Salivary Proteins of Whitefly Bemisia tabaci Modulate Plant Defenses by Activating SA Signaling Pathway

ABSTRACT

The whitefly Bemisia tabaci is an infamous agricultural pest that inflicts significant harm on crops globally by directly feeding on them and indirectly transmitting viruses. Although it is believed that whiteflies inject saliva into their host plants to modulate plant defenses, however, due to their small size, study on the salivary proteome of whiteflies is still sparse and the roles of many salivary proteins remain elusive. This research employed four‐dimensional data‐independent acquisition (4D DIA) based proteomic techniques to analyze whitefly secreted saliva. In total, 2625 salivary proteins were identified. Bioinformatic analysis results showed that the secreted saliva proteins may play roles in hydrolysis, transport, protein binding and metabolism, and significantly increased the levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities as well as malondialdehyde (MDA), soluble sugars (SS), soluble protein (SP), and proline (PRO) in cucumbers. The expression of the salicylic acid (SA) response genes (PR1a, PR3, and PR5) were significantly induced. Additionally, exogenous methyl‐salicylate (MeSA) treatment of cucumbers can significantly reduce the reproductive capacity and survival rate of whiteflies, and decrease the preference of whiteflies for host plants. This study's outcomes provide valuable insights into the interaction between plants and insects. It has been proved that the salivary proteins of whitefly participate in host plant defense responses by activating the SA signaling pathway, and provides basic data on the functional study of whitefly saliva elicitors and effectors, which can be used for development of novel strategies for pest management.

Summary

• A total of 2625 salivary proteins of whitefly Bemisia tabaci were identified by 4D‐DIA proteomics.

• The saliva proteins participate in host plant defense responses by activating the SA signaling pathway.

• MeSA treatment of cucumbers can significantly reduce the reproductive capacity and survival rate of whiteflies, and decrease the preference of whiteflies for host plants.

Salivary proteins of whitefly participate in host plant defense responses by activating the salicylic acid (SA) signaling pathway. Feeding by whitefly significantly increased the levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in cucumbers, reduce the host's adaptability, and decrease the preference of whiteflies for host plants.

1. Introduction

Salivary proteins of insect herbivores indeed contribute to the intricate relationship between insects and plants (Howe and Jander 2008; Hogenhout and Bos 2011). During feeding, the mixture of salivary proteins is delivered into plants (Miles 1999). The bioactive ingredients in saliva are involved in degrading plant cell walls, promoting digestion and absorption, forming salivary sheaths, regulating plant defenses, and transmitting viruses (van Bel and Will 2016; Huang et al. 2019; Chi et al. 2024). Numerous salivary proteins and chemical compounds have been discovered in insect herbivores, such as glucose oxidase (GOX), calcium‐binding proteins (C002), endo‐β‐1,4‐glucanase (NlEG1), Vitellogenin (Vg), HARP1, Me47, Armet and CarE10 (Musser et al. 2002; Will et al. 2007; Mutti et al. 2008; Wu and Baldwin 2010; Sharma et al. 2014; Wang et al. 2015; Kettles and Kaloshian 2016; Ji et al. 2017, 2021; Chen et al. 2019; Du et al. 2022). A recent study showed that the salivary protein BPH14‐interacting salivary protein (BISP) from the brown planthopper (Nilaparvata lugens) can be perceived by a plant immune receptor conferring host plant resistance (Guo et al. 2023). The glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) of leafhopper and other hemipterans can inhibit the outbreak of H₂O₂ in plants, thus promoting insect feeding and virus transmission (Wang et al. 2024). Sfapyrase of fall armyworm (Spodoptera frugiperda) functions as a salivary elicitor, triggering the jasmonic acid (JA) defense responses of maize (Yao et al. 2025). These findings suggest that insect salivary effectors are crucial for both feeding behaviors and the regulation of plant defense mechanisms. The salicylic acid (SA) signaling pathway is a crucial regulatory mechanism in plant immune responses. When plants are subjected to insect feeding stress, they will induce the synthesis of SA, trigger systemic signals, and activate the expression of defense‐related genes, thereby enhancing the plants’ resistance to insects. The activation of the SA signaling pathway by insect salivary proteins is an important mechanism in the interaction between insects and plants (Xu et al. 2019; Erb and Reymond 2019). Nevertheless, the molecular processes that govern how salivary proteins influence insect feeding and plant defense are not yet fully comprehended.

