So, detecting TSWV abundance in larval thrips by RT-qPCR can indirectly indicate the ability of thrips larvae to acquire TSWV. The experimental process is shown in Fig. 2A.

Results

cDNA cloning and characterization

After sequencing, the full-length coding sequence of F. occidentalis UBR7 was obtained (Fig. 3). The UBR7 gene contained an unbroken open reading frame of 1,246 nucleotides, encoding 414 amino acids. The amino acid sequence of UBR7 was compared with that of FOCC003013-RA, and there were only two site differences in the sequences of the first 300 amino acids. Based on the deduced AAs, the theoretical molecular weight of UBR7 was predicted to be 46.55 kDa with a theoretical pI of 4.89 (Table S3). Protein BLAST and identity analyses suggested that the UBR7 gene belongs to the E3 ubiquitin–protein ligase family that recognizes N-degrons and structurally related molecules for ubiquitin-dependent proteolysis or related processes through the UBR box motif. In addition, UBR7 harbors a PHD finger domain (142–193 AAs) and a conserved zinc finger domain (51–115 AAs) (Fig. 3), which can specifically recognize histone modifications and some DNA sequences that participate in plant life processes including plant autoimmunity (Mouriz et al., 2015). Therefore, we suspect that F. occidentalis UBR7 is similar to UBR7 in plants. Compared to F. occidentalis UBR7-domains (51–193 AAs) within TSWV-susceptible plants, the UBR7 AAs in this region were highly conserved (Protein sequence identity NaN% = 0.56). The larger the letter in Fig. 4, the more conserved the amino acid site. Within the neighbor-joining phylogenetic tree, F. occidentalis UBR7 belonged to “the E3 ubiquitin–protein ligase UBR7 F. occidentalis” clade, It was clustered with T. palmi, C. secundus, and P. humanus corporis in the same branch (Fig. 5).

Expression of UBR7 in F. occidentalis

To investigate whether the expression of the UBR7 gene in F. occidentalis (V⁻) was related to the developmental period, RT-qPCR and Western blotting were used to detect the expression levels of the UBR7 in L1s, L2s, pupae, female adults, and male adults. The protein expression results were consistent with the mRNA levels. The expression levels in the adult stages were significantly higher than those in other stages, with no gender difference (Figs. 6A and 6B). Furthermore, there was no significant difference between the L1s, L2s, and pupal stages. Meanwhile, there was no significant difference in the expression of the UBR7 between female and male adults, but there were substantial differences compared with other instars. This result indicated that UBR7 may be more involved in its biological functions during the adult stage of thrips.

The transmission ability of male western flower thrips was higher than that of F. occidentalis (Ogada & Poehling, 2015; Stafford, Walker & Ullman, 2011). E3 ubiquitin-protein ligases are associated with the infection of hosts by viruses and transmission of viruses by insect vectors (Snippe, Goldbach & Kormelink, 2005). Therefore, we questioned whether the expression of UBR7 varies depending on the sex of F. occidentalis. To explore whether TSWV influences the UBR7 gene of F. occidentalis in different sexes, we used RT-qPCR and Western blotting to detect the expression level of UBR7 in female and male thrips with and without TSWV (Figs. 6C and 6D). The results of UBR7 at the mRNA transcript level were generally consistent with those at the protein level. When male and female thrips did not carry TSWV (♀V⁻, ♂V⁻), there was no significant difference in UBR7 expression. There were also no significant differences between sexes when exposed to TSWV when both male and female thrips carried TSWV (♀V⁺, ♂V⁺).

To investigate whether UBR7 was differentially expressed in different insect tissues, we divided F. occidentalis into three parts: head, thorax, and abdomen, and examined the relative expression of UBR7 by RT-qPCR and Western blotting (Figs. 6E and 6F). The protein detection results were consistent with the changes in mRNA levels. UBR7 was highly expressed in thoracic tissue and was significantly differentially expressed compared to its expression in the head and abdomen.

