BtRDP is a salivary protein and secreted into plants.

(a) Detection of BtRDP in Nicotiana tabacum plants. The untreated and Bemisia tabaci-infested tobacco plants are collected for western-blotting assays. Six independent B. tabaci-infested plants are tested. The presence of lower intensity band (arrow) in each replicate is summarized in the bracket. Asterisk indicates non-specific binding. Coomassie brilliant blue (CBB) staining is conducted to visualize the sample loading. (b, c) Detection of BtRDP in different tissues by western-blotting and qRT-PCR assays. Ov, ovary; SG, salivary gland; Ca, carcass; FB, fat body. Silver staining and anti-actin serum are used to visualize the sample loading. (d) Expression patterns of BtRDP in different development stages. Ny, nymph; Ps, pseudopupa; Fe, female; Ma, male. B. tabaci tubulin is used as an internal control. The relative quantitative method (2-ΔΔCt) is used to evaluate the quantitative variation. Data are presented as mean values ± SEM (n= 3 independent biological replicates). Different lowercase letters indicate statistically significant differences at the P < 0.05 level according to one-way ANOVA test followed by Tukey’s multiple comparisons test. (e) Immunohistochemical staining of BtRDP. The salivary gland and its nearby tissues are dissected and incubate with anti-BtRDP serum or pre-immune serum conjugated with Alexa Fluor™ 488 NHS Ester (green) and actin dye phalloidinrhodamine (red). The nucleus is stained with DAPI (blue). PSG, principal salivary gland; ASG, accessory salivary gland. Experiments are repeated thrice for (b), while twice for (e). Similar results are observed and representative images are displayed.

Effects of BtRDP on Bemisia tabaci.

(a) Treating B. tabaci with dsBtRDP significantly reduces the transcript and protein level of target gene. The dsGFP-treated B. tabaci is used as a control. qRT-PCR data are presented as mean values ± SEM (n= 6 independent biological replicates). (b–c) Effects of BtRDP knockdown on insect reproduction (b) and feeding behavior (c). Electrical penetration graph (EPG) is used to monitor the insect feeding behavior, which can be classified into nonpenetration (np), pathway duration (C), phloem salivation (E1) and phloem ingestion (E2) phases. All EPG recordings are performed for 8 h. Typical EPG waveforms are displayed in Fig. S8. (d-f) Insect performance on transgenic plants overexpressing BtRDP (oeBtRDP). (d) Detection of BtRDP in transgenic Nicotiana tabacum overexpressing a complete coding region of BtRDP. The empty vector (EV) plants are used as the control. The flag tag is fused to the C-terminal ends of recombinant proteins. (e) Comparison of insect reproduction on oeBtRDP#1 and EV plants. Five B. tabaci individuals are confined to indicated plants for 3 days, and the oviposited eggs are counted. (f) Attraction of oeBtRDP#1 and EV leaves to B. tabaci in a two-choice equipment. A group of 40 female B. tabaci are released into a device containing oeBtRDP#1 and EV leaves. The number of insects settling on each leaf is counted. After 48 h, the number of eggs on each leaf is counted. (g-i) Insect performance on transgenic plants overexpressing BtRDP-sp (oeBtRDP-sp). (g) Detection of BtRDP-sp in transgenic N. tabacum overexpressing BtRDP without a signal peptide. The insect reproduction (h) and settlement (i) on oeBtRDP-sp transgenic plants are recorded. Data are presented as mean values ± SEM. The EPG data are first checked for normality and homogeneity of variance, and data not fitting a normal distribution are subjected to log10 transformation. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant. Western-blotting assays are repeated thrice with the similar results.

NtRLP4 interacts with BtRDP and confers plant resistance to Bemisia tabaci.