The whitefly Bemisia tabaci is an agricultural super pest, endangering more than 600 host plants, transmitting more than 400 plant viruses, and causing serious economic losses to agricultural production around the world (De Barro et al. 2011; Liu et al. 2007; Xia et al. 2021). The secretion of protein effectors by whitefly may inhibit plant defense mechanisms, thereby enhancing the adaptability of the host (Wang et al. 2017; Lee et al. 2018; Xu et al. 2019). Recent studies show that the substantial harm caused by B. tabaci may be facilitated by its saliva. Such as, the salivary protein Fer1 enhances whitefly feeding by lowering H₂O₂ levels, promoting callose deposition, and inhibiting defense mechanisms mediated by JA (Su et al. 2019). The salivary effector protein Bsp9 of whitefly may inhibit plant immune signaling, thus enhancing the attraction and fitness of whiteflies, consequently boosting the spread of viruses (Wang et al. 2019). The protein Armet was identified as a salivary effector that enhances the performance of whiteflies on tobacco plants (Du et al. 2022). The salivary proteins Bt56 and BtE3 of whitefly can activate the salicylic acid (SA) signaling pathway of tobacco, reduce plant resistance and promote whitefly survival and fecundity (Xu et al. 2019; Su et al. 2019; Wang et al. 2019; Peng et al. 2023). Due to the small size of the whitefly individual, a large number of salivary proteins have not been discovered so far, or rather it is still a technical challenge to identify its complete salivary proteome.

Four‐dimensional data‐independent acquisition (4D DIA) is an advanced proteomics technique that combines data‐independent acquisition (DIA) with four‐dimensional separation technology (Meier et al. 2020; Yang et al. 2021, 2022). 4D DIA extends the traditional three‐dimensional (3D) separation (retention time, m/z, and ion intensity) by adding a fourth dimension, ion mobility, which significantly improves scan speed, detection sensitivity, and overall performance in protein identification and quantification (Chen et al. 2023). In this study, the 4D DIA proteomics technique provided a comprehensive profile of whitefly salivary proteins, including those that are challenging to identify because of their scarcity. The results of this study offer fresh perspectives on the molecular mechanisms and the evolutionary dynamics of the relationships between plants and insects. Salivary proteins of insect herbivores are multifunctional, playing critical roles in insect feeding, survival, and the modulation of plant defenses. Gaining insight into these interactions may result in progress in both basic research and practical applications, including the creation of new methods for pest management.

2. Materials and Methods

2.1. Sample Preparation

The whitefly B. tabaci (MED cryptic species) was initially collected in 2023 from cucumbers (Cucumis sativus) in Zhengzhou, and subsequently fed on cucumber plants (Bojie‐18A) in a greenhouse at 26 ± 1°C, under a 14/10 h (light/dark) photoperiod and relative humidity 65 ± 5%. The colony was tested by sequencing a mtCOI gene (Khasdan et al. 2005; Shatters et al. 2009; He et al. 2023). About 1000 whiteflies were transferred to a new cucumber plant with 3–4 leaves, after feeding for 2‐d, all the whiteflies and eggs were carefully removed using a small brush, the cucumber leaves were collected for protein extraction and analysis.

2.2. Protein Extraction and Trypsin Digestion

Cucumber leaves were first grinded with liquid nitrogen, subsequently, 5 volumes of lysis buffer (containing 4% sodium dodecyl sulfate, 100 mmol/L Tris‐HCl, 10 mmol/L dithiothreitol, 1% protease inhibitor, pH 7.6) was added for ultrasonic cracking. Add the appropriate amount of Tris‐saturated phenol and vortexed, then centrifuged at 5500 g at 4°C for 10 min. Take the supernatant and 5 volumes of 0.1 M ammonium acetate/methanol were added for precipitation overnight. The precipitation was rinsed with methanol at a low temperature, followed by a rinse with acetone. After that, the precipitates were dissolved in 200 mM TEAB for dispersion through ultrasonication. An overnight digestion was performed by adding trypsin at a ratio of 1:50 (w/w). After sample reduction and alkylation, purification of the peptides was carried out using a Strata X SPE column, they were subsequently dehydrated through vacuum freeze‐drying (Jiao et al. 2024; Fan et al. 2024).

