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

Abstract

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.

Keywords: chemosensory protein; grain aphid; molecular basis; plant immunity; salicylic acid; salivary elicitor.

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. β‐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. β‐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 μm.

Figure 2

SmCSP4 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

SmCSP4 activated salicylic acid‐mediated signalling defence pathway in plant. (a) Phenotype and H₂O₂ 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 MgCl₂, pEDV6:DsRed or pEDV6:AvrRpt2 were set as blank, negative and positive controls, respectively. (b) Content of H₂O₂ 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 MgCl₂ and pEDV6:DsRed were set as controls. Bar = 330 μm. 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 MgCl₂ 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 MgCl₂ 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 ± SE.

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 ± SE.

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 μm. (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

SmCSP4‐silenced aphid via nanocarrier‐mediated 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 aphid‐resistant 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 ± 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).

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‐α‐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 μm. (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 μm. (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.

Figure 8

Silencing 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. β‐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 post‐feeding 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 ± SE. Different letters above the bars indicate significant differences among treatments (P < 0.05, One‐way ANOVA followed by Duncan's multiple range test).

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 ± 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 ± 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 ± 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. CaMV35S‐empty vector was used for normalization between samples. Data are means of three biological replicates (±SE). 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 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 SA‐mediated plant resistance against S. miscanthi.