SmCSP4 from aphid saliva stimulates salicylic acid‐mediated defence responses in wheat by interacting with transcription factor TaWKRY76

Summary Aphid salivary proteins are critical in modulating plant defence responses. Grain aphid Sitobion miscanthi is an important wheat pest worldwide. However, the molecular basis for the regulation of the plant resistance to cereal aphids remains largely unknown. Here, we show that SmCSP4, a chemosensory protein from S. miscanthi saliva, is secreted into wheat plants during aphid feeding. Delivery of SmCSP4 into wheat leaves activates salicylic acid (SA)‐mediated plant defence responses and subsequently reduces aphid performance by deterring aphid feeding behaviour. In contrast, silencing SmCSP4 gene via nanocarrier‐mediated RNAi significantly decreases the ability of aphids to activate SA defence pathway. Protein–protein interaction assays showed that SmCSP4 directly interacts with wheat transcriptional factor TaWRKY76 in plant nucleus. Furthermore, TaWRKY76 directly binds to the promoter of SA degradation gene Downy Mildew Resistant 6 (DMR6) and regulates its gene expression as transcriptional activator. SmCSP4 secreted by aphids reduces the transcriptional activation activity of TaWRKY76 on DMR6 gene expression, which is proposed to result in increases of SA accumulation and enhanced plant immunity. This study demonstrated that SmCSP4 acts as salivary elicitor that is involved in activating SA signalling defence pathway of wheat by interacting with TaWRKY76, which provide novel insights into aphid‐cereal crops interactions and the molecular mechanism on induced plant immunity.


Introduction
Plants have evolved complex and accurate defence strategies to prevent or limit damage from herbivorous insects and microbial pathogens (Jones and Dangl, 2006).During pathogen or insect attack, plants respond by producing phytohormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), which act as signalling molecules to stimulate downstream defence responses in plants (Berens et al., 2017).In general, chewing insects and necrotrophic pathogens mainly activate JA/ET-mediated defences, whereas hemipterans and biotrophic pathogens primarily induce SA-mediated defence responses (Z€ ust and Agrawal, 2016).
Plants can rapidly and precisely perceive herbivore cues to activate defences against their attack.Herbivore-associated molecular patterns (HAMPs) or elicitors in the oral secretions of herbivores play important roles in eliciting plant defences (Acevedo et al., 2015).Several HAMPs or elicitors have been identified in the regurgitant and/or saliva of chewing insects including fatty acid conjugates (e.g., volicitin), caeliferins, bruchins and the plant-derived peptides called inceptins (Alborn et al., 1997(Alborn et al., , 2007;;Schmelz et al., 2006).
Aphids are important agricultural and forest pests that cause damage by directly drawing phloem sap from sieve tubes and by acting as vectors to transmit various plant viruses (Blackman and Eastop, 2000).During the penetration of plant cells, the stylets of aphids essentially follow an intercellular pathway to reach the sieve elements (Silva-Sanzana et al., 2020;Tjallingii and Esch, 1993).At the early stage of feeding, aphids secrete gelling saliva, which hardens to form a protective sheath around the stylets; subsequently aphids secrete watery saliva containing a number of proteins, which are injected into the plant cells (Cherqui and Tjallingii, 2000;Miles, 1999).To date, many studies have demonstrated that aphid saliva performs important roles in mediating the interactions between aphid and host plant (Elzinga and Jander, 2013;Yates and Michel, 2018).For example, the infiltration of salivary components in the 3-10 kDa fraction obtained from green peach aphid (Myzus persicae) induced the expression of defence-related genes in Arabidopsis thaliana and activated resistance against aphids, demonstrating the defenceeliciting activity of aphid salivary components (De Vos and Jander, 2009).Moreover, green peach aphid Myzus persicae salivary proteins Mp10, Mp42, Mp56, Mp57 and Mp58 decreased aphid reproduction when transiently expressed in tobacco plants Nicotiana benthamiana, highlighting their potential roles in activating plant defences (Bos et al., 2010;Rodriguez et al., 2014).Similarly, overexpression of salivary protein Armet secreted by pea aphid Acyrthosiphon pisum and infiltration of its purified form in N. benthamiana primarily induced SA-mediated defence responses, which enhanced plant resistance to the bacterial pathogen Pseudomonas syringae (Cui et al., 2019).Buchnera aphidicola-derived molecular chaperone proteins GroEL and GroES were also identified as elicitors that betray aphids by triggering the plant defence response (Chaudhary et al., 2014;Li et al., 2022).However, little is known about the mechanisms adopted by host plants to recognize the HAMP-like molecules or elicitors secreted by aphids.
Like pathogen effectors, salivary effectors have also been identified in aphid saliva, which suppress plant defence responses and contribute to plant susceptibility, thus promoting aphid virulence.The salivary protein C002 of A. pisum is the first wellcharacterized aphid effector, which is essential for continuous phloem feeding (Mutti et al., 2006(Mutti et al., , 2008)).In addition, transient overexpression of genes encoding the MpC002 protein of M. persicae and the Me10 and Me23 proteins of potato aphid Macrosiphum euphorbiae in N. benthamiana resulted in a significant increase in aphid fecundity, implying that these effectors suppress plant defence (Atamian et al., 2013;Bos et al., 2010;Elzinga et al., 2014;Kettles and Kaloshian, 2016).Ectopic expression of bird cherry-oat aphid Rhopalosiphum padi genes encoding candidate effectors RpC002 and Rp1 in barley Hordeum vulgare enhanced susceptibility to aphids by inhibiting the expression of defence-related genes (Escudero-Martinez et al., 2020).
The chemosensory proteins (CSPs) of insects mainly function as carriers of pheromones and odorants and are typically expressed in diverse chemosensory organs, including antennae, mouthparts, and other chemosensory structures (Pelosi et al., 2018).CSPs have been identified in various body parts of insects, including the thorax, legs and reproductive organs, and play a role in insect development, mating, leg regeneration, immune responses, pesticide resistance, and mediating interactions with the defence-related molecules of the host plant (Peng et al., 2021;Stathopoulos et al., 2002).CSPs have also been identified in the salivary glands and watery saliva of several hemipteran insects, such as M. persicae, R. padi, black cherry aphid Myzus cerasi and brown planthopper Nilaparvata lugens (Ji et al., 2013;Thorpe et al., 2016).When transiently expressed in N. benthamiana, the gene encoding Mp10, a CSP detected in M. persicae saliva, inhibited flg22-induced defence responses but induced chlorosis and local cell death, resulting in a significant reduction in aphid fecundity (Bos et al., 2010;Rodriguez et al., 2014).
Grain aphid Sitobion miscanthi, which was wrongly designated previously as English grain aphid Sitobion avenae in China, is one of the major insect pests of cereal crops worldwide and the most dominant aphid species in the major wheat Triticum aestivumgrowing regions of China (Jiang et al., 2019;Zhang, 1999).S. miscanthi can cause huge yield losses by directly drawing phloem sap and transmitting various plant viruses, such as barley yellow dwarf virus (Blackman and Eastop, 2000).S. miscanthi infestation mainly activates the SA-associated defence signalling pathway in wheat (Huang et al., 2019), and the watery saliva secreted by S. miscanthi induces the expression levels of several SA-related genes, thus enhancing resistance against aphids (Zhang et al., 2017a).Four CSP genes have been identified in S. miscanthi based on the transcriptome profiling of its salivary glands (Zhang et al., 2017b).However, the roles and molecular mechanisms of SmCSPs in modulating wheat defence responses remain unknown.Here we identified a secreted CSP of S. miscanthi, SmCSP4, which can effectively activate SA-mediated plant defence response and enhance wheat resistance against aphids.We demonstrate that SmCSP4 interacts with the TaWRKY76 transcription factor of wheat in the plant cell nucleus and suppresses the SA degradation pathway by inhibiting the binding of TaWRKY76 to the downy mildew resistant 6 (DMR6) gene promoter.

Identification and characterization of SmCSP4
SmCSP4 is 441 bp in length and is predicted to encode a 146amino acid protein, with a molecular weight of 16.54 kDa and a signal peptide (SP) at its N terminus.Phylogenetic analysis revealed that SmCSP4 is the most closely related to the A. pisum CSP4 (ApisCPS4) protein (Figure S1A).Sequence alignment showed that SmCSP4 contains four conserved cysteine residues (CX 6 CX 18 CX 2 C) and exhibits high similarity to the homologues from A. pisum, M. persicae, and A. gossypii (Figure S1B).The results of RT-PCR and RT-qPCR indicated that SmCSP4 was expressed in different tissues of aphids, including the whole body, head, salivary gland, leg, abdomen and thorax, and its expression was significantly higher in legs compared with the other tissues (Figure 1a,b).The transcript level of SmCSP4 in aphids significantly increased after feeding on wheat plants, but the gene exhibited a higher fold change after feeding on the aphid-resistant than on the aphid-susceptible wheat variety (Figure 1c).
A nuclear localization signal (NLS) sequence from 126 to 142 amino acids was predicted at the C-terminal end of SmCSP4 (Figure 1d).To further confirm the subcellular localization of SmCSP4, the SmCSP4-GFP fusion, SmCSP4 NLSm -GFP with mutant NLS fusion or GFP alone (control) was transiently expressed in N. benthamiana leaves.In the control group expressing only GFP under the control of the cauliflower mosaic virus (CaMV) 35S promoter, fluorescence was detected throughout the cytoplasm and nucleus.However, in leaves transformed with the SmCSP4-GFP fusion, fluorescence was observed only in the nucleus, indicating that SmCSP4 localizes to the nucleus.However, fluorescence was observed in both cytoplasm and nucleus of leaves expressing SmCSP4 NLSm -GFP (Figure 1d).

