Figures and data
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, as well as the salivary gland samples, are collected for western-blotting assays. Asterisk indicates non-specific binding. Rubisco staining (RbcL) is conducted to visualize the amount of 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, f) Immunohistochemical staining of BtRDP in salivary glands (e) and salivary sheath secreted from B. tabaci (f). 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. The lower images represent the enlarged images of the boxed area in the upper image. Experiments are repeated thrice for (a and b), while twice for (e and f). 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. (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. S7. (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. The EPG data are first checked for normality and homogeneity of variance, and data not fitting a normal distribution are subjected to log10 transformation. Data are presented as mean ± SEM. Statistical significance in panels (a), (b), (c), (e), (f), (h), and (i) was assessed using two-tailed unpaired Student’s t-tests. ***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 NlSP104 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 NlSP104. All genes are expressed with a complete coding region in Co-IP assays, while NlSP104 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 NlSP104 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 dsNlSP104-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 NlSP104 on the accumulation of transient-expressed OsRLP4. OsRLP4-myc is agro-injected into Nicotiana benthamiana together with different concentrations of NlSP104-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 NlSP104 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.
Characteristic of BtRDP.
(a) Deduced amino acid sequence of BtRDP. Arrow indicates the signal peptide cleavage site. (b-g) Mass spectrums of the identified unique peptides in watery saliva of Bemisia tabaci.
Analysis of insect RDP.
(a) Phylogenetic tree of RDP sequences from Aleyrodidae species. The trees are constructed by RAxML v0.9.0 using the maximum likelihood method with 1000 bootstrap replicates. Nodes with bootstrap values greater than 50 are displayed. (b) Sequence alignments of RDPs. The amino acid sequence of RDPs used for phylogenetic tree construction are aligned using ClusterX software. Black shades indicate the conserved regions.
Expression patterns of Bemisia tabaci BtRDP.
Transcripts per million (TPM) expression values of BtRDP in different tissues (a) and at different developmental stages (b) are determined based on the transcriptomic data. Data are presented as mean values ± SEM (n=3 independent biological replicates).
Expression patterns of BtActin and Bt18s rRNA..
The transcript level of each gene in different tissues (a) and at different developmental stages (b) is quantified by qRT-PCR. Bemisia tabaci tubulin is used as an internal control. The relative quantitative method (2-ΔΔCt) is used to evaluate the quantitative variation. Ov, ovary; SG, salivary gland; Ca, carcass; FB, fat body; Ny, nymph; Ps, pseudopupa; Fe, female; Ma, male. 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.
BtRDP is efficiently and specifically suppressed by dsBtRDP.
(a) Relative transcript level of BtRDP, BtFTSP, BtE3, and Bt56 after dsRNA treatments. Bemisia tabaci are treated with dsGFP and dsBtRDP. The expression pattern of each gene is determined 4 days post treatment using qRT-PCR methods. 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) (b) Protein level of BtRDP after dsRNA treatments. Actin is used as an internal control. Two independent biological replicates are displayed. (c) Immunohistochemical staining of BtRDP after dsRNA treatment. The salivary gland (SG) and its nearby tissues are collected from dsGFP- and dsBtRDP-treated Bemisia tabaci. The samples are incubated with anti-BtRDP serum conjugated with Alexa Fluor™ 488 NHS Ester (green) and actin dye phalloidinrhodamine (red), and examined by Leica SP8. The nucleus is stained with DAPI (blue). The lower image in each treatment represents the enlarged images of the boxed area in the upper image. Experiments are repeated twice, with each inspecting more than 10 salivary glands.
Effects of dsRNA treatment on insect survivorship and salivary sheath formation.