2.3. Mass Spectrometer

The peptides were solubilized in solvent A (2% acetonitrile, 0.1% formic acid), then injected into the reversed‐phase analytical column. The gradient for separating the peptides is outlined as follows: 0–9 min, 6%–24% solvent B (0.1% formic acid in acetonitrile); 9–11 min, 24%–35%B; 11–13 min, 35%–80%B; 13–15 min, 80%B. The peptides were continuously delivered using a NanoElute UHPLC system and analyzed by the timsTOF Pro 2 mass spectrometry. The full MS scan was set as 300–1500 and 20 PASEF‐MS/MS scans were acquired per cycle (Jiao et al. 2024; Fan et al. 2024; Fang et al. 2025).

2.4. Database Search

The DIA data were analyzed using the DIA‐NN (v1.8). The tandem mass spectra were searched against the Whitefly Genome Database (http://www.whiteflygenomics.org/ftp/MED/), which contains 19,996 entries, in conjunction with a concatenated reverse decoy database. Trypsin/P was specified as the cleavage enzyme, with up to one missed cleavage permitted. Fixed modifications included the excision of N‐terminal methionine and carbamidomethylation of cysteine residues. The false discovery rate (FDR) was controlled at less than 1% (Fan et al. 2024; Fang et al. 2025).

2.5. GO Annotation

Gene Ontology (GO) analysis encompasses three primary domains: (1) Cellular Component: This category encompasses specific cellular components recognized as integral elements within larger cellular architectures in the GO framework. Examples include distinct cellular structures like the rough endoplasmic reticulum and nucleus, as well as assemblies of gene products forming complex entities such as ribosomes or protein dimers; (2) Molecular Function: This aspect primarily concerns the chemical activities of molecules that can be observed at the molecular level, such as catalytic actions or binding interactions; (3) Biological Process: This term refers to the coordinated and precise execution of a specific function within an organism by a network of interacting molecules, detailing how these processes contribute to the overall biological functionality. The GO annotation procedure entails utilizing the eggnog‐mapper tool, and drawing upon the EggNOG database. Subsequently, functional classification and annotation analysis are conducted on these proteins, categorized by their involvement (Jiao et al. 2024; Fang et al. 2025; Wang et al. 2023).

2.6. Domain Annotation

A protein's structural domain refers to a particular region within the protein that exhibits sequence conservation and can typically function autonomously. This domain functions as a critical structural component of molecular entities, typically comprising 25 to 500 amino acids. These regions exhibit spatial compactness and structural stability, enabling them to fold independently into functional units. Proteins can possess multiple domains, and individual domains may be present across various proteins. In this study, we performed structural domain annotation of the identified proteins by utilizing the Pfam database in conjunction with the PfamScan tool (Jiao et al. 2024; Fan et al. 2024; Fang et al. 2025; Wang et al. 2023).

2.7. KEGG Pathway Annotation

The Kyoto Encyclopedia of Genes and Genomes (KEGG) compiles existing knowledge on protein‐protein interaction networks, including pathways, genes, gene products, complexes, and biological complexes. KEGG pathways primarily cover metabolism, genetic information processing, and cellular processes. We annotate protein pathways based on the KEGG pathway database, identifying proteins via BLAST comparisons, and the annotation is determined by the highest‐scoring match (Jiao et al. 2024; Fan et al. 2024; Fang et al. 2025).

2.8. Functional Enrichment

Fisher's exact test was used to analyze the significance of functional enrichment of identified proteins (using all proteins in the species database as the background). Functional terms exhibiting a fold enrichment greater than 1.5 and a p value of less than 0.05 were regarded as statistically significant (Jiao et al. 2024; Fan et al. 2024; Fang et al. 2025).

2.9. Protein‐Protein Interaction Network

Proteins identified through their database accession numbers or sequences were analyzed within the STRING database. Interactions were exclusively considered among the proteins in our dataset, omitting any external candidates. The database employs a “confidence score” to gauge the interaction reliability, and we selected interactions with scores exceeding 0.7, indicating high confidence. Visualization of this interaction network was achieved using the R package “visNetwork” (Szklarczyk et al. 2023).