(a) Domain organization of NtRLP4. NtRLP4 contains a predicted N-terminal signal peptide (SP), a malectin-like domain, a LRR domain, and a transmembrane domain (TM). (b) The protein level of NtRLP4 in response to B. tabaci infestation. The untreated Nicotiana tabacum is used as a negative control. Four independent biological replicates are performed, and the representative images are displayed. The band density is measured using ImageJ. The density values from 4 biological replicates are calculated and the mean value in the controls is set at 1.0. (c) Yeast two hybrid assays showing the interaction between BtRDP and NtRLP4. The different combinations of constructs are transformed into yeast cells, and are grown on the selective medium SD/-Trp/-Leu (DDO), and the interactions are tested with SD/-Trp/-Leu/-His/-Ade (QDO). (d) Co-immunoprecipitation assay showing the interaction between BtRDP and NtRLP4. Total proteins are extracted from N. benthamiana leaves transiently co-expressing NtRLP4-myc/NtCf9-myc with BtFTSP-flag/BtRDP-flag. All genes are expressed with a complete coding region, and the myc/flag tags are fused at the C-terminus. Precipitation is performed using flag beads. The samples are probed with anti-flag and anti-myc antibodies for immunoblotting analysis. (e–h) Analysis of transgenic N. tabacum overexpressing NtRLP4 (oeRLP). (e) Detection of NtRLP4 level in oeRLP plants. The empty vector (EV) plant is used as a control. Two independent oeRLP lines are selected. The samples are probed with an anti-NtRLP4 antibody. Rubisco staining (RbcL) is used to visualize the amount of sample loading. (f) Attraction of oeRLP#1 and EV leaves to B. tabaci in a two-choice equipment. A group of 40 female B. tabaci are released into a device containing oeRLP#1 and EV leaves. The number of insects settling on each leaf is counted at each time point. After 48 h, the number of eggs on each leaf is counted. (g) Comparison of insect reproduction on oeRLP#1 and EV plants. Five B. tabaci individuals are confined to indicated plants for 3 days, and the oviposited eggs are counted. (h) Relative transcript levels of salicylic acid (SA)- and jasmonic acid (JA)-associated genes in oeRLP#1 and EV plants. PAL, phenylalanine ammonia lyase; NPR1, nonexpressor of pathogenesis-related protein 1; FAD7, fatty acid desaturase 7; PDF1.2, plant defensin 1.2. Two independent biological replicates are performed in (d) and (e). Data in (f), (g), and (h) are presented as mean values ± SEM. For insect bioassays in (f) and (g), n= 10 independent biological replicates. For qRT-PCR in (h), n= 3 independent biological replicates. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

BtRDP suppress plant defenses by promoting NtRLP4 degradation.

(a) Effects of dsBtRDP suppression on insect reproduction (n= 10 independent biological replicates) when feeding on empty vector (EV) and NtRLP4-silenced (RNAi-RLP) transgenic Nicotiana tabacum. (b) Transient overexpressing BtRDP-mCherry attenuates H2O2 accumulation caused by NtRLP4-GFP overexpression. N. tabacum co-expressing RFP-mCherry and NtRLP4-GFP is used as a control. All genes are expressed with a complete coding region. The experiment is repeated 5 times with the similar results. (c) Relative transcript level of salicylic acid (SA)- and jasmonic acid (JA)-associated genes in BtRDP-mCherry/NtRLP4-GFP and RFP-mCherry/NtRLP4-GFP plants (n=3 independent biological replicates). PAL, phenylalanine ammonia lyase; NPR1, nonexpressor of pathogenesis-related protein 1; FAD7, fatty acid desaturase 7; PDF1.2, plant defensin 1.2. (d) Comparison of insect reproduction (n= 16 independent biological replicates) on transient-expressed BtRDP-mCherry/NtRLP4-GFP and RFP-mCherry/NtRLP4-GFP plants. (e) Attraction of BtRDP-mCherry/NtRLP4-GFP and RFP-mCherry/NtRLP4-GFP leaves to B. tabaci in a two-choice equipment (n= 16 independent biological replicates). The number of eggs on each leaf is counted at 48 h post insect release. (f) Effects of BtRDP and BtRDP-sp overexpression on NtRLP4 accumulation. Transgenic oeBtRDP (left) and oeBtRDP-sp (right) plants are probed with anti-NtRLP4 and anti-flag antibodies for immunoblotting analysis. The experiments are repeated twice with the similar results. (g) Degradation of NtRLP4 by BtRDP in N. benthamiana leaves. NtRLP4-myc and BtRDP-flag are transiently co-expressed in N. benthamiana plants through Agrobacterium infiltration. Agrobacterium carrying NtCf9-myc and GUS-flag are used as negative controls. (h) Effects of 26S proteasome inhibitor (MG132) on NtRLP4 accumulation. Co-infiltrated leaves are treated with MG132 at 24 h post injection. The samples are probed with anti-flag and anti-myc antibodies for immunoblot analysis. Rubisco staining (RbcL) is conducted to visualize the amount of sample loading. The small triangle indicates the different concentrations of Agrobacterium (OD600 = 0.05, 0.3, and 1.0). (i) NtRLP4 is ubiquitinated in planta. NtRLP4-myc is co-expressed transiently with HA-UBQ in N. benthamiana leaves. Extracted total proteins are immunoprecipitated by anti-myc beads and immunoblotted with anti-myc or anti-HA antibody. Experiments are repeated thrice for (g) and (h), while twice for (i). Band density is measured using ImageJ. The density values from three biological replicates are calculated and the mean value in the first lane is set at 1.0. Data are presented as mean values ± SEM. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