Aphids secrete SmCSP4 into the plant cell during feeding
SmCSP4 is believed to be a component of aphid watery saliva.Western blotting was performed to verify that the SmCSP4 was secreted into plant cells.As shown in Figure 2, the results revealed that SmCSP4 was present in the whole body of S. miscanthi and in aphid-infested wheat leaves, but was not detected in wheat leaves without aphid infestation, these results suggested that SmCSP4 is secreted by aphids into wheat plants during feeding.

SmCSP4 induces SA-mediated plant immunity
Wheat is the major host plants of S. miscanthi.To directly determine whether SmCSP4 of S. miscanthi is capable of modulating wheat plant defence, the SmCSP4 or SmCSP4 NLSm was delivered into wheat leaves using the P. fluorescens EtAnH-mediated delivery system.The infiltration of EtAnH expressing SmCSP4 induced slight chlorotic symptoms and resulted in a significant H 2 O 2 accumulation in the infiltrated region of leaves, and also, the H 2 O 2 content in SmCSP4-treated ª 2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407 leaves was significantly higher than that in SmCSP4 NLSminfiltrated groups (Figure 3a,b).In addition, significantly more callose deposits were detected in wheat leaves expressing SmCSP4 or SmCSP4 NLSm when compared to controls, but the number of callose deposits in SmCSP4 NLSm -treated leaves was lower than that in leaves delivered with SmCSP4 (Figure 3c).
Gene expression analysis showed that the JA-responsive gene FAD was significantly down-regulated in SmCSP4 or SmCSP4 NLSm -delivered wheat leaves at 2 dpi, and the expression levels of LOX had no significant changes when compared to control.However, the SA biosynthesis gene PAL and SA pathway marker gene PR1 were all significantly up-regulated in wheat leaves delivering with SmCSP4 or SmCSP4 NLSm (Figure 3d).In addition, the delivery of SmCSP4 or SmCSP4 NLSm into wheat leaves significantly increased the SA levels at 2 dpi compared with the control, but no significant change was observed in the JA levels.Moreover, the expression levels of PR1 and SA levels in SmCSP4-treated leaves were significantly higher than those in SmCSP4 NLSm -treated leaves (Figure 3e).The delivery of SmCSP4 and SmCSP4 NLSm into wheat leaves was confirmed using Western blot (Figure S2).These results suggest that SmCSP4 is involved in the activation of SA-associated defence pathways in wheat plants, and NLS domain of SmCSP4 is important for the induction of wheat SA-mediated plant defence.

SmCSP4 expression in wheat enhances plant resistance against S. miscanthi
The survival rate of aphids on SmCSP4-infiltrated wheat leaves was significantly lower than that on control leaves (Figure 4a).In addition, the fecundity and weight of aphids feeding on SmCSP4infiltrated wheat leaves were significantly decreased compared with the control groups (Figure 4b,c).
Moreover, the feeding behaviour of aphids on wheat leaves infiltrated with SmCSP4 was negatively affected (Figure 4d,e).The duration of phloem ingestion (E2) was significantly lower in wheat leaves infiltrated with SmCSP4 than in leaves infiltrated with the DsRed (control).Contrarily, the durations of stylet probing activity (C) waveforms in SmCSP4-expressed leaf areas were significantly higher than those in the control groups.These results indicate that SmCSP4 reduces aphid performance and enhances host resistance against aphids.
We also found that the SA content showed a significant increase in wheat leaves infested with S. miscanthi; however, this increase in SA content was greater in the aphid-resistant than in the aphid-susceptible wheat variety (Figure S3A).Exogenous SA application up-regulated the transcript levels of SA-responsive genes (PAL, PR1 and PR5) and induced significant callose deposition in wheat leaves (Figure S3B,C).No significant differences were observed in the survival rates of aphids feeding on SA-treated wheat leaves compared to the control, but the number of nymphs produced by aphids was significantly reduced after feeding on SA-treated plants (Figure S3D).These results suggest that the SmCSP4-induced increase in SA levels enhanced wheat resistance against aphids.
CSP4 orthologs from other aphid species enhance SA levels and plant resistance More callose deposits were observed in wheat leaves expressing CSP4 orthologs from M. persicae (MpCSP4), A. pisum (ApCSP4) and cotton aphid (Aphis gossypii) (AgCSP4) (Figure 5a).In addition, the SA content significantly increased in wheat leaves delivering with MpCSP4, ApCSP4 and AgCSP4 at 2 dpi (Figure 5b).Moreover, aphid fecundity significantly decreased after feeding on wheat leaves expressing these three CSP4 compared with the controls (Figure 5c).This suggests that aphid CSP4 orthologs possess conserved functions that elicit plant defence.
Silencing of SmCSP4 via nanocarrier-mediated RNAi activates weaker SA-associated defence responses but reduces aphid fecundity A schematic representation of nanocarrier star polycation (SPc)based transdermal dsRNA delivery is presented in Figure 6a.The transcript level of SmCSP4 was significantly reduced in S. miscanthi treated with 500 ng/lL dsRNA of SmCSP4 for 24 h.After 48 h of dsRNA treatment, the expression level of SmCSP4 further decreased to 0.28-fold, which was significantly lower than the transcript levels observed in the control (Figure 6b).SmCSP4-silenced aphids induced less SA production in wheat leaves compared with the control groups; however, infestation by SmCSP4-silenced aphids had no significant effects on the JA content of wheat leaves (Figure 6c).
Effects of SmCSP4 silencing on aphid survival, fecundity and olfactory behaviour were also investigated.As presented in Figure 6d,e, SmCSP4 knockdown had no significant effects on S. miscanthi survival rate but significantly reduced their fecundity compared with the control (dsGFP).Furthermore, the olfactory behaviour assay results indicated SmCSP4 is not involved in the recognition of chemical cues that the repellent effect of aphid alarm pheromone (E)-b-farnesene (EBF) and attractive activity of wheat plant volatiles on S. miscanthi was not affected by SmCSP4 knockdown (Figure S4).These results suggested that SmCSP4 is essential for aphid fitness but also involved in the induction of SA defence in wheat as elicitor.

SmCSP4 interacts with TaWRKY76 in plant cell nucleus
To identify the SmCSP4-interacting proteins, a Yeast two-hybrid (Y2H) assay was conducted using SmCSP4 as the bait to screen an aphid-infested wheat cDNA library.Ten CDS full sequences of putative positive targets of SmCSP4 were obtained preliminary.Sequence analysis revealed that all these identified candidate SmCSP4-interacting proteins were 26S proteasome non-ATPase regulatory subunit 14 homologue (GenBank accession no.XM_044500535) and transcription factor TaWRKY76 (GenBank accession no.XM_044541645).As SmCSP4 localizes to the nucleus, we focused on the transcriptional factor of wheat TaWRKY76.Phylogenetic analysis of the predicted amino acid sequences of Arabidopsis WRKY proteins revealed that TaWRKY76 clustered into group IIa of the WRKY superfamily (Figure S5).Full-length TaWRKY76 CDS was used to generate the prey construct to confirm the interaction between TaWRKY76 and SmCSP4 via Y2H assays (Figure 7a).Subcellular localization of TaWRKY76 was examined by transiently expressing the   TaWRKY76:GFP fusion in N. benthamiana leaves.The fluorescence signal of TaWRKY76-GFP was observed only in the nucleus of N. benthamiana cells (Figure 7b), indicating that TaWRKY76 localizes to the nucleus.
The SmCSP4-TaWRKY76 interaction in plant cells was verified by performing bimolecular fluorescence complementation (BiFC) assays.SmCSP4-nYFP and TaWRKY76-cYFP were co-expressed in N. benthamiana protoplasts.YFP signal was observed in the nucleus of N. benthamiana protoplasts but not in the cytoplasm (Figure 7c), indicating that SmCSP4-nYFP and TaWRKY76-cYFP are localized to and interact in the nucleus of plant cells.We further verified the interaction between SmCSP4 and TaWRKY76 via in vivo co-immunoprecipitation (Co-IP) assays in N. benthamiana leaves agroinfiltrated with SmCSP4-HA and TaWRKY76-MYC or with HA and TaWRKY76-MYC (control).As presented in Figure 7d, in leaves co-infiltrated with SmCSP4-HA and TaWRKY76-MYC, the SmCSP4-HA and TaWRKY76-MYC recombinant proteins could be immunoprecipitated by anti-HA and anti-MYC agarose beads, respectively; however, no target protein was immunoprecipitated in the control groups.These results further confirmed the interaction between SmCSP4 and TaWRKY76 in vivo.