(a, b) Effects of dsRNA treatment on salivary sheath formation. Newly emerged Bemisia tabaci adults are injected with dsGFP and dsBtRDP. The dsRNA-treated B. tabaci are fed on artificial diets. The salivary sheaths left on parafilm are inspected by scanning electron microscopy (a), and the length of salivary sheath is measured from the top to base of salivary sheath (b). Data are presented as mean values ± SEM. Twenty salivary sheaths from each treatment are measured. P-value is determined by two-tailed unpaired Student’s t test. *P < 0.05. (c) Effects of dsRNA treatment on insect survivorship. A group of 20-30 B. tabaci are placed in a leaf cage, and their mortality is recorded for ten consecutive days. Three independent biological replications are performed. Differences in survivorship between the two treatments are tested by log-rank test. ns, not significant.
Typical EPG waveforms for Bemisia tabaci feeding on Nicotiana tabacum.
The B. tabaci feeding behavior can be classified into nonpenetration (np), pathway duration (C), phloem salivation (E1) and phloem ingestion (E2).
Effects of empty vector (EV) transgenic plants on Bemisia tabaci.
Two-choice experiment is performed to investigate the attraction of wild type (WT) and EV transgenic plants to B. tabaci. A group of 40 female B. tabaci are released into a device containing EV and WT leaves. The number of insects settling on each leaf is counted at 3, 6, 12, 24, 36, and 48 h (a). After 48 h, the number of eggs on each leaf is counted (b). Data are presented as mean values ± SEM. Ten independent biological replicates are performed. P-values are determined by two-tailed unpaired Student’s t test. ns, not significant.
Effects of BtRDP and BtRDP-sp overexpression on Bemisia tabaci.
Insect performance on oeBtRDP#2 (a, b) and oeBtRDP-sp#2 (c, d) transgenic plants are tested. (a, c) Comparison of insect reproduction on empty vector (EV) and oeBtRDP#2/oeBtRDP-sp#2 transgenic plants. Five B. tabaci individuals are confined to indicated plants for 3 days, and the oviposited eggs are counted. Twelve independent biological replicates are performed. (b, d) Attraction of EV and oeBtRDP#2/oeBtRDP-sp#2 leaves to B. tabaci in a two-choice equipment. A group of 40 female B. tabaci are released into a device containing indicated leaves. The number of insects settling on each leaf is counted at 3, 6, 12, 24, 36, and 48 h. After 48 h, the number of eggs on each leaf is counted. Data are presented as mean values ± SEM. Ten independent biological replicates are performed. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001.
Expression patterns of NtRLP4 in response to Bemisia tabaci infestation.
Relative transcript level of NtRLP4 in response to B. tabaci infestation are determined by qRT-PCR. Data are presented as mean values ± SEM. 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. Three independent biological replicates are performed.
Sequence alignments of SlCf9 and NtCf9.
The amino acid sequence of SlCf9 and NtCf9 are aligned using ClusterX software. Black shades indicate the conserved regions.
BtRDP interacts with NtRLP4.
(a) Yeast two hybrid assays showing the interaction between NtRLP4 and different whitefly salivary proteins (BtFTSP-sp, BtSP16.3-sp and BtSP37.4-sp, without signal peptides), as well as BtRDP-sp and truncated 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). (b) Expression of three control salivary proteins at the protein level. The target bands are indicated by asterisks. (c) Bimolecular fluorescence complementation assay showing the specific interaction between BtRDP and NtRLP4. YFP fluorescence is observed when co-expressing the N-terminal nYFP tag fused NtRLP4 and N-terminal cYFP tag fused BtRDP-sp. Bar =40 μm. (d) Co-immunoprecipitation (Co-IP) assays on oeBtRDP transgenic plants. oeBtRDP transgenic plants that overexpressing BtRDP-flag are incubated with anti-flag beads. Endogenous NtRLP4 can be immunoprecipitated by BtRDP-flag in oeBtRDP transgenic plants, while the flag tag alone in EV plants fails to immunoprecipitate endogenous NtRLP4. Rubisco staining (RbcL) is used to visualize the amount of sample loading.
NtSOBIR1 interacts with NtRLP4 but not BtRDP.