2.10. Analysis of SA, MeSA, Stress Factors, and Genes Expression

For the defense response assay, we placed one cucumber plant with 3–4 leaves in the cage, 200 whiteflies were released and feed for 2‐d, and one plant was not subjected to any treatment and was designated as control. Three replicates were performed. The wounding treatment was carried out by using needle punctures to simulate the mechanical damage caused by insect feeding. The phytohormones salicylic acid (SA), methyl salicylate (MeSA) and stress factor superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities as well as malondialdehyde (MDA), soluble sugars (SS), soluble protein (SP), proline (PRO), H₂O₂, and O₂ ⁻ were analyzed by Wuhan ProNets Testing Technology Co. Ltd. Briefly, after different treatments, phytohormone extraction was performed and analyzed with a high‐performance liquid chromatography‐tandem mass spectrometry system (QTRAP5500). The stress factors were extracted and analyzed with a Spectra Max Single Mode Reader (ABS Plus), by measuring the absorbance value of the extract at a specific wavelength, the enzymatic activity and content can be calculated (Wang et al. 2024; Xu et al. 2019). To measure the expression levels of marker genes in SA pathway, the TB Green Premix Ex Taq Ⅱ (Takara) was used for the qRT‐PCR, with the following protocol: 94°C for 3 min, 94°C for 20 s of 40 cycles, and 60°C for 34 s in Real‐Time PCR system (Thermo). Fold change was calculated by 2^−ΔΔCt method (Livak and Schmittgen 2002). Primers are shown in supporting materials (Table S1).

2.11. Host Plant Susceptibility Bioassays

Seedlings of cucumber (aged 5 weeks) were cultured in a greenhouse and sprayed with the different concentrations (0.01, 0.1, and 1 mmol/L) of methyl‐salicylate (MeSA) solutions. The seedlings were sprayed with water as controls. Clip cages were installed on the abaxial surface of a cucumber leaf. Then, 10 female adult whiteflies were released into the clip cages on MeSA‐treated and control plants. Each treatment was repeated 6 times. Two days after the release of the whiteflies, the number of live whiteflies and eggs deposited on the leaf within the clip cages were counted.

For the choice test, the insect cage (80 × 60 × 60 cm) was used, with one MeSA treatment and one control cucumber plants placed on cross sides. Then, 100 adult whiteflies were released between the two plants, and after 10 min the number of adults that landed on each plant was counted. Six replicates were performed (He et al. 2023).

3. Results and Discussion

3.1. Protein Extraction and Identification

The SDS‐PAGE profiles of protein samples of cucumber leaves after feeding by whitefly B. tabaci extracted from trypsin digestion showed no degradation of proteins during proteomic analysis, and high‐abundance proteins were evident (Figure 1A). We used 4D DIA proteomic method to further understand the proteomic profile of cucumber leaves after feeding by whitefly B. tabaci. In the present study, DIA data were retrieved using DIA‐NN (v1.8). The database is Whitefly Genome Database (http://www.whiteflygenomics.org/ftp/MED/). The false discovery rates (FDR) of total peptides, unique peptides and identified proteins were 1%, with quantities of 10,541, 9416, and 2625, respectively (Figure 1B). 10,569 (99.4%) peptides were distributed in 7–20 amino acids in length (Figure 1C), which conforms to the general rules of enzymatic hydrolysis and mass spectrometry cleavage. As shown in Figure 1D, 1853 (70.6%) of proteins were comprised of at least 2 peptides. A protein corresponding to multiple specific peptide segments (or corresponding to multiple spectra) is conducive to increasing the reliability of protein identification and the accuracy of protein intensity values. Overall, 2625 whitefly salivary proteins were identified by the DIA approach (Table S2). The distribution of molecular weight and peptide length is consistent with the properties of trypsin digestion. In addition, the identified proteins had effective sequence coverage, with 923 proteins (35.2%) having sequence coverage greater than 10% (Figure 1E). The distribution of peptide segment lengths, the peptide segment quantities, and the protein coverage all meet the quality control requirements. Compared with the previous research results, we identified more saliva proteins, which might be related to the differences in sampling and identification methods. Furthermore, insects feed on different hosts, and the types and quantities of their saliva secretions also vary significantly (Huang et al. 2021). These results show that the method applied to DIA analysis in this study has good feasibility and validity.

Figure 1.