Silencing of TaWRKY76 induces the SA defence response pathway in wheat and reduces aphid performance
To determine the function of TaWRKY76 in modulating SAassociated plant defence, the transcription of TaWRKY76 in the aphid-susceptible wheat cultivar Mingxian169 was downregulated by virus-induced gene silencing (VIGS) using the BSMV vector.Wheat leaves infiltrated with the BSMV:TaPDS recombinant vector exhibited severe photobleaching at 10 dpi but no phenotypic change, suggesting that the VIGS system effectively worked in this study (Figure 8a).Gene expression analysis revealed that the transcript level of TaWRKY76 was significantly reduced at 24 hpi and further decreased to 0.36-fold at 48 hpi (Figure 8b).
SA-responsive genes, including PAL, PR1 and PR5 were significantly up-regulated (Figure 8c), but the expression levels of DMR6, an enzyme involved in SA hydroxylation, the main pathway that mediates SA degradation in plants (Van Damme et al., 2008;Zhang et al., 2017), was significantly decreased in TaWRKY76 knockdown plants.And the SA content of TaWRKY76-silenced wheat leaves was significantly increased compared with the control (Figure 8d).In addition, the fecundity and weight of aphids were significantly reduced after being fed on wheat leaves inoculated with BSMV:TaWRKY76 compared with wild-type plants and wheat leaves inoculated with BSMV:00 (Figure 8e,f), indicating that TaWRKY76 is involved in the suppression of SA-mediated wheat defence responses against S. miscanthi.
SmCSP4 suppresses the transcriptional activation activity of TaWRKY76 on DMR6 expression DMR6 was significantly down-regulated in TaWRKY76-silenced wheat plants as described above, and two potential WRKY binding regions were also identified in the promoter sequence of DMR6 gene (Figure S6).To investigate the transcriptional activity of TaWRKY76 in regulating DMR6 gene expression, we performed Y1H assays.In the transcriptional activity assay, yeast cells harbouring pGBKT7-TaWRKY76 could grow on SD/-Trp/-Leu/-His medium, indicating that TaWRKY76 acts as a transcriptional activator (Figure 9a).Yeast cells co-transformed with AD and pHIS2-proDMR6 or with AD-TaWRKY76 and pHIS2-proDMR6 could grow on SD/-Trp/-His/-Leu medium.However, on SD/-Trp/-His/-Leu medium containing 50-mM 3-AT, positive controls and yeast cells containing AD-TaWRKY76 and pHIS2-proDMR6 could grow well, whereas those carrying AD and pHIS2-proDMR6 failed to grow (Figure 9b), indicating that DMR6 is transcriptionally regulated by TaWRKY76.Dualluciferase assay was then performed to confirm the binding of TaWRKY76 to the DMR6 promoter.The promoter sequence of DMR6 was fused with the LUC reporter gene to construct the proDMR6-LUC vector, and the TaWRKY76 CDS was cloned into pGreenII62-SK to generate the effector plasmid.The relative LUC/REN ratio significantly increased in N. benthamiana leaves infiltrated with pGreenII0800-proDMR6-LUC and pGreenII62-TaWRKY76-SK at a rate nearly sevenfold greater than that of control plants, suggesting that TaWRKY76 specifically binds to the DMR6 promoter in vivo (Figure 9c).
As shown in Figure 9d,e, when SmCSP4 was delivered into wheat leaves, the expression levels of DMR6 were significantly down-regulated.Moreover, the transcript levels of DMR6 were Figure 7 SmCSP4 interacted with TaWRKY76 of wheat.(a) Yeast two-hybrid assays to detect the interaction between SmCSP4 (Bait) and TaWRKY76 (Prey).Gradient concentrations of the yeast cells co-transformed with SmCSP4 and TaWRKY76 were assayed for growth on SD-Trp-Leu-His-Ade LB plates containing X-a-gal and Aureobasidin A (QDO/X/A).Yeast cells co-transformed with pGBKT7-53+pGADT7-T and pGBKT7-Lam+pGADT7-T are used as positive and negative controls, respectively.(b) Subcellular localization of TaWRKY76 in N. benthamiana.The images were taken 2 days after agroinfiltration using confocal microscopy.Bar = 20 lm.(c) BiFC assays for the interaction of SmCSP4 with TaWRKY76 in N. benthamiana protoplasts were introduced with a mixture of SmCSP4-nYFP and TaWRKY76-cYFP constructs.YFP signals were observed at 20 h post-incubation using laser scanning confocal microscope.Infiltration with Agrobacterium expressing the SmCSP4 or TaWRKY76 alone was used as controls.Scale bar = 20 lm.(d) Confirmation of the interaction between SmCSP4 and TaWRKY76 using Co-IP assays.Western blots of total proteins from N. benthamiana leaves transiently expressing the labelled proteins eluted from anti-HA or anti-MYC magnetic beads were detected with the anti-MYC or anti-HA antibody.IP with IgG antibody served as negative control.The sizes of SmCSP4: HA and TaWRKY76: MYC bands were 34 and 55 kDa, respectively.significantly higher in wheat leaves infested with SmCSP4-knockdown aphids when compared to controls.These results indicated that SmCSP4 was involved in the regulation of DMR6 expression in wheat.To further examine whether the (Figure 9f).To verify the histochemical staining, the GUS activity was examined in the injection area of each leaf.The results were consistent with the GUS phenotypic observations (Figure 9f).
Furthermore, the promoter region of DMR6 was fused with the LUC reporter gene to construct the proDMR6-LUC vector, and the CDS of SmCSP4 or TaWRKY76 was cloned into pGreen II 62-SK to generate effector plasmids (Figure 9g).The interaction of TaWRKY76 with proDMR6-LUC led to 6.2-fold increase in the relative LUC/REN ratio in N. benthamiana leaves (Figure 9g).However, the LUC/REN ratio was significantly reduced to only 2.3-fold when SmCSP4 was co-infiltrated with TaWRKY76 and proDMR6-LUC.In summary, these results indicate that TaWRKY76 is a transcriptional activator of DMR6, but SmCSP4 secreted by aphids reduces the transcriptional activation activity of TaWRKY76 on DMR6 expression.