Effects of RNAi on F. occidentalis

Effects of RNAi on UBR7 gene expression in F. occidentalis without TSWV

Compared with that in the CK and ds-EGFP groups, the UBR7 gene expression in the ds-UBR7 experimental group was significantly reduced (Fig. 7A). Furthermore, the result indicated that this dsRNA fragment could effectively down-regulate the expression of the UBR7 gene in thrips (V⁻).

Effects of RNAi on the survival rate of F. occidentalis without TSWV

To understand whether UBR7 affected the vital activity of thrips, we recorded the survival rate of thrips 24 h after RNAi feeding. Compared with the CK group and the ds-EGFP group, the survival rate of F. occidentalis (V⁻) in the ds-UBR7 group decreased significantly (Fig. 7B). Therefore, it was hypothesized that the UBR7 gene might be involved in F. occidentalis (V⁻) vital activity.

Effect of RNAi on UBR7 gene expression and viral abundance in F. occidentalis with TSWV

RNAi was performed on adult thrips carrying TSWV (Fig. 1A). As the time of RNAi was extended, the expression of the UBR7 gene gradually decreased and then slowly recovered, with an overall U-shape pattern (Fig. 1B). Similarly, the abundance of TSWV was consistent with the expression of UBR7, which also decreased and then recovered (Fig. 1C). This implied a link between the expression of UBR7 and the abundance of TSWV in thrips with TSWV.

Effect of RNAi on acquiring TSWV of F. occidentalis

To explore the effect of UBR7 on the ability of thrips (V⁻) to acquire TSWV, RT-qPCR was used to detect TSWV in thrips larvae after feeding on leaves containing the virus. Compared with those in the CK group and the ds-EGFP group, there was no significant difference in the ability of F. occidentalis in the ds-UBR7 group after RNAi. Fig. 2B shows that UBR7 did not affect the ability of F. occidentalis to acquire TSWV. In this study, UBR7 was only highly expressed in adult stages and at low levels in the larval stage (Figs. 6A and 6B), indicating that it was less important in the larval stage.

Effect of RNAi on transmitting TSWV of F. occidentalis

UBR7 was highly expressed in the adult stage of F. occidentalis (Figs. 6A and 6B), and E3 ubiquitinase is related to the spread of the virus (Snippe, Goldbach & Kormelink, 2005). Therefore, we used ELISA to detect the protein concentration of TSWV in leaves to explore the effect of UBR7 on the ability of F. occidentalis (V⁺) to transmit TSWV. After RNAi with F. occidentalis (V⁺), the absorbance value in the ds-UBR7 group (experimental group) was significantly lower than that in the CK group (blank control) (P < 0.0001) and ds-EGFP group (negative control group) (P < 0.001) (Fig. 2D). This result demonstrated that down-regulation of the UBR7 gene impaired the ability of thrips to transmit TSWV.

UBR7 protein interacts directly with the TSWV N protein

The proteins interacting with UBR7-domain were screened using SPR and LC-MS/MS and then compared with the NCBI datasets. The results showed the top five proteins with the highest scores (Table 2). Most matched proteins were from the viral host N. benthamiana and viruses in the genus Orthotospovirus such as TSWV and Chrysanthemum stem necrosis orthotospovirus. Among these, the highest score was the N protein of TSWV (GenBank accession number: gi|284810746); this explained why UBR7 bound to the TSWV N protein with high efficiency. Detailed peptide information can be found in Figs. S6–S9.

Table 2:

The top five most strongly interacting proteins with UBR7-domino.

Accessiona	Description	Mass	Score	Matchesb	Sequencesc	emPAI	Coverage
gi|284810746	nucleocapsid protein [Tomato spotted wilt orthotospovirus]	29,094	650	34 (23)	16 (12)	4.08	51%
gi|729042213	glyceraldehyde 3-phosphate dehydrogenase-A [Nicotiana benthamiana]	42,945	115	10 (5)	7 (5)	0.45	19%
gi|926663240	heat shock protein 90-1 [Nicotiana benthamiana]	80,443	94	6 (3)	5 (2)	0.08	6%
gi|1219878403	Gc-Gn glycoprotein precursor [Chrysanthemum stem necrosis orthotospovirus]	130,189	89	6 (4)	5 (4)	0.10	4%
gi|660450867	domains rearranged methyltransferase 1 [Nicotiana benthamiana]	69,106	78	4 (4)	1 (1)	0.05	0%

DOI: 10.7717/peerj.15385/table-2

Notes:

a The protein number in NCBI.

b The total number of peptides matched, in brackets is the number of matches higher than the significance threshold.

c The total number of sequences matched, in brackets is higher than the significance threshold sequence number.