SDS‐PAGE and proteome analysis of cucumber leaves after feeding by whitefly. (A) Protein pattern of protein samples. The protein bands are clear, evenly distributed, and there is no degradation; (B) Identified peptides and proteins. Based on the specific peptide segments of the protein, the protein information is identified; (C) Distribution of peptides length distribution. 99.4% peptides were distributed in 7–20 amino acids in length, which conforms to the general rules of enzymatic hydrolysis; (D) Distribution of peptide segments. 70.6% proteins were comprised of at least two peptides; (E) Distribution of protein coverage. 923 proteins (35.2%) having sequence coverage greater than 10%.

The presence of these secreted proteins suggests they might play an important role in insect saliva functions, including digestion and modulating plant defense mechanisms (Kehr 2006; Carolan et al. 2011; Huang et al. 2021). We identified 2 glucosidases (BTA029276.1 and BTA025527.1) and 9 proteases (BTA001332.1, BTA011326.1, BTA013048.1, etc.) that may participate in carbohydrate and protein digestion. Furthermore, 22 dehydrogenases, such as Glyceraldehyde‐3‐phosphate dehydrogenase (BTA002410.2), NADH dehydrogenase (BTA022664.1), Short‐chain specific acyl‐CoA dehydrogenase (BTA003357.1), and 3 disulfide‐isomerases (BTA020552.1, BTA020952.3 and BTA002408.1) were identified. Among them, the dehydrogenases were closely related to the biosynthesis of abscisic acid, as well as inhibiting the outbreak of H₂O₂ in hosts (Wang et al. 2024; Cheng et al. 2002; Smith and Boyko 2007). In fungi‐plant interactions, disulfide‐isomerases have been hypothesized to have chaperone effects and participate in plant disease signaling pathways (Ray et al. 2003). Enzymes found in whitefly secretions could potentially influence plant cell signaling pathways and serve as elicitors of stress responses. In addition, we have also identified some previously reported effectors, such as Fer1 (BTA011657.1), Vg (BTA017585.2), and GAPDH (BTA002410.2), etc. The presence of these secreted proteins suggests they might play an important role in insect saliva functions, including digestion and modulating plant defense mechanisms (Su et al. 2019; Huang et al. 2021; Wang et al. 2024).

3.2. GO Functional Enrichment Analysis

For GO analysis, including three major categories were used to distinguish differentially abundant proteins (DAPs) (Figure 2A, Table S3). Regarding biological process (BP), the organic substance metabolic processes, cellular metabolic processes, and primary metabolic processes were the top three GO terms, which are primarily related to biochemical metabolism. The most abundant terms in the cellular component (CC) category were intracellular anatomical structure, organelles, and cytoplasm. In addition, the protein binding was the richest term in the molecular function (MF) category. Further clustering analysis based on the functional classification of DAPs was conducted using Fisher's exact test. The differences in functional enrichment among the BP, CC, and MF categories are illustrated in Figures 2B, 2C, and 2D, respectively. Specifically, the BP category is predominantly associated with ribonucleoside monophosphate metabolic processes, RNA metabolic processes, catabolic processes, and certain biosynthetic processes. In the CC category, significant enrichment is observed for functions related to ribonucleoprotein complexes, mitochondria, ribosomes, and other membrane‐associated functions. Functions significantly enriched in the MF category include RNA binding, structural molecule activity, ribosomal structural constituent, and other nucleoside binding‐related activities. These findings collectively reinforce previous research suggesting that the primary salivary glands may actively participate in metabolism, transport, and the synthesis of binding proteins (Su et al. 2012).

Figure 2.

Gene ontology (GO) analysis of the salivary proteins. (A) Functional classification of the proteins, where the X‐axis shows the number of proteins and the Y‐axis lists the GO terms; (B) Enrichment analysis of GO terms related to biological processes; (C) cellular components; and (D) molecular functions. In the bubble charts, the vertical axis provides the functional description of GO terms, while the horizontal axis indicates the log₂‐transformed fold enrichment, with higher values signifying greater enrichment. The color of the points reflects the significance of the enrichment p value. The size of the points represents the number of proteins identified in the GO function.