Discussion
The salivary elicitors of aphids, especially those that attack cereal crops, and their mechanism of eliciting plant defence responses have rarely been characterized to date.In this study, SmCSP4, a salivary protein of S. miscanthi was found to be secreted into plant cells during aphid feeding, and the expression level of SmCSP4 was significantly up-regulated during aphid infestation on wheat plants.Previous studies found that Mp10, the CSP4 homologue of M. persicae, was detected in cytoplasm and nucleus of N. benthamiana (Rodriguez et al., 2014).However, the subcellular localization assay in our study proved that SmCSP4 is translocated to the plant cell nucleus.Furthermore, the subcellular localization of SmCSP4 with mutant NLS changed that were detected in both cytoplasm and nucleus of cells, confirming the nuclear localization of SmCSP4.SmCSP4 with NLS mutations could also be detected in plant nucleus, it is proposed that SmCSP4 is a small protein which could be positively diffused into plant nucleus when NLS was mutated.
Sitobion miscanthi is an oligophagous insect and targets wheat as its main host (Blackman and Eastop, 2000).To verify the roles of SmCSP4 in regulating wheat defence, SmCSP4 was delivered into wheat cells using the EtHAn-mediated delivery system, which has been successfully applied to study the functions of effectors of the wheat stripe rust fungus Puccinia striiformis f. sp.tritici (Bai et al., 2021;Cheng et al., 2017;Upadhyaya et al., 2014;Xu et al., 2019b).The delivery of SmCSP4 in wheat leaves induced H 2 O 2 accumulation and increased the number of callose deposits.Moreover, the expression levels of SA pathway-associated genes PAL and PR1 were significantly up-regulated in SmCSP4infiltrated wheat leaves, whereas the expression of JA-responsive genes FAD was inhibited.In addition, the SA content of wheat leaves was significantly increased after SmCSP4 infiltration, whereas the JA content showed no significant change.
Additionally, we found that SmCSP4 with NLS mutant activated lower SA levels, indicating that NLS domain is vital for the eliciting activity of SmCSP4.
RNAi has been commonly used to characterize the function of hemipteran genes encoding salivary proteins (Guo et al., 2020;Xu et al., 2019a).Recently, nanocarrier-mediated RNAi has become a practical strategy for silencing target genes in insects and holds great potential as a pest control strategy (Niu et al., 2021;Yan et al., 2020).In this study, to further examine the potential roles of SmCSP4 in activating SA-mediated plant defence, the SmCSP4 gene was silenced after the application of the nanocarrier-mediated transdermal dsRNA delivery system.Our results indicated that the SmCSP4 expression significantly decreased after the application of dsSmCSP4/nanocarriers/detergent, and its transcript levels reduced by more than 60% compared with the controls, suggesting that nanocarriermediated RNAi is an effective strategy for testing gene function in aphids.After the silencing of SmCSP4, lower SA levels were activated in wheat leaves.Therefore, we concluded that SmCSP4 induces SA-mediated wheat defence responses.However, aphid fecundity was significantly decreased after the silencing of SmCSP4, suggesting that SmCSP4 is essential for aphid fitness on host plants.Previous studies have demonstrated that chemosensory proteins (CSPs) are related to insect xenobiotic detoxification.CSP2 and CSP3 from whitefly B. tabaci show a high binding affinity to a-pentyl-cinnamaldehyde, which is a common chemical in plant oil with toxic effects upon direct contact (Liu et al., 2016).Recently, the high-affinity binding of pyrethroid insecticides revealed that the leg-enriched sensory appendage protein, SAP2, exhibited pyrethroid resistance to malaria mosquito Anopheles gambiae (Ingham et al., 2020).CSP5 protein of A. gossypii and the CSP4 and CSP6 proteins of R. padi play significant roles in improving aphid resistance to insecticides (Li et al., 2021;Peng et al., 2021).A previous study showed that the contents of 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3 (4H)-one (DIMBOA), a toxic plant secondary metabolite in aphidresistant wheat variety Zhong4wumang were significantly higher than those in aphid susceptible wheat varieties (Liu et al., 2002).In our study, SmCSP4 was expressed higher on Zhong4wumang than on susceptible one Mingxian169.It is speculated that S. miscanthi secreted more SmSCP4 to bind specific toxic metabolites in resistant wheat to improve the fitness on hosts.Meanwhile, a coevolutionary arm race between aphid and plant may occur that plant can perceive the secreted SmCSP4 to active stronger SA-mediated defence response to enhance plant resistance against aphids.Whether SmCSP4 is involved in insecticide resistance or other physiological processes of S. miscanthi is worthy of further investigation.
The survival rate and fecundity of S. miscanthi were significantly decreased after feeding on SmCSP4-infiltrated wheat The second leaves wheat cultivar Mingxian169 were inoculated with barley stripe mosaic virus BSMV:00 (Control) and recombinant BSMV:TaPDS, BSMV:TaWRKY76 at two-leaf stage, and the phenotypes were observed and photographed 10 days after inoculation.(b) Expression levels of TaWRKY76 in wheat leaves inoculated with BSMV:TaWRKY76 at 5 days (n = 3).Wide type (WT) and wheat leaves inoculated with BSMV:00 were set as control groups.(c) Expression levels of SA signalling pathway associated genes PAL, PR1, PR5 and DMR6 after silencing of TaWRKY76 in wheat leaves.b-Actin was set as internal reference gene.Three replicates were performed for each treatment.(d) Content of SA in wide type (WT) and wheat leaves inoculated with BSMV:00 or BSMV:TaWRKY76 at 5 days (n = 5).(e) Number of nymphs produced by each aphid in 5 days after feeding on TaWRKY76-silenced wheat leaves (n = 15).(f) Aphid weight at 7 days postfeeding on TaWRKY76-silenced wheat leaves (n = 15).WT and wheat leaves inoculated with BSMV:00 were set as control groups.All data shown are mean AE SE.Different letters above the bars indicate significant differences among treatments (P < 0.05, One-way ANOVA followed by Duncan's multiple range test).leaves, and its feeding behaviour was also negatively affected, with shorter durations of phloem ingestion phases.It has been proposed that the SA signalling defence pathway elicited by SmCSP4 decreases aphid performance.Consistently, we found that the exogenous application of SA to wheat plants induced plant defence and reduced S. miscanthi fecundity.Our findings are consistent with those of previous studies showing that SA can contribute to plant resistance against aphids.The accumulation of SA and exogenous application of SA analogues in tomato and Arabidopsis are associated with reduced aphid performance and feeding preferences (Avila et al., 2012;Boughton et al., 2006;Cooper et al., 2004).Elimination of SA accumulation increased aphid longevity on tomato plants expressing NahG and completely abolished aphid resistance in plants carrying the Mi-1 resistance gene, demonstrating that SA is essential for potato aphid resistance (Li et al., 2006).Interestingly, the expression of the CSP4 homologues of M. persicae, A. pisum and A. gosspyii induced callose deposition and SA-responsive defence responses in wheat, resulting in a significant reduction in aphid fecundity.These findings demonstrated the conserved role of aphid CSP4 homologues in activating plant immunity.M. euphorbiae effector Me10 and its homologue in A. gossypii, Ag10k, have been proven to promote aphid virulence in a conserved pathway by interacting with TFT7 in planta and interfering with the function of the TFT7 immune complex (Chaudhary et al., 2019).Sequence alignment analysis revealed that the CSP4 homologues of four different aphid species exhibit high sequence identity and conserved functional domains.Therefore, whether the regulatory mechanisms involved in activating plant defence are conserved among these homologues should be investigated in the future.
WRKY transcription factors comprise one of the largest families of transcriptional regulators exclusively found in plants and play important roles in plant resistance to various pathogens and insect pests.Several WRKYs have been shown to interact with the salivary effectors of insects to regulate host plant immunity.For example, the effector Bsp9 of whitefly (Bemisia tabaci) interacts with Arabidopsis WRKY33 and interferes with the interaction between WRKY33 and MPK6, thus suppressing the plant defence responses (Wang et al., 2019).Vitellogenin protein secreted by small brown planthopper (Laodelphax striatellus) directly interacts with the rice (Oryza sativa) transcription factor OsWRKY71 and weakens H 2 O 2 -mediated plant defence (Ji et al., 2021).In our study, full-length SmCSP4 (containing the NLS) interacted with TaWRKY76, which has not been functionally characterized in wheat, in the nucleus.A previous study reported that the CathB3 salivary elicitor of M. persicae suppresses aphid feeding by interacting with plant EDR1-like in phloem and triggering the accumulation of reactive oxygen species (Guo et al., 2020).To the best of our knowledge, salivary elicitors targeting WRKY transcription factors and eliciting plant immunity have not been reported in aphids to date.
Yeast transcriptional activation verification assays indicated that TaWRKY76 possesses transcriptional activation activity.Furthermore, silencing of TaWRKY76 via VIGS up-regulated the SA content of wheat plants, implying that TaWRKY76 is involved in the inhibition of SA biosynthesis or the promotion of SA degradation as a transcriptional activator.In Arabidopsis, DMR6 encodes a putative 2OG-Fe (II) oxygenase, which is involved in SA hydroxylation and is required for the susceptibility to downy mildew through the regulation of SA degradation (Van Damme et al., 2008;Zhang et al., 2017).In apple (Malus domestica), WRKY17 is essential for SA degradation and fungal disease susceptibility by directly binding to the promoter of DMR6 and promoting its expression (Shan et al., 2021).But how SA biosynthesis is transcriptionally regulated by TaWRKY76 is hitherto unknown.Herein, the expression of TaWRKY76 increased LUC expression when transiently transformed in N. benthamiana leaves under the control of the DMR6 promoter, confirming that TaWRKY76 directly activates DMR6 expression in vivo.The DMR6 orthologs were highly up-regulated in the aphidsusceptible wheat variety after infestation by Puccinia graminis f. sp.tritici (Henningsen et al., 2021).Given its essential role in plant disease susceptibility, the loss of function of DMR6 by RNAi and genome editing in crops are promising strategies for controlling plant pathogens.For instance, mutations in the Arabidopsis DMR6 gene confer resistance to hemibiotrophic pathogens Pseudomonas syringae and Phytophthora capsici as well as sldmr6-1 tomato mutants display enhanced broad-spectrum resistance against different classes of pathogens, such as bacteria, oomycetes, and fungi (Thomazella et al., 2021;Zeilmaker et al., 2015).We also found that silencing of TaWRKY76 induced SA accumulation and caused detrimental effects on aphid performance and feeding behaviour, suggesting that TaWRKY76 is a susceptible gene in wheat.Previously, the CRISPR-Cas9mediated inactivation of the susceptible gene TaPsIPK1 in wheat targeted by P. striiformis f. sp.tritici (Pst) effector PsSpg1 conferred broad-spectrum resistance against Pst without affecting the important agronomic traits of wheat plants (Wang et al., 2022).Therefore, susceptible genes TaWRKY76 and DMR6 could be promising targets for engineering wheat plants with broad-spectrum resistance against aphids and pathogens.
Figure 9 SmCSP4 inhibits the transcriptional activation activity of TaWRKY76 on DMR6 expression.(a) Yeast assays showing the transcriptional activation activity of TaWRKY76.Yeast cells co-transformed with pGBKT7-53+pGADT7-T and pGBKT7-Lam+pGADT7-T are used as positive and negative controls, respectively.(b) Yeast one-hybrid assay (Y1H) examined the interaction of the TaWRKY76 with DMR6 promoter.AD-TaWRKY76 and pHIS2-proDMR6 constructs were co-transformed into yeast Y187, and recombined cells were grown on SD/-Trp/-His or SD/-Trp/-Leu/-His containing 50 mM 3-AT.Yeast cells co-transformed with pGBKT7-p53 and pGAD53m are used as positive control.(c) Dual-luciferase reporter assays detected the binding activity of TaWRKY76 in the promoter of DMR6.pGreenII0800-proDMR6-LUC and pGreenII62-TaWRKY76-SK were transiently transformed into N. benthamiana leaves to detect the luminescence.The relative luciferase activities in different samples were calculated by normalizing the LUC values against REN (n = 6).All data presented in the boxplots were shown as mean AE SE.Different letters above the bars indicate significant differences among treatments (P < 0.05, One-way ANOVA followed by Duncan's multiple range test).(d) The expression levels of DMR6 on wheat leaves infiltrated with pEDV6:SmCSP4 or pEDV6: DsRed (control) at 24 h and 48 h.(e) The expression levels of DMR6 on wheat leaves infested with SmCSP4-silenced S. miscanthi at 24 h and 48 h.Wheat leaves infested with aphids treated with dsGFP were used as control.Three replicates were performed for each treatment.All data shown are mean AE SE. 'ns' indicates no significant differences between controls and treatments.Asterisks above bars and lines indicate significant differences between controls and treatments (**P < 0.01; ***P < 0.001; Student's t test).(f) Effects of SmCSP4 on binding activity of TaWRKY76 to the promoter of DMR6 by GUS staining assays.N. benthamiana leaves were infiltrated with Agrobacterium harbouring 35S:GUS, proDMR6:GUS, 35S:TaWRKY76-GFP, 35S:SmCSP4-GFP alone, or co-infiltrated with various Agrobacterium mixture.Leaf disk was stained using GUS staining at 2 days post-infiltration, and then decolorized with 70% ethanol.( 1 In GUS staining and dual-luciferase assays, SmCSP4 secreted by aphids could suppress the binding of TaWRKY76 to the DMR6 promoter, which decreased gene expression.The SmCSP4induced reduction in DMR6 expression level may subsequently cause an increase in the SA content and greater plant resistance against aphids.Similarly, a previous study reported that the salivary effector vitellogenin of L. striatellus negatively affects the transcriptional activity of the interacting protein OsWRKY71 and influences the expression of OsGF14b, suppressing H 2 O 2mediated plant defence (Ji et al., 2021).However, further research is warranted to elucidate the molecular mechanism of SmCSP4-mediated inhibition of the transcription activation activity of TaWRKY76.
In summary, our study demonstrates that the salivary elicitor SmCSP4 of S. miscanthi activates SA-mediated wheat defence responses by interacting with TaWRKY76.SmCSP4 reduced the transcriptional activity of TaWRKY76 to regulate the expression of the SA degradation gene DMR6, resulting in enhanced SA levels and greater plant immunity against aphids (Figure 10).