(a) Domain organization of NtSOBIR1. NtSOBIR1 contains a predicted N-terminal signal peptide (SP), a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain, and a kinase domain. (b) Co-immunoprecipitation assay showing the interaction between NtSOBIR1 and NtRLP4. (c) Co-immunoprecipitation assay showing the interaction between NtRLP4 and BtRDP, but not NtSOBIR1 and BtRDP. The complete coding region of NtSOBIR1 and NtRLP4 are fused with flag and myc tags at the C-terminal ends. Total proteins are extracted from N. benthamiana leaves co-expressing NtRLP4-myc with GFP-flag or NtSOBIR1-flag. Precipitation is performed using flag beads. The samples are probed with anti-flag and anti-myc antibodies for immunoblot analysis. Experiments are repeated twice with the similar results.
Effect of NtRLP4 overexpression on Bemisia tabaci performance.
(a) Attraction of empty vector (EV) and oeNtRLP4#2 (oeRLP#2) transgenic Nicotiana tabacum to B. tabaci in a two-choice equipment. A group of 40 female B. tabaci are released into a device containing oeRLP#2 and EV leaves. The number of insects settling on each leaf is counted at 3, 6, 12, 24, 36, and 48 h. After 48 h, the number of eggs on each leaf is counted. (b) Comparison of insect reproduction on EV and oeRLP#2 transgenic plants. Five B. tabaci individuals are confined to indicated plants for 3 days, and the oviposited eggs are counted. (c) Attraction of leaves transiently overexpressing GFP and NtRLP4-GFP to B. tabaci in a two-choice equipment. (d) Comparison of insect reproduction on N. tabacum leaves transiently overexpressing GFP and NtRLP4-GFP. (e) Relative transcript level of salicylic acid (SA)- and jasmonic acid (JA)-associated genes in N. tabacum leaves transiently overexpressing GFP and NtRLP4-GFP. PAL, phenylalanine ammonia lyase; NPR1, nonexpressor of pathogenesis-related protein 1; FAD7, fatty acid desaturase 7; PDF1.2, plant defensin 1.2. (f) Transiently overexpressing NtRLP4-GFP induces H2O2 accumulation. For bioassays in (a), (b), (c), and (d), ten independent biological replicates are performed. For qPCR analysis in (e), three independent biological replicates are performed. Data are presented as mean values ± SEM. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001. The experiment in (f) is repeated five times with the similar results.
Transcriptomic comparison of empty vector (EV) and oeNtRLP4#1 (oeRLP#1) transgenic plants.
(a) Principal component analysis (PCA) of gene expression patterns in EV and oeRLP#1 transgenic Nicotiana tabacum plants. The first two principal components (PC1 and PC2) based on transcriptomic results are shown. Four independent biological replicates are performed. (b, c) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) that significantly up-regulated (b) and down-regulated (c). Enriched P-values are calculated according to one-sided hypergeometric test using TBtools software. (d, e) Expression pattens of DEGs that annotated as RLK/RLP (d) or WRKY transcription factor (e). Gene expression patterns are illustrated by a heatmap. The max transcripts per million value of each gene is set as 100.
Effect of NtSOBIR1 overexpression and silencing on Bemisia tabaci performance.
(a) Validation of NtSOBIR1 transient overexpression using qRT-PCR (n= 4 biological replicates). (b) NtSOBIR1 transient overexpression induces cell death phenotype five days post agro-injection. (c) Comparison of insect reproduction on Nicotiana tabacum transiently overexpressing GFP and NtSOBIR1-GFP. Five B. tabaci individuals are confined to indicated plants for 3 days, and the oviposited eggs are counted. (d) Attraction of GFP and NtSOBIR1-GFP leaves to B. tabaci in a two-choice equipment. A group of 40 female B. tabaci are released into a device containing GFP and NtSOBIR1-GFP leaves. The number of insects settling on each leaf is counted at 3, 6, 12, 24, 36, and 48 h (left). After 48 h, the number of eggs on each leaf is counted (right). (e) Silencing efficiency of hairpin NtSOBIR1 using qRT-PCR (n= 3 independent biological replicates). (f) Transiently silencing NtSOBIR1 affect growth of infiltrated leaves. (g) Comparison of insect reproduction on Nicotiana tabacum leaves infiltrated with hairpin empty vector (EV) and NtSOBIR1. (h) Attraction of hairpin EV and NtSOBIR1-treated leaves to B. tabaci in a two-choice equipment. The experiments in (b) and (f) are repeated ten times with the similar results. In (c), (d), (g), and (h), ten independent biological replicates are performed. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001; **P < 0.01.