To further verify the results of SPR and LC-MS/MS, GST pull-down and Co-IP assays were used to analyze the interaction between UBR7-domain and TSWV N. In the GST pull-down assay, TSWV N, GST and UBR7 could be detected in the input (Fig. 8A), indicating that the GST pull-down system was functional. Fig. 8B shows the result after the beads pull down. UBR7-domain could be detected after co-incubation with TSWV N-GST but not after co-incubation with GST (Fig. 8B). In the Co-IP assay (Fig. 8C), TSWV N and UBR7 could be detected in the input, meaning the Co-IP system usually works. After immunoprecipitation, no bands existed in the lgG-negative control group, while TSWV N and UBR7 bands could be detected in the TSWV N group. In brief, GST pull-down and Co-IP confirmed the direct interaction between TSWV N and UBR7 in vitro and in vivo, respectively, which was consistent with the results of SPR.

Discussion

Ubiquitin is an essential protein in PTM widely involved in regulating innate immune signaling pathways. Ubiquitin is highly conserved in all eukaryotes (Alejandro et al., 2019; Heaton, Borg & Dixit, 2016; Schinz & Littlefield, 1985). Ubiquitination regulates protein homeostasis, cell cycle progression, gene transcription, receptor transport, and immune response (Haglund & Dikic, 2014). Ubiquitination regulation is not limited to eukaryotic cells but is also found in viruses (Gao et al., 2021; Liu, Cai & Gao, 2018). Many viral proteins can mimic or usurp key regulators that affect the binding of ubiquitin-like protein modifiers and ubiquitin-proteins in the host and interfere with the corresponding enzymatic cascades to effectively promote viral replication (Gao et al., 2021; Stukalov et al., 2021). For the virus–host interaction, previous studies have shown that the virus has evolved strategies to exploit specific PTM processes during the immune evasion (Wimmer, Schreiner & Dobner, 2012).

Through NCBI sequence alignment analysis, the highly expressed gene FOCC003013-RA in F. occidentalis response to TSWV belongs to PREDICTED: Frankliniella occidentalis putative E3 ubiquitin-protein ligase UBR7 (NCBI Reference Sequence: XM_026422690.1), and we also named it UBR7. In the previous study, FOCC003013-RA was described as a hemocyanin subunit type 1 precursor. However, when the amino acid sequences of UBR7 and FOCC003013-RA were compared with the “hemocyanin subunit 1 precursor” of insect origin, we found that the amino acid sequences of UBR7 and FOCC003013-RA showed very little consistency with other sequences and no conserved structural domain of hemocyanin subunit 1 precursor (Fig. S8). UBR7 belongs to the E3 ubiquitin–protein ligase family with typical RING-type family characteristics and encodes a UBR box-containing protein, with a PHD in the C-terminus and a zinc finger in the N-recognin (Figs. 3 and 5) (Dasgupta et al., 2022). Also, plant E3 ubiquitin–protein ligase is essential in regulating hormone responses, morphogenesis, disease resistance, and abiotic stress response. Overexpression of the plant E3 ubiquitin–protein ligase BnTR1 in rapeseed and rice enhanced the resistance to heat stress (Liu et al., 2013). CaRING1 silencing increased the infection rate of plants to bacterial spots in red pepper (Dong, Choi & Hwang, 2011). The UBR7 protein in the E3 ubiquitin–protein ligase family in tobacco directly interacted with the N TIR-NLR, and UBR7 downregulation enhanced TMV resistance (Zhang et al., 2019). Thus, we hypothesized that if UBR7 of F. occidentalis also has a similar immune function to plant UBR7, their amino acid sequences have certain similarities. By comparing AAs, we confirmed the high similarity between F. occidentalis and Solanaceae plant UBR7, which made us more confident on our speculation (Fig. 4). Therefore, we hypothesized that the UBR7 protein in F. occidentalis may be closely related to TSWV. SPR (Table 2, Figs. S6–S9), GST pull-down and Co-IP experiments (Fig. 8) all demonstrated that western flower thrips UBR7 interacts with the TSWV N protein, validating our hypothesis.