Aphids and plants
Winter wheat (Triticum aestivum) cultivars Mingxian169 (aphidsusceptible) and Zhong4wumang (aphid-resistant) were used in this study (Xu et al., 2011).Seeds were incubated in sterilized Petri dishes containing distilled water for 3-4 days at 25 AE 1 °C to induce germination.Healthy seedlings were carefully transferred into plastic pots containing organic soil (peat: vermiculite = 3 : 1) and grown in a climate chamber at 20 AE 1 °C under 16 h light/8 h dark photoperiod for 12 days (i.e., until reaching the two-leaf stage).Tobacco plants Nicotiana tabacum and N. benthamiana were grown in an environmentally controlled greenhouse at 23 AE 1 °C under 16 h light/8 h dark photoperiod.After 4-5 weeks, the N. benthamiana plants were used in transient transformation experiments using Agrobacterium tumefaciens.
Clones of S. miscanthi, A. pisum and A. gossypii were initially established from a single aphid collected from a field in Langfang City, Hebei Province, Northern China, and clones of M. persicae were established from a single aphid collected from a tobacco field in Kunming City, Yunnan Province, Southwestern China.No pesticides were applied on the collection sites.All aphid clones were maintained under laboratory conditions (16 h light/8 h dark photoperiod and 20 AE 1 °C) on the corresponding host plants, including wheat, broad bean Vicia faba, cotton Gossypium hirsutum, and N. tabacum.

Subcellular localization of SmCSP4 in N. benthamiana
To perform subcellular localization assays, the SmCSP4 CDS excluding the signal peptide (SmCSP4) and SmCSP4 with mutant nuclear localization signal (SmCSP4 NLSm ) were cloned into the pCAMBIA1300 vector using the In-Fusion Cloning Kit (Takara, Japan) according to the manufacturer's protocols.An A to C single base mutation was introduced in base 422 of the coding sequence of SmCSP4 NLSm .A. tumefaciens GV3101 cells carrying the pCAMBIA1300:SmCSP4-GFP, pCAMBIA1300:SmCSP4 NLSm -GFP or pCAMBIA1300:GFP (control) construct were resuspended in infiltration buffer (10 mM MgCl 2 , 10 lM acetosyringone, 10 mM 2-(N-morpholino) ethanesulfonic acid [pH 5.6]) at a final optical density OD 600 of 0.5 and then infiltrated into 4-week-old N. benthamiana leaves as described previously (Bos et al., 2010).Then, GFP signal was detected via Confocal Laser Scanning Microscope (LSM700, Zeiss) at an excitation wavelength of 488 nm at 24 h post-infiltration (hpi).DAPI was used as a counterstain to observe the nuclei.

Delivery of SmCSP4 into wheat via the type III secretion system (T3SS)
The SmCSP4 or SmCSP4 NLSm sequence was cloned into the pEDV6 vector via Gateway recombination (Thermo Fisher, USA) according to the manufacturer's protocols, and the resultant construct (pEDV6:SmCSP4 or pEDV6:SmCSP4 NLSm ) was transformed into Pseudomonas fluorescens strain EtHAn via electroporation.The recombinant EtHAn strain carrying pEDV6:SmCSP4 or pEDV6:SmCSP4 NLSm was grown in KB liquid medium (25 lg/ mL rifampicin, 30 lg/mL gentamicin) for 24 h, and then cells were washed with 10 mM MgCl 2 solution for three times and resuspended in 10 mM MgCl 2 solution.EtHAn cell suspension (OD 600 = 1.0) was infiltrated into the second leaf of 12-day-old wheat seedlings (var.Zhong4wumang) at the two-leaf stage using a needleless syringe.Wheat leaves infiltrated with 10 mM MgCl 2 solution or EtHAn carrying pEDV6:DsRed or pEDV6: AvrRpt2 were used as blank, negative, and positive controls respectively (Xu et al., 2019b).The infiltrated plants were grown and maintained in a climate chamber at 20 AE 1 °C for 2 days.

Western blot
Wheat leaves (12-day-old, var.Mingxian 169) were infested with 600 S. miscanthi for 2 days.Aphids were then removed carefully from each leaf using soft brush and the leaves were immediately washed with distilled water for 3 times.Western blot was performed to detect SmCSP4 protein in S. miscanthi whole body, aphid-infested wheat leaves and aphid-uninfested leaves using purified SmCSP4 rabbit polyclonal antibody (ABclonal, China).Wheat leaves without aphid infestation were used as control.
SmCSP4-or SmCSP4 NLSm -expressed wheat leaves were collected at 2hpi, and the SmCSP4 or SmCSP4 NLSm protein was also detected by Western blot using anti-SmCSP4 (ABclonal, China) or anti-GFP polyclonal antibody (Cat. No. D110008, Sango Biotech, China).Protein was extracted from aphid whole body and plant leaves using RIPA Lysis Buffer for Western and IP (Beyotime, China) and then quantified using BCA protein assay kit (Bio-Rad, USA).Equal amount of protein of each sample was separated by 10% SDS-PAGE and transferred onto a Polyvinylidene Fluoride (PVDF) membrane.PVDF membrane was blocked with 5% BSA for 2 h at room temperature and then incubated with purified polyclonal antibody (1 : 1000 dilution) overnight.Anti-actin polyclonal antibody for plant (Cat.No. D110007, Sango Biotech, China) and insect (Cat.No. bs-8778R, Beijing Biosynthesis Biotechnology Co., Ltd.China) were used as loading controls for Western blot analysis.The antigen-antibody complexes were visualized using a goat anti-rabbit IgG-conjugated horseradish peroxidase (HRP) antibody (Cat. No. sc-2054, Santa Cruz Biotechnology, Inc., USA) at a 1 : 10 000 dilutions.Clarity ECL solution (Bio-Rad, USA) was added for detection, and the chemiluminescence signal was then captured with a ChemiDoc XRS + imaging system.
RNAi using the nanocarrier-mediated delivery system Double-stranded RNA (dsRNA) of SmCSP4 was prepared using TranscriptAid T7 High-Yield Transcription Kit (Thermo Fisher Scientific, CA) according to the manufacturer's protocols, with the primers listed in Table S2.The dsRNA of SmCSP4 or GFP was mixed with the SPc nanocarrier at a 1 : 1 ratio (w/w).Then, 0.05% detergent (surfactant and softened water) was added to the solution to reduce the surface tension of the hydrophilic nanocomplexes (Li et al., 2019;Yan et al., 2020).Subsequently, 0.1 lL of the SmCSP4 dsRNA/nanocarrier/detergent formulation or GFP dsRNA/nanocarrier/detergent formulation (control) with a dose of 500 ng/lL dsRNA was dispensed on the notum of apteral adult aphids.After the solution was completely dried, all aphids were carefully transferred onto wheat plants using a soft brush.The dsRNA-treated aphid samples were collected at 24 or 48 h post-infestation on wheat and washed three times with distilled water to remove any residues of nanocarrier/detergent.Real-Time Quantitative PCR (RT-qPCR) was performed to detect the transcript levels of SmCSP4.Each treatment was performed in triplicate, and each replicate contained five dsRNA-treated aphids.

Y-tube olfactometer assay
Behavioural assays were performed using a glass Y-tube olfactometer (2.7 cm diameter, 10 cm trunk length, and 16.5 cm branch length) as described previously (Zhang et al., 2015).The air was purified by passing through granular activated carbon at a flow rate of 0.1 L/min.One arm of the Ytube was introduced with EBF (dissolved in hexane at a concentration of 10 ng/lL) or volatiles of whole wheat plants, whereas the other arm (control) was introduced with hexane or clean air, respectively.A Y-shaped copper wire was placed at the centre of the Y-tube to facilitate aphid mobility.Aphids treated with dsSmCSP4/nanocarrier/detergent, dsGFP/nanocarrier/detergent or nanocarrier/detergent (negative control) formulations at 48 h as mentioned above were then placed at the end of the copper wire in the trunk.The number of aphids in each arm exceeded 5 cm was recorded after 10 min.A total of 15 replicates (with 30 apterous aphids per replicate) were conducted for each treatment.