Rice RLP4 are targeted by salivary protein NlSP694 from Nilaparvata lugens.

(a) Domain organization of Oryza sativa RLP4 (OsRLP4). (b, c) Yeast two-hybrid and Co-IP assays showing the interaction between OsRLP4 and NlSP694. All genes are expressed with a complete coding region in Co-IP assays, while NlSP694 without a signal peptide is used in yeast two-hybrid assays. Experiments in (b) are repeated twice with the similar result. (d) The expression patterns of NlSP694 in different N. lugens tissues. Te, testis; Ov, ovary; SG, salivary gland; Ca, carcass; FB, fat body. Data are presented as mean values ± SEM (n= 3 independent biological replicates). Different lowercase letters indicate statistically significant differences at P < 0.05 level according to one-way ANOVA test followed by Tukey’s multiple comparisons test. (e-g) Effects of dsRNA treatment on insect survivorship (e), reproduction (f), and honeydew excretion (g). For survivorship analysis, a group of 30 N. lugens are reared in a cage. Three independent biological replications are performed. Differences in survivorship between the two treatments are tested by log-rank test. ns, not significant. For reproduction analysis, n= 10 and 13 individuals are tested in dsGFP- and dsNlSP694-treatment, respectively. The sterile females are excluded from data analysis. For honeydew analysis, n= 10 independent biological replicates. P-values in (f) and (g) are determined by two-tailed unpaired Student’s t test. ***P < 0.001; **P < 0.01. (h) Effect of NlSP694 on the accumulation of transient-expressed OsRLP4. OsRLP4-myc is agro-injected into Nicotiana benthamiana together with different concentrations of NlSP694-flag and NlSP7-flag. NtCf9-myc is used as a negative control. The small triangle indicates the different concentrations (OD600 = 0.05, 0.3, and 1.0) of Agrobacterium. Rubisco staining (RbcL) is conducted to visualize the amount of sample loading. Experiments are repeated three times with the similar results. Band density is measured using ImageJ. The density values from three biological replicates are calculated with the mean value in the first lane being set at 1.0.

The proposed model for the suppression of receptor-like proteins (RLPs)-mediated plant defenses by salivary effectors.

Host plants employ pattern-recognition receptors (PRRs) to detect various damage-associated molecular patterns (DAMPs) and herbivore-associated molecular patterns (HAMPs) triggered by insect feeding. The RLP4/SOBIR1 complex plays a vital role in initiating pattern-triggered immunity (PTI), including H2O2 burst, upregulation of jasmonic acid (JA), and downregulation of salicylic acid (SA), which hinders insect feeding. The whitefly Bemisia tabaci and planthopper Nilaparvata lugens independently evolved salivary proteins that targeted plant RLP4. B. tabaci salivary sheath protein BtRDP interacts with the leucine-rich repeat (LRR) domain of RLP4 from Nicotiana tabaci and Solanum lycopersicum, while N. lugens NlSP694 targets both the LRR domain and the malectin-like domain of Oryza sativa RLP4. These interactions promote the ubiquitin-dependent degradation of RLP4, thereby disrupting the stability of the RLP4/SOBIR1 complex. The presence of salivary effectors causes a hormonal shift and suppresses the H2O2 burst, finally favoring insect feeding.