Most of the current research on thrips interactions with TSWV have focused solely on TSWV (Gupta, Kwon & Kim, 2018; Montero-Astúa et al., 2014), while studies on thrips have stayed at the ecological (Gupta, Kwon & Kim, 2018; Sarwar, 2020; Stumpf & Kennedy, 2005), transcriptomic (Schneweis, Whitfield & Rotenberg, 2017), proteomics levels (Badillo-Vargas et al., 2019; Zheng et al., 2020). There are few studies on the biology and gene function of TSWV affecting thrips (Badillovargas et al., 2012). In F. occidentalis, the high expression of the UBR7 gene primarily occurred in the adult stage, and UBR7 (FOCC003013-RA) was also the most up-regulated gene in transcriptome data; these all indicated the temporal specificity of UBR7. Given the differences between the transcriptome and proteome at the tissue level in the larval and adult stages, and the fact that F. occidentalis can only transmit TSWV in adult stages after the thrips acquire the virus in the larval stages (Wetering, Goldbach & Peters, 1996), we again hypothesized that UBR7 is strongly associated with TSWV transmission in F. occidentalis. In addition, TSWV induced high expression of UBR7 in F. occidentalis (Figs. 6C and 6D), mainly in the thorax of the thrips (Figs. 6E and 6F). Arboviruses of plants circulate and multiply within the insect vector; the vector serves as an alternative host for the plant pathogen (Geetanchaly & John, 2021). Usually, the vector acquires the plant pathogen by feeding on infected plants. It has been hypothesized that once the virus enters the insect, it will cross intestinal barriers, internal organs, and visceral muscles and can be found throughout the hemolymph (Perilla-Henao & Casteel, 2016). The salivary gland is an essential organ for the persistent transmission of viruses in insect vectors; it is the last line of defence that plant viruses need to overcome to circulate in the insects (Stafford-Banks et al., 2014). For vector insects to transmit pathogens to new plant hosts, the virus must spread from the hemolymph to the salivary glands (Whitfield, Falkb & Rotenberga, 2015). At the same time, salivary glands are found mainly in the thorax of insects (Hiroaki et al., 2020; Ohnishi et al., 1996). TSWV circulates and proliferates in F. occidentalis and is spread by adult thrips (Gupta, Kwon & Kim, 2018). Therefore, UBR7 was highly expressed in the thoracic tissue of F. occidentalis. We hypothesize that this was because TSWV was enriched in the salivary glands of adult thrips, inducing high expression of UBR7.

Salivary glands, one of the essential organs of vector-borne viruses, are closely related to virus transmission. Thus, we speculated that UBR7 participates in transmitting TSWV by F. occidentalis. From this, we decided to measure changes in the transmission of F. occidentalis after RNA interference. The transmission of TSWV can be influenced by host plants, virus load, sex, age, susceptibility to pesticides, and behavior of its insect vector, thrips (Kumm & Moritz, 2010; Maris et al., 2004; Pappu, Jones & Jain, 2009; Stafford, Walker & Ullman, 2011; Whitfield & Oliver, 2016; Zhang et al., 2015; Zhao et al., 2016). Carrying the virus could improve the reproduction rate of F. occidentalis (Maris et al., 2004) and affect the sex ratio of thrips, increasing the number of male thrips, sex with a greater dispersal and virus transmission capability (Wan et al., 2020). At the same time, TSWV could also alter the feeding behavior of F. occidentalis, in which virus carriers fed significantly more than the non-carriers (Stafford, Walker & Ullman, 2011).