Detection of callose deposition and H 2 O 2 accumulation in leaves
Aniline blue staining was performed to visualize callose deposition in wheat leaves at 2 days post-infiltration (dpi), as described previously (Hood and Shew, 1996).Callose deposition was observed and photographed under an Echo Revolve Hybrid Microscope (Echo Laboratories, USA) using a DAPI (4, 6diamidino-2-phenylindole) filter.The number of callose deposits was counted at 15 different sites (1 mm 2 area per site) on each infiltrated leaf.Five biological replicates were performed for each treatment.
The accumulation of H 2 O 2 in wheat leaves was detected via DAB staining according to the protocol described previously (Zhao et al., 2018).The stained leaves were imaged using a Canon digital camera or Olympus SZX-16 (Olympus Corporation, Japan).The endogenous H 2 O 2 levels in wheat leaves were determined using hydrogen peroxide (H 2 O 2 ) Content Assay Kit (Sangon Biotech, China) according to the manufacturer's protocols.Six biological replicates for each treatment were conducted, and five leaf samples with the same treatment were pooled as one replicate.

Analyses of SA and JA contents
Phytohormone contents were examined as described previously (Xiang et al., 2011), with minor modifications.Briefly, 0.5 g of wheat leaves infested with twenty apterous S. miscanthi adults or infiltrated with MgCl 2 , P. fluorescens EtHAn carrying pEDV6: DsRed, pEDV6:SmCSP4, pEDV6:SmCSP4 NLSm , pEDV6:ApCSP4, pEDV6:MpCSP4 or pEDV6:AgCSP4 was harvested at 2 dpi and homogenized in liquid nitrogen mixed with labelled internal standards ( 2 D 4 -SA, 2 D 6 -JA).Then, 10 mL of ethyl acetate was added to the sample.This was followed by ultrasound extraction for 20 min and centrifugation at 14 000 g for 20 min at 4 °C.After vacuum-drying, the final extracts were re-dissolved in methanol and filtered through a 0.2 lm nylon filter.The SA and JA contents in wheat leaves were measured via liquid chromatography-tandem mass spectrometry (LC-MS/MS).At least five biological replicates were performed.

RT-qPCR
Total RNA isolation and RT-qPCR assays were performed as described previously (Zhang et al., 2022).Briefly, total RNA was extracted from aphid tissues, tobacco or wheat leaves using TRIzol Reagent (Thermo Fisher, USA) according to the manufacturer's protocols.First-strand cDNA synthesis was conducted using the HiScript III First-Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, China), and RT-qPCR was performed on the ABI Prism 7500 Real-Time PCR System using SYBR Premix Ex Taq Kit (TaKaRa, Japan) and sequence-specific primers (Table S2).Three replicates were conducted for each treatment, and each replicate contains three technical replicates.

Aphid bioassays
To investigate the effects of gene silencing on S. miscanthi performance, 10 dsRNA-treated apterous adult aphids were transferred onto wheat leaves (var.Zhong4wumang) and confined using a clip cage (2.5 cm 9 2.5 cm 9 2.5 cm).The number of surviving aphids was counted every other day for 8 days, and a total of 15 replicates were performed for each treatment.Five apterous adult aphids were also treated with dsRNA as mentioned above, and the number of nymphs produced by aphids was recorded daily for 5 days.Aphids treated with dsGFP were included as a control.A total of 15 replicates were performed for each treatment.
After P. fluorescens EtHAn infiltration, exogenous SA application at 2 days, or TaWRKY76 silencing, 10 apterous adult aphids were transferred onto the treated wheat leaves (var.Zhong4wumang) and confined using clip cages as described above.The number of surviving aphids was counted every other day for 8 days, and a total of 15 replicates were performed for each treatment.Five apterous adult aphids were transferred onto the treated wheat leaves as mentioned above, and the number of nymphs produced by aphids was recorded daily for 5 days.Ten newborn nymphs were reared on wheat leaves infiltrated with the SmCSP4 delivering or TaWRKY76 silencing construct, and each aphid was weighed on a microbalance (Sartorius, GPC, Germany) after 7 days of feeding.Fifteen replicates were performed for each treatment.Wheat plants treated with SA solvent, P. fluorescens EtHAn carrying pEDV6:DsRed, or BSMV::00 were used as control, respectively.
Assessment of aphid feeding behaviour using electronic penetration graph (EPG) The feeding behaviour of adult apterous aphids on wheat leaves (var.Zhong4wumang) expressed with SmCSP4 or DsRed (control) was recorded continuously for 6 h in a Faraday cage using EPG (DC-EPG Giga-8) in an environmentally controlled room (20 °C AE 1 °C, 16 h light/8 h dark photoperiod, 70% relative humidity).EPG waveforms were recorded and analysed using the Stylet+ software (EPG system; www.epgsystems.eu).The characteristics of aphid feeding waveform patterns were identified as previously reported (Prado and Tjallingii, 1994).Ten replicates were conducted for each treatment.

Y2H assay
Sitobion miscanthi-infested wheat leaves were harvested at 6, 12, 24, 48 and 96 hpi, and cDNA libraries were constructed by OE Biotech.Co., Ltd.(Shanghai, China) using the CloneMiner II cDNA Library Construction Kit (Thermo Fisher, USA) according to the manufacturer's protocols.The CDS of SmCSP4 corresponding to the mature protein was cloned into the pGBKT7 vector to generate SmCSP4-BD (bait vector), and putative SmCSP4interacting proteins were screened using the Matchmaker Gold Yeast Two-Hybrid System (Clontech, USA) according to the manufacturer's protocols.To further confirm the interaction between SmCSP4 and TaWRKY76, the yeast two-hybrid (Y2H) gold yeast strain was co-transformed with SmCSP4-BD and TaWRKY76-AD constructs, and serial dilutions of the transformed cells were plated onto appropriate selective dropout media.Yeast strains co-transformed with pGBKT7-53 and pGADT7-T, pGBKT7-Lam and pGADT7-T were set as positive and negative control mating, respectively.

BiFC assay
The SmCSP4 sequence and TaWRKY76 gene were cloned into pSPYNE-35S and pSPYCE-35S, respectively.Protoplasts were isolated from two young leaves of 4-week-old N. benthamiana plants that were cut in small pieces and vacuum infiltrated with 5% (w/v) cellulase Onozuka R-10 and 0.4% (w/v) macerozyme R.10 (Yakult, Japan) in 0.4 M mannitol, and incubated for 3 h followed by shaking at 40 rpm in dark.The homogenates were filtered through a 40 lm nylon mesh and resuspended in W5 solution after centrifuge.The cells were finally resuspended in mannitol-magnesium buffer to get a density of 2 9 10 5 protoplasts per mL.For transformation, 10 lg of SmCSP4-nYFP and TaWRKY76-cYFP constructs were co-transformed into 200 lL of protoplast suspension by using 40% (v/v) PEG.The cells were resuspended in 200 lL W1 buffer and incubated at room temperature for 20 h after centrifuging at 200 g for 1 min.Images were captured by a Confocal Laser Scanning Microscope (LSM700, Zeiss).Upon excitation of 488 nm, yellow fluorescent protein fluorescence and chlorophyll autofluorescence were recorded at 493-531 nm and 644-800 nm, respectively.Plant cell nucleus were stained with DAPI for 30 min prior to detection.

Co-IP assay
SmCSP4-HA and TaWRKY76-MYC were co-expressed in N. benthamiana leaves, and total protein was extracted from these leaves using the RIPA Lysis Buffer (Beyotime, China).Protein extracts were centrifuged at 15000 g at 4 °C for 30 min and the supernatants were incubated with anti-HA magnetic beads or anti-MYC magnetic beads (Thermo Fisher, USA) for 3 h at 4 °C.The Co-IP assay was performed using the Pierce Magnetic HA-Tag IP/Co-IP Kit and Pierce Magnetic MYC-Tag IP/Co-IP Kit (Thermo Fisher, USA).The immunoprecipitated proteins were detected via Western blotting using anti-MYC (1 : 1000) antibody or anti-HA antibody (1 : 1000) (antibodies-online Inc., Germany).The luminescent signal was visualized using BIO-RAD ChemiDoc TM XRS + .N. benthamiana leaves co-expressed with TaWRKY76-MYC and HA were set as controls, and IP with IgG antibody served as negative control.Barley stripe mosaic virus (BSMV)-mediated gene silencing A TaWRKY76 fragment was cloned into the BSMV-c vector.The recombinant vector and the vector containing the tripartite viral genome were linearized and transcribed using the mMESSAGE mMACHINEâ T7 Transcription Kit (Thermo Fisher, USA) according to the manufacturer's protocols.Transcripts of BSMV-a, BSMV-b, and BSMV-c/BSMV-c: phytoene desaturase (TaPDS)/ BSMV-c:TaWRKY76 were mixed in 1 : 1 : 1 ratio and then added to FES buffer for inoculation.The BSMV:00 and BSMV:TaPDS constructs were used as negative and positive controls for BSMV infection, respectively.The treated leaves were collected at 10 dpi for RNA extraction, and silencing efficiency was detected via RT-qPCR.Three replicates were conducted.