Effects of RLP silencing on Bemisia tabaci performance.
(a) Detection of NtRLP4 protein in empty vector (EV) and NtRLP4-silenced (RNAi-RLP) transgenic Nicotiana tabacum. Rubisco staining (RbcL) is conducted to visualize the amount of sample loading. (b, c) Attraction of EV and RNAi-RLP plants to B. tabaci in a two-choice equipment. Two NtRLP4-silenced plants (RNAi-RLP#1 in b; RNAi-RLP#2 in c) are assayed. A group of 40 female B. tabaci are released into a device containing two leaves. The number of insects settling on each leaf is counted at 3, 6, 12, 24, 36, and 48 h. After 48 h, the number of eggs on each leaf is counted. Ten independent biological replicates are performed. Data are presented as mean values ± SEM. P-values are determined by two-tailed unpaired Student’s t test. ***P < 0.001.
Influence of BtRDP on NtRLP4 accumulation by fluorescent analysis.
The NtRLP4-GFP is transiently co-expressed with red fluorescent protein (RFP)-mCherry, BtFTSP-mCherry, or BtRDP-mCherry via agroinfiltration. The samples are imaged by confocal microscopy at 48 h post injection. RFP-mCherry and BtFTSP-mCherry are used as negative controls. Three independent biological replicates are performed, and three representative images are taken in each biological replicate. Fluorescence intensity is measured using ImageJ. The intensity values from three biological replicates are calculated and the mean value in the upper left image is set at 1.0. Representative fluorescence images are displayed. The small triangle indicates the different concentrations (OD600 = 0.1 and 1.0) of Agrobacterium. Bar= 40 μm.
Effects of BtRDP on suppressing NtRLP4-associated plant defenses.
(a) Comparison of insect reproduction on Nicotiana tabacum plants transiently overexpressing GFP along and BtRDP-mCherry/NtRLP4-GFP. Five Bemisia tabaci individuals are confined to indicated plants for 3 days, and the oviposited eggs are counted. (b) Attraction of GFP- and BtRDP-mCherry/NtRLP4-GFP-expressed leaves to B. tabaci in a two-choice equipment. A group of 40 female B. tabaci are released into a device containing two leaves. The number of insects settling on each leaf is counted at 3, 6, 12, 24, 36, and 48 h. After 48 h, the number of eggs on each leaf is counted. Data are presented as mean values ± SEM. Ten independent biological replicates are performed. P-values are determined by two-tailed unpaired Student’s t test. *P < 0.05; ns, not significant.
Degradation of NtRLP4 by purified BtRDP-sp in Nicotiana benthamiana leaves.
Recombinant BtRDP-sp-his and GFP-his proteins are expressed in Escherichia coli, and purified by Ni-NTA. N. benthamiana plants overexpressing NtRLP4-myc or NtCf9-myc are then infiltrated with different concentrations of purified BtRDP-sp-his and GFP-his. The samples are probed with anti-myc antibodies for immunoblot analysis. Rubisco staining (RbcL) is conducted to visualize the amount of sample loading. Coomassie brilliant blue (CBB) staining is conducted to visualize the amount of recombinant BtRDP-sp-his and GFP-his proteins. Experiments are repeated three times with the similar results. 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.
Effect of BtRDP on NtRLP4 and NtSOBIR1.
NtRLP4-myc and NtSOBIR1-flag were transiently co-expressed with different concentration of BtRDP-flag in Nicotiana benthamiana plants through Agrobacterium infiltration. 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 amount of BtRDP were controlled by infiltrating different concentrations of Agrobacterium (OD600 = 0.1, 0.3, and 1.0).