As a result, the transmission efficiency of F. occidentalis varies with gender (Liao et al., 2015). In the field, we could not determine the number of thrips carrying TSWV in thrips populations. There is also no way to determine the virus load in each thrips. Therefore, to reduce experimental error due to differences in sex and individual viral load, we selected thrips at random, regardless of sex, to ensure consistent sex ratios and consistent viral loads in each group of thrips. The results of RNAi experiments confirmed our hypothesis, in which the down-regulation of UBR7 attenuated TSWV transmission. Simultaneously, UBR7 expression decreased and then increased with increasing RNAi time. This was caused by the gradual degradation of dsRNA, which can no longer interfere with the UBR7 gene. And we did not add additional artificial feed containing dsRNA throughout the RNAi experiment. As UBR7 expression recovered, the abundance of TSWV also started to rise, presumably because UBR7 influenced the proliferative effect of TSWV and thus reduced the ability of western flower thrips to transmit the virus. The abundance of the virus in vector insects is also a critical factor in the success of TSWV transmission by thrips Nagata et al. (2002). Rotenberg and colleagues found that virus titer was positively associated with the frequency of transmission events (Rotenberg et al., 2009). With low URB7 expression, the efficiency of TSWV transmission by thrips decreased.

Meanwhile, RNAi had little effect on the ability of thrips to acquire TWSV. The typical expression of UBR7 in the larval stage suggests that it has a minor physiological role in the larval stage. Thrips acquire the virus at L1 and L2 (Wetering, Goldbach & Peters, 1996), with a lower expression of UBR7 in the instar stages. As the expression level of UBR7 is low in the thrips larval stage, RNAi does not significantly affect UBR7 expression, so it will not have a significant biological impact. Therefore, we speculate that UBR7 could interfere with virus replication and thus influence the transmission efficiency of F. occidentalis, while the efficiency of TSWV acquisition was unaffected. The movement of the virus from the salivary glands of thrips to the new host plant may not be affected by protein silencing, which still requires further investigation.

Meanwhile, many proteins interacting with UBR7-domain were screened by SPR (Table 2), among which TSWV N was the most closely involved. At the same time, we verified the direct interaction between UBR7 protein and TSWV N by GST pull-down (Figs. 8A and 8B) and Co-IP (Fig. 8C) in vitro and in vivo respectively. It was proved that the UBR7 domain site (51-193AA) in F. occidentalis can directly interact with the N protein of TSWV. These results are consistent with our initial hypothesis that TSWV N directly interacts with UBR7.

The N protein is a phosphorylated nucleocapsid protein of the virus, and the epitope of N protein can induce the body to produce an effective immune response (Shi et al., 2017; Zhou et al., 2019). N protein also protects internal nucleic acids from damage by nucleases in the external environment (Afrasiabi et al., 2020). UBR7 is highly expressed in adult thrips carrying TSWV (Figs. 6A–6D). We hypothesize that RNAi reduced the expression of UBR7, thus preventing the interaction between UBR7 and TSWV-N, breaking the homeostasis in thrips, and resulting in increased mortality, reduced activity and decreased transmission ability of thrips. However, the exact mechanism still needs to be further explored and verified.

Conclusions

In conclusion, our study found that the UBR7 gene was involved in TSWV transmission by thrips. The results demonstrated the direct interaction between the F. occidentalis UBR7 protein and the TSWV N protein (51–199 AAs domain). UBR7 was expressed and functional in the adult stage, mainly in thrips’ thoracic tissue. Down-regulation of UBR7 expression reduced the transmission efficiency of thrips. These results suggested that UBR7, an E3 ubiquitin–protein ligase family member, is closely related to TSWV transmission by F. occidentalis. The pathway through which the UBR7 protein affects TSWV proliferation needs further characterization. The molecular structure where the UBR7 protein interacts with TSWV N in F. occidentalis remains to be studied. Moreover, we will continue to explore whether UBR7-targeted pesticides will help TSWV transmission and F. occidentalis control. Our study provides insight into the mechanism of TSWV transmission by F. occidentalis.

Supplemental Information