Transcriptional activation assays in yeast
To perform the transcriptional activation assay in yeast, the TaWRKY76 CDS was introduced into the pGBKT7 vector, as described previously (Shan et al., 2021).The primers used in this experiment are listed in Table S2.
Yeast One-Hybrid (Y1H) assay Y1H assay was carried out using the MATCHMAKER One-Hybrid System (Clontech, CA, USA), according to the manufacturer's protocols.The TaWRKY76 CDS was cloned into pGADT7-Rec2 to generate the prey vector, and the regulatory elements of the DMR6 promoter (proDMR6) were cloned into pHIS2 to construct the reporter (bait) vector.Then, the Y187 yeast strain was cotransformed with AD-TaWRKY76 and pHIS2-proDMR6 and cultured on SD/-Trp/-Leu/-His medium supplemented with 50-mM 3-amino-1,2,4-triazole (3-AT) to verify the interaction.Yeast cells co-transformed with pHIS2-p53 and pGAD53m served as a positive control.

Dual-luciferase reporter assay
The TaWRKY76 CDS or proDMR6 sequence were cloned into the pGreenII-062SK and pGreenII0800-LUC vectors, respectively.To assess the effects of SmCSP4 on the regulatory activity of TaWRKY76, the SmCSP4 sequence was cloned into pGreenII-062SK to generate the effector plasmid.Then, the 35S: TaWRKY76 or 35S:SmCSP4 effector plasmid and the proDMR6 reporter plasmid were co-transformed into A. tumefaciens strain GV3101, which was then infiltrated into N. benthamiana leaves (as described above).Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured using the Dual-Luciferase Assay System (Promega, USA).Six replicates were performed for each treatment.

b-Glucuronidase (GUS) staining assay
The proDMR6 sequence was cloned into pBI121-GUS (which lacks the 35S promoter) to generate the proDMR6:GUS construct.The primer sequences used are listed in Table S2.The recombinant vectors were individually transformed into A. tumefaciens strain GV3101.Equal volumes of Agrobacterium cultures harbouring proDMR6:GUS+35S:TaWRKY76-GFP, proDMR6: GUS+35S:SmCSP4-GFP and proDMR6:GUS+35S:TaWRKY76-GFP+35S:SmCSP4-GFP were mixed and infiltrated into N. benthamiana leaves.Agrobacterium cells harbouring 35S:GUS served as a positive control, whereas those harbouring proDMR6: GUS, 35S:TaWRKY76-GFP or 35S:SmCSP4-GFP alone served as negative controls.The infiltrated sections of leaves were collected at 2 dpi, immersed in the GUS staining solution overnight at 37 °C and destained with 70% ethanol.The treated N. benthamiana leaves were also collected to detect the GUS activity 2 days after the infiltration as described previously (Jefferson et al., 1987).Each treatment was conducted with six replicates.

Statistical analysis
All statistical tests were conducted using the SPSS 20.0 software.To compare two independent groups, statistical significance was determined using Student's t-test.When comparing multiple groups, statistical significance was determined via one-way analysis of variance and Duncan's multiple range test.EPG data were analysed by a Mann-Whitney U test.P values less than 0.05 were considered to be statistically significant.Figure S6 The potential binding sites of transcriptional factor TaWRKY76 on the promoter of DMR6 gene.Table S1 All CSP amino acid sequences used for phylogenetic tree construction.Table S2 All primers used in this study.

Figure 1
Figure 1 Characterization of SmCSP4 from S. miscanthi.(a) Detection of SmCSP4 in various aphid tissues using RT-PCR.(b) Detection of SmCSP4 in different aphid tissues by RT-qPCR.b-actin and NADH hydrogenase were used as internal reference genes.Standard error (SE) is represented by the error bar.Different lower-case letters above the bars indicate significant differences among treatments (P < 0.05, one-way ANOVA followed by Duncan's multiple range test).Abbreviation for tissues: Whole body of apterous adults (WB), heads (HE), antennae (AN), thorax (TH), abdomen (AB), salivary glands (SG), and legs (LG).(c) The expression levels of SmCSP4 in S. miscanthi after feeding on aphid-susceptible wheat variety (S) Mingxian169 and aphid-resistant (R) wheat variety Zhong4wumang at different time points.b-actin and NADH dehydrogenase were used as internal reference genes.Three replicates were conducted for each treatment.Asterisks above bars indicate significant differences between controls and treatments (*P < 0.05; **P < 0.01; Student's t test).(d) Subcellular localization of SmCSP4 or SmCSP4 NLSm in N. benthamiana.GFP was used as control groups.The images were taken 24 h after agroinfiltration using confocal laser scanning microscopy.Bar = 25 lm.

Figure 2
Figure2SmCSP4 is a secreted salivary protein.Western blot analysis of SmCSP4.Lane 1, protein extracted from aphid whole body; lane 2, protein extracted from wheat leaves without aphid feeding; lane 3, protein extracted from wheat leaves fed upon by aphids.Anti-actin antibody of plants or insects was used to detect the loading of lanes 1-3.M: Protein marker.The asterisk denotes an unspecific band.

Figure 3
Figure3SmCSP4 activated salicylic acid-mediated signalling defence pathway in plant.(a) Phenotype and H 2 O 2 accumulation in wheat leaves (var.Zhong4wumang) after infiltration with P. fluorescens EtAnH carrying pEDV6: SmCSP4 or pEDV6: SmCSP4 NLSm at 2 days.Leaves infiltrated with MgCl 2 , pEDV6:DsRed or pEDV6:AvrRpt2 were set as blank, negative and positive controls, respectively.(b) Content of H 2 O 2 in wheat leaves expressed with SmCSP4 or SmCSP4 NLSm after 2 days of infiltration (n = 6).Different lower-case letters above the bars indicate significant differences between controls and treatments (P < 0.05; one-way ANOVA followed by Duncan's multiple range test).(c) Aniline blue staining was performed to examine callose deposition in the wheat leaves infiltrated with EtAnH carrying pEDV6: SmCSP4 or pEDV6:SmCSP4 NLSm at 2 days using epifluorescence microscopy.Leaves treated with MgCl 2 and pEDV6:DsRed were set as controls.Bar = 330 lm.Five replicates were used for analysis.(d) The expression levels of JA synthesis-related genes FAD and LOX, and SA signalling pathway-associated genes PAL and PR1 in wheat leaves infiltrated with MgCl 2 solution or P. fluorescens EtAnH carrying pEDV6:DsRed, pEDV6:SmCSP4 or pEDV6:SmCSP4 NLSm at 2 days (n = 3).(e) Endogenous JA and SA contents in wheat leaves inoculated with MgCl 2 solution or P. fluorescens EtAnH carrying pEDV6:DsRed, pEDV6:SmCSP4 or pEDV6:SmCSP4 NLSm .Six replicates were conducted for each treatment.Data in the bar chart are represented as mean AE SE.

Figure 4
Figure 4 Expression of SmCSP4 in wheat-reduced aphid performance.(a) The survival rate of S. miscanthi when feeding on wheat leaves (var.Zhong4wumang) delivered with SmCSP4.Fifteen biological replicates were performed for each group.(b) Number of nymphs produced by S. miscanthi after feeding on wheat leaves infiltrated with pEDV6:DsRed (control) or pEDV6: SmCSP4 for 5 days.Fifteen biological replicates were performed for each group.(c) Body weight of S. miscanthi after feeding on wheat leaves inoculated with pEDV6:DsRed (control) or pEDV6:SmCSP4 for 7 days.Fifteen biological replicates were performed for each group.Asterisks above the bars indicate significant differences between controls and treatments (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test).(d) Representative EPG waveforms of S. miscanthi feeding on wheat leaves delivered with DsRed (control) and SmCSP4, respectively.(e) Feeding behaviour parameters of S. miscanthi, including durations of nonprobing (np), stylet probing (C), salivation (E1) and phloem ingestion (E2) when feeding on wheat leaves treated with pEDV6: DsRed (control) or pEDV6: SmCSP4.Ten biological replicates were performed for each treatment.Asterisks above the bars indicate significant differences between controls and treatments (*P < 0.05; **P < 0.01; ***P < 0.001; Mann-Whitney U test.).All data are represented as mean AE SE.

Figure 5
Figure 5 SmCSP4 orthologs from M. persicae, A. pisum and A. gosspyii enhanced SA levels and wheat resistance.(a) Callose deposition in wheat leaves (var.Zhong4wumang) expressed with CSP4 orthologs from A. pisum (ApCSP4), M. persicae (MpCSP4) and A. gosspyii (AgCSP4) at 2 days using aniline blue staining.Bar = 330 lm.(b) Endogenous SA contents in wheat leaves inoculated with P. fluorescens EtAnH carrying pEDV6: MpCSP4, pEDV6: ApCSP4 or pEDV6: AgCSP4 respectively.Five replicates were conducted for each treatment.Leaves infiltrated with pEDV6: DsRed was used as control.(c) Number of nymphs produced by S. miscanthi after feeding on wheat leaves delivered with DsRed, ApCSP4, MpCSP4 or AgCSP4, respectively, for 5 days.Fifteen biological replicates were performed for each treatment.Standard error of the mean (SE) is represented by the error bar.Different lower-case letters above the bars indicate significant differences between controls and treatments (P < 0.05, one-way ANOVA followed by Duncan's multiple range test).