3.3. KEGG Pathway Enrichment Analysis

KEGG pathway analysis revealed a total of 33 pathways associated with the identified protein species (Figure 3A, Table S4). This indicates a broad range of biological processes and functions that these proteins are involved in. Notably, key pathways included transport and catabolism, signal transduction, protein folding, sorting and degradation, translation, transcription, carbohydrate metabolism, energy metabolism, and endocrine system processes (Figure 3A). KEGG pathway enrichment analysis indicated that the proteasome (map03050), ribosome (map03010), nucleocytoplasmic transport (map03013), DNA replication (map03030), and spliceosome (map03040) were the most significantly enriched pathways, characterized by a higher number of expressed proteins. Among them, map03050 proteasome have the highest enrichment levels and lower p‐values (Figure 3B, Table S5). The main function of the salivary glands is saliva secretion, leading to an abundance of ontologies and pathways related to transport, metabolism, and secretion processes. Hemipteran saliva mainly comprises water, electrolytes, lipids, amino acids, and proteins (Miles 1999). The secretion of fluids and electrolytes involves multiple types of transporters, such as Na⁺/K⁺/Cl^– co‐transporters and V‐ATPases (Turner and Sugiya 2002; Walz et al. 2006). Consequently, the enrichment of transport‐related proteins is crucial for regulating secretion. In addition to fluid and electrolyte secretion, macromolecular secretion is also important. Our findings highlight the prominence of membrane transport and nucleocytoplasmic transport, which may be integral to intracellular transport and the extracellular secretion of vesicles. Proteins enriched in these categories could also facilitate the spread of plant viruses (Su et al. 2012). Future studies into how these proteins contribute to viral transmission might lead to new strategies for virus prevention and control.

Figure 3.

Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the salivary proteins. (A) KEGG pathway enrichment analysis of the proteins; (B) The analysis of enriched KEGG pathways. The pathway descriptions on the Y‐axis and the log₂‐transformed fold enrichment on the X‐axis, with higher numbers indicating higher enrichment levels.

3.4. Analysis of Proteins Related to Metabolism

The primary salivary gland is a special organ that secretes salivary macromolecules and has high metabolic activity. This feature was illustrated by observing the dense cytoplasm in salivary gland cells and the well‐organized whorl structures of the rough endoplasmic reticulum (Ghanim et al. 2001). Coincidentally, GO terms and KEGG pathways enrichment analysis indicated that salivary proteins significantly contribute to metabolic processes and regulatory functions (Tables S3 and S5). For instance, protein processing within the endoplasmic reticulum is crucial for the proper folding of newly synthesized proteins. The proteasome, which serves as the primary molecular machinery for protein degradation in eukaryotic cells, exhibits a range of proteolytic activities, and participates in almost most of the life activities of organisms through specific degradation of target proteins (Goldberg 2003). The 26S proteasome consists mainly of 19S regulatory particles and 20S core particles. The 19S regulatory particle is tasked with recognizing protein substrates labeled with ubiquitin chains for de‐folding, and ultimately delivering the de‐folded protein substrate to the 20S core particle for degradation (Baugh et al. 2009). Among these, 19S regulatory particles‐related proteins Rpn1, Rpn2, Rpn3, Rpn4, Rpn5, Rpn6, Rpn7, Rpn9, Rpn12, and Rpn13, as well as 20S core particles proteins such as α1, α2, α5, α6, α7, β1, β3, β4, β5, β6, and β7 were highly expressed in the saliva (Figure 4). These results suggest that the types of identified proteins on salivary proteins are closely related to hydrolase activity and metabolic pathways, which was consistent with the results of previous studies (Huang et al. 2021).

Figure 4.

The differentially abundant proteins (DAPs) involved in proteasome of salivary proteins of whitefly Bemisia tabaci. The total proteins were identified in the colored background frame. The red fill in the figure indicates the identified protein. (With the update of the KEGG website, the color of the comment may be different from the box in the image).

3.5. Protein‐Protein Interaction (PPI) Network Analysis

The identified proteins were compared against the STRING database (http://string-db.org/), visualization of this interaction network was achieved using the R package “visNetwork.” As shown in Figure 5. Each circle symbolizes an identified protein, with its size reflecting the number of interactions it has. To clearly show the interactions between proteins, we selected the top 50 proteins with the closest interactions and drew the protein interaction network (Table S6). The results of this study indicated that the salivary proteins of whiteflies have functional diversity and can participate in the interaction process of multiple proteins, which will contribute to understanding the important role of salivary proteins in insect‐host interactions.