Figure 6
Figure 6 SmCSP4-silenced aphid via nanocarriermediated RNAi activated weaker defence responses.(a) Schematic diagram for applying the nanocarrier-mediated transdermal dsRNA delivery system.The dsRNA/nanocarrier/detergent droplet was dropped on the notum of S. miscanthi using microinjector.(b) Relative expression levels of SmCSP4 at 24 and 48 h after dsSmCSP4/ nanocarrier/detergent or dsGFP/nanocarrier/ detergent (control) treatments.Three biological replicates were conducted for each group.(c) Content of SA and JA in wheat leaves after infestation of aphids treated with dsGFP or dsSmCSP4 at 12 and 24 dpi.Five replicates were performed for each treatment.(d) Survival rate of SmCSP4-silenced aphids after feeding on aphidresistant wheat variety Zhong4wumang at four different time points.Fifteen biological replicates were conducted (n = 15).(e) Number of nymphs produce by each aphid treated with dsSmCSP4 or dsGFP after feeding on wheat plants (var.Zhong4wumang) for 5 days.Fifteen biological replicates were conducted.All data shown are mean AE SE. 'ns' indicates no significant differences between controls and treatments.Asterisks above bars and lines indicate significant differences between controls and treatments (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test).
transcriptional activation of DMR6 by TaWRKY76 was affected by SmCSP4, b-Glucuronidase (GUS) staining and dual-luciferase reporter assays were performed.In the GUS staining assay, no blue colour was observed in N. benthamiana leaves infiltrated ª 2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407 with 35S:TaWRKY76 or 35S:SmCSP4, N. benthamiana leaves coinfiltrated with proDMR6:GUS and 35S:TaWRKY76-GFP stained more intensely than those infiltrated with proDMR6:GUS, suggesting that TaWRKY76 acts as an activator of DMR6.However, GUS staining was significantly lighter in leaves coinfiltrated with 35S:SmCSP4, proDMR6:GUS, and 35S:TaWRKY76 ª 2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407

Figure 8
Figure8Silencing of TaWRKY76 activated salicylic acid-mediated wheat defence responses.(a) The second leaves wheat cultivar Mingxian169 were inoculated with barley stripe mosaic virus BSMV:00 (Control) and recombinant BSMV:TaPDS, BSMV:TaWRKY76 at two-leaf stage, and the phenotypes were observed and photographed 10 days after inoculation.(b) Expression levels of TaWRKY76 in wheat leaves inoculated with BSMV:TaWRKY76 at 5 days (n = 3).Wide type (WT) and wheat leaves inoculated with BSMV:00 were set as control groups.(c) Expression levels of SA signalling pathway associated genes PAL, PR1, PR5 and DMR6 after silencing of TaWRKY76 in wheat leaves.b-Actin was set as internal reference gene.Three replicates were performed for each treatment.(d) Content of SA in wide type (WT) and wheat leaves inoculated with BSMV:00 or BSMV:TaWRKY76 at 5 days (n = 5).(e) Number of nymphs produced by each aphid in 5 days after feeding on TaWRKY76-silenced wheat leaves (n = 15).(f) Aphid weight at 7 days postfeeding on TaWRKY76-silenced wheat leaves (n = 15).WT and wheat leaves inoculated with BSMV:00 were set as control groups.All data shown are mean AE SE.Different letters above the bars indicate significant differences among treatments (P < 0.05, One-way ANOVA followed by Duncan's multiple range test).

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2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407 Yong Zhang et al.
Figure9SmCSP4 inhibits the transcriptional activation activity of TaWRKY76 on DMR6 expression.(a) Yeast assays showing the transcriptional activation activity of TaWRKY76.Yeast cells co-transformed with pGBKT7-53+pGADT7-T and pGBKT7-Lam+pGADT7-T are used as positive and negative controls, respectively.(b) Yeast one-hybrid assay (Y1H) examined the interaction of the TaWRKY76 with DMR6 promoter.AD-TaWRKY76 and pHIS2-proDMR6 constructs were co-transformed into yeast Y187, and recombined cells were grown on SD/-Trp/-His or SD/-Trp/-Leu/-His containing 50 mM 3-AT.Yeast cells co-transformed with pGBKT7-p53 and pGAD53m are used as positive control.(c) Dual-luciferase reporter assays detected the binding activity of TaWRKY76 in the promoter of DMR6.pGreenII0800-proDMR6-LUC and pGreenII62-TaWRKY76-SK were transiently transformed into N. benthamiana leaves to detect the luminescence.The relative luciferase activities in different samples were calculated by normalizing the LUC values against REN (n = 6).All data presented in the boxplots were shown as mean AE SE.Different letters above the bars indicate significant differences among treatments (P < 0.05, One-way ANOVA followed by Duncan's multiple range test).(d) The expression levels of DMR6 on wheat leaves infiltrated with pEDV6:SmCSP4 or pEDV6: DsRed (control) at 24 h and 48 h.(e) The expression levels of DMR6 on wheat leaves infested with SmCSP4-silenced S. miscanthi at 24 h and 48 h.Wheat leaves infested with aphids treated with dsGFP were used as control.Three replicates were performed for each treatment.All data shown are mean AE SE. 'ns' indicates no significant differences between controls and treatments.Asterisks above bars and lines indicate significant differences between controls and treatments (**P < 0.01; ***P < 0.001; Student's t test).(f) Effects of SmCSP4 on binding activity of TaWRKY76 to the promoter of DMR6 by GUS staining assays.N. benthamiana leaves were infiltrated with Agrobacterium harbouring 35S:GUS, proDMR6:GUS, 35S:TaWRKY76-GFP, 35S:SmCSP4-GFP alone, or co-infiltrated with various Agrobacterium mixture.Leaf disk was stained using GUS staining at 2 days post-infiltration, and then decolorized with 70% ethanol.(1) 35S:GUS; (2) proDMR6:GUS; (3) proDMR6:GUS+35S:TaWRKY76-GFP; (4) proDMR6:GUS+35S:TaWRKY76-GFP+35S:SmCSP4-GFP; (5) 35S:SmCSP4-GFP; (6) 35S:TaWRKY76-GFP.The experiments were conducted with six biological replicates.Scale bar = 2 mm.The values are presented as mean AE SE.Different letters above the bars indicate significant difference among treatments (P < 0.05, one-way ANOVA followed by Duncan's multiple range test).(g) The effect of SmCSP4 on TaWRKY76 activated DMR6 expression in N. benthamiana leaves using dual-luciferase reporter assays.CaMV35Sempty vector was used for normalization between samples.Data are means of three biological replicates (AESE).Different letters above the bars indicate significant difference among treatments (P < 0.05, one-way ANOVA followed by Duncan's multiple range test).

Figure 10 A
Figure 10 A schematic summary of the roles of SmCSP4 secreted by Sitobion miscanthi during aphid-wheat interactions.(a) In wheat plants, TaWRKY76 can bind to the promoter of DMR6 and induce its gene expression as transcriptional activator, causing SA degradation.(b) During the probing and feeding of Sitobion miscanthi, salivary protein SmCSP4 is secreted into plant cells which can be interacted with TaWRKY76 in nucleus.Meanwhile, the regulation process of TaWRKY76 on DMR6 gene expression is inhibited by interacting with SmCSP4, which resulted in increases of SA accumulation and enhanced SAmediated plant resistance against S. miscanthi.

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2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407

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2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407

Figure S1
Figure S1 Sequence analysis of SmCSP4 from S. miscanthi.Figure S2 Western blot analysis of SmCSP4 or SmCSP4 NLSm protein in wheat leaves post-2 days infiltration.Figure S3 Salicylic acid improved wheat resistance against aphids.Figure S4 Olfactory responses of SmCSP4-silenced aphids to aphid alarm pheromone (E)-b-farnesene and wheat plant volatiles.Figure S5 Phylogenetic tree constructed by comparing the amino acid sequences of TaWRKY76 with WRKYs identified from Arabidopsis thaliana.FigureS6The potential binding sites of transcriptional factor TaWRKY76 on the promoter of DMR6 gene.TableS1All CSP amino acid sequences used for phylogenetic tree construction.TableS2All primers used in this study.

Figure S2
Figure S1 Sequence analysis of SmCSP4 from S. miscanthi.Figure S2 Western blot analysis of SmCSP4 or SmCSP4 NLSm protein in wheat leaves post-2 days infiltration.Figure S3 Salicylic acid improved wheat resistance against aphids.Figure S4 Olfactory responses of SmCSP4-silenced aphids to aphid alarm pheromone (E)-b-farnesene and wheat plant volatiles.Figure S5 Phylogenetic tree constructed by comparing the amino acid sequences of TaWRKY76 with WRKYs identified from Arabidopsis thaliana.FigureS6The potential binding sites of transcriptional factor TaWRKY76 on the promoter of DMR6 gene.TableS1All CSP amino acid sequences used for phylogenetic tree construction.TableS2All primers used in this study.

Figure S3
Figure S1 Sequence analysis of SmCSP4 from S. miscanthi.Figure S2 Western blot analysis of SmCSP4 or SmCSP4 NLSm protein in wheat leaves post-2 days infiltration.Figure S3 Salicylic acid improved wheat resistance against aphids.Figure S4 Olfactory responses of SmCSP4-silenced aphids to aphid alarm pheromone (E)-b-farnesene and wheat plant volatiles.Figure S5 Phylogenetic tree constructed by comparing the amino acid sequences of TaWRKY76 with WRKYs identified from Arabidopsis thaliana.FigureS6The potential binding sites of transcriptional factor TaWRKY76 on the promoter of DMR6 gene.TableS1All CSP amino acid sequences used for phylogenetic tree construction.TableS2All primers used in this study.

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2023 The Authors.Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 21, 2389-2407