Figure 5.

Protein‐protein interaction (PPI) network analysis revealed the salivary proteins interaction network. Each protein can interact with one or more other proteins, forming a complex network. The interaction network reveals that salivary proteins have diverse functions and can participate in multiple interactions.

3.6. Whitefly Infestation Promotes Plant Defense

To investigate the roles of salivary proteins of whitefly in cucumber plants, we measured the defense level of cucumbers against the whitefly after a 2‐d exposure. POD, SOD, and CAT are important protective enzymes in plants. They regulate the system through feedback mechanisms, maintaining normal levels of free radicals within the plant and thereby enhancing its resistance to pests. H₂O₂, as one of the most abundant reactive oxygen species (ROS) in cells, is a key signaling molecule for plant growth and development as well as for resisting adverse stress. MDA (a metabolite of H₂O₂), PRO, SS, SP, and O₂ ⁻ are commonly used indicators in the physiological research of plant resistance. It was found that exposure to whitefly significantly increased the levels of SOD (Figure 6A; F _1,4 = 6.604, p < 0.001), POD (Figure 6B; F _1,4 = 0.434, p < 0.001), CAT (Figure 6C; F _1,4 = 0.063, p < 0.001) activities as well as MDA (Figure 6D; F _1,4 = 4.997, p < 0.001), SS (Figure 6E; F _1,4 = 0.007, p < 0.001), SP (Figure 6F; F _1,4 = 0.019, p = 0.043), and PRO (Figure 6G; F _1,4 = 0.000, p = 0.039) in cucumber plants, whereas the levels of H₂O₂ (Figure 6H; F _1,4 = 0.099, p = 0.142) and O₂ ⁻ (Figure 6I; F _1,4 = 5.017, p = 0.658) were not significantly changed. These data suggest that cucumber defense against whitefly, after a 2‐d exposure, involves both the activities of SOD, POD, and CAT, as well as the levels of MDA, SS, SP, and PRO in cucumber plants metabolism responses.

Figure 6.

Whitefly infestation promotes plant defense. Defense response induced by whitefly infestation in cucumber plants, as determined by the contents of SOD (A), POD (B), CAT (C) activities as well as MDA (D), SS (E), SP (F), PRO (G), H₂O₂ (H), and O₂ ⁻ (I). Values in bar plots represent mean ± SE. Asterisks indicate significant difference using a Student's t‐tests (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference).

3.7. Whitefly Infestation Improves Cucumber Plants Defense by Activating SA Signaling

To further confirm the effect of whitefly infestation on cucumber defenses, and exploring the functional relationship between whitefly saliva and the SA pathway induction. we measured the levels of SA, and MeSA in cucumber plants after wounding treatment, whitefly infested and wounding treatment plus whitefly infested. After treatment for 2‐d, the levels of SA (Figure 7A; F _3,8 = 1423.05, p < 0.001) and MeSA (Figure 7B; F _3,8 = 231.28, p < 0.001) in whitefly infested cucumber and wounding treatment plus whitefly infested cucumberwere significantly higher than that in control and wounding treatment cucumber. The expression of the SA response genes pathogenesis‐related protein 1a (PR1a) (Figure 7C; F _1,4 = 10.108, p = 0.004), pathogenesis‐related 3 (PR3) (Figure 7C; F _1,4 = 12.705, p = 0.003), and pathogenesis‐related 5 (PR5) (Figure 7C; F _1,4 = 9.971, p = 0.009) were significantly induced in infested cucumber, but not nonexpresser of PR genes 1 (NPR1) (Figure 7C; F _1,4 = 8.523, p = 0.998). It is possible that NPR1 levels fluctuate rapidly and are not detected at the specific time point examined or the induction of PR genes could be a result of crosstalk, where other signaling molecules or pathways compensate for the lack of NPR1 activation.

Figure 7.

Whitefly infestation improves plant resistance to whitefly by activating SA signaling. The levels of SA (A), and MeSA (B) in the control, wounding treatment, wounding treatment plus whitefly infested and whitefly infested cucumber plants were measured, whiteflies were allowed to feed on cucumber plants for 2 d. (C) Expression levels of marker genes in SA signaling pathway of control and infested cucumber plants: pathogenesis‐related protein 1a (PR1a), pathogenesis‐related 3 (PR3), pathogenesis‐related 5 (PR5), and nonexpresser of PR genes 1 (NPR1). Data were calculated using the 2^−ΔΔCt method. The effect of exogenous MeSA application on whitefly fecundity (D), survival (E) and preferences choice (F). Values in bar plots represent mean ± SE. Asterisks indicate significant difference using a Student's t‐tests (A–C) (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference). Statistical analysis was performed using a one‐way ANOVA with the least significant difference (LSD) test (p < 0.05) (D, E) and χ ² tests (F).

To examine whether the increased MeSA in cucumber affects the performance of the whitefly, we examined the effect of exogenous MeSA treatment on whitefly survival and fecundity. Following MeSA treatment, the fecundity (Figure 7D; F _3,20 = 347.515, p < 0.001) and survival (Figure 7E; F _3,20 = 39.656, p < 0.001) of whiteflies on the MeSA‐treated cucumber were significantly lower than that on control cucumber plants. We next compared whitefly attractiveness in exogenous MeSA‐treated cucumber and control plants, we found that they showed no significantly difference in whitefly preference when spraying 0.01 mmol/L MeSA (Figure 7F; x ² = 0.195, p = 0.659), but there were fewer whiteflies chose to cucumber plants spray 0.1 mmol/L MeSA (Figure 7F; x ² = 4.154, p = 0.042) and 1 mmol/L MeSA (Figure 7F; x ² = 9.720, p = 0.002). Collectively, these data suggest that whitefly infestation increases the resistance of cucumber probably by activating the SA signaling pathway.

It is worth noting that different plants adopt different defense strategies when infested by the insects. This might be related to the differences in the components of the saliva secreted. Whitefly is a polyphagous pest that can damage various plants. Our research has provided potential clues suggesting that saliva proteins may play a role in the ability of whitefly to feed on different plants, which deserves further investigation.

4. Conclusions

Whitefly B. tabaci is an agricultural super pest, requiring extensive researches and innovative management strategies to minimize its impact on global food security and agricultural productivity. With the use of pesticides, whiteflies have developed high resistance to pesticides, people urgently need new methods to control the damage caused by the whiteflies. Defense mechanisms involved in the production of callose and reactive oxygen species (ROS) and gene expression associated with SA and JA pathway have been identified as potential control strategies. SA is an important signaling molecule that regulates the stress response of plants. It can induce various stress resistances of plants, including pest resistance. Salivary proteins from herbivorous insects are central to their interaction with plants, mediating processes from nutrient acquisition to manipulation of plant defenses. These proteins can inhibit plant defense as effectors or induce plant defense as elicitors. In this study, the DIA‐based proteomic methodologies analysis has proven effective in identifying and characterizing whitefly secreted saliva proteins. A total of 2625 salivary proteins were identified.

The results from GO and KEGG analyses suggest that these proteins may play roles in hydrolysis, transport, protein binding and metabolism, and participate in host plant defense responses by activating the SA signaling pathway. Exogenous MeSA treatment of cucumber plants can significantly reduce the reproductive capacity and survival rate of whiteflies, and also decrease the preference of whiteflies for host plants. This approach not only reveals the complexity of the whitefly salivary proteome but also provides valuable insights into the molecular mechanisms underlying the interaction between whiteflies and their host plants. These findings are crucial for understanding the biology of whiteflies and could potentially lead to the development of novel pest control strategies.

Author Contributions

Haifang He: conceptualization, methodology, investigation, data curation, software, funding acquisition, writing – original draft. Long Liu: methodology, data curation, software. Shuixiang Xie: investigation, validation. Baozheng Shi: investigation, validation. Jialei Liu: investigation, software. Guangliang Lu: methodology, software. Jingjing Li: methodology, software. Chenchen Zhao: software. Lin Niu: data curation. Rune Bai: project administration, funding acquisition. Caiyan Lei: software. Menghan He: methodology, software, writing – original draft. Qingbo Tang: conceptualization, methodology, writing – review and editing. Fengming Yan: funding acquisition, supervision, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

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He, H. , Liu L., Xie S., et al. 2026. “Salivary Proteins of Whitefly Bemisia tabaci Modulate Plant Defenses by Activating SA Signaling Pathway.” Archives of Insect Biochemistry and Physiology 122: e70202. 10.1002/arch.70202.

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.