Effect of BtRDP on the transcript level of NtRLP4.
NtRLP4-myc is agroinfiltrated together with different concentration of BtRDP-flag or GFP-flag into Nicotiana benthamiana leaves. The transcript level of NtRLP4 is determined by qRT-PCR. Data are presented as mean values ± SEM. Three independent biological replicates are performed. The same lowercase letters “a” indicate no statistically significant differences at P < 0.05 according to the one-way ANOVA test followed by Tukey’s multiple comparisons test.
Effects of autophagy inhibitor on NtRLP4 accumulation.
NtRLP4-myc and BtRDP-flag were transiently co-expressed in Nicotiana benthamiana plants through Agrobacterium infiltration. Co-infiltrated leaves are treated with autophagy inhibitor BAF and 3-MA 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 = 0.1 and 1.0). Experiments are repeated three times with the similar results. 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.
Sequence alignment and phylogenetic tree of RLP4 homologs.
(a) The amino acid sequence of RLP4s from Nicotiana tabacum, N. benthamiana, Solanum lycopersicum, and Oryza sativa are aligned using ClusterX software. Black shades indicate the conserved regions. (b) Phylogenetic analysis of RLP4 and Cf9-associated genes used in this study. The unrooted phylogenetic trees are constructed by RAxML v0.9.0 using the maximum likelihood method with 1000 bootstrap replicates.
Influence of BtRDP on RLP4 homology in Solanum lycopersicum.
(a) Domain organization of S. lycopersicum RLP4 (SlRLP4). SlRLP4 contains a predicted N-terminal signal peptide (SP), a malectin-like domain, a LRR domain, and a transmembrane (TM) domain. (b, c) Yeast two-hybrid and co-immunoprecipitation (Co-IP) assays showing the interaction between BtRDP and SlRLP4. In (b), BtRDP is expressed without a signal peptide (BtRDP-sp), while SlRLP4 and SlCf9 are expressed without a signal peptide and transmembrane domain (SlRLP4(23-546) and SlCf9(24-524)). (d) Effect of BtRDP on the accumulation of SlRLP4. SlRLP4-myc and SlCf9-myc are agro-injected together with different concentration of BtRDP-flag or GFP-flag. In (c, d), the complete coding region of BtRDP, SlRLP4, SfCf9 are fused with flag or myc tags at C-terminal ends, respectively. 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. For SlRLP4-myc bands (lane 1-6), the lane 1 is set at 1.0. For SlCf9-myc bands (lane 7-9), the lane 7 is set at 1.0.
Yeast two hybrid assays showing the interaction between OsRLP4 and salivary proteins from planthopper species.
OsRLP4(29-551) (OsRLP4 without signal peptides and transmembrane domains) is fused to pGADT7 vector, while salivary proteins without signal peptides are fused to pGBKT7 vector, respectively. 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). Twenty salivary proteins are selected. The accession number of each protein is listed as follow: (1) MF278694.1, (2) XP_039291719.1, (3) KU365967.1, (4) MF278706.1, (5) MF278711.1, (6) MF278714.1, (7) MF278715.1, (8) MF278720.1, (9) XP_022195702.1, (10) XP_022196818.2, (11) XP_039284081.1, (12) XP_022192221.2, (13) XP_022207944.2, (14) KT764973,15) RZF44823.1, (16) RZF42644.1, (17) RZF33006.1, (18) RZF48570.1, (19) RZF42817.1, (20) RZF33751.1.
Three-dimensional structure of BtRDP and NlSP104.
AlphaFold2 is used to predicate the protein structure. The structures are colored as a spectrum from N-terminus (blue) to C-terminus (red).
Expression patterns of Nilaparvata lugens NlSP104.
Transcripts per million (TPM) expression values of NlSP104 in different tissues are determined based on the transcriptomic data. The head that contains salivary glands exhibits highest NlSP104 expression in male and female N. lugens. Data are presented as mean values ± SEM (n=3 independent biological replicates).
Original images for blots in Figures 5 and Supplementary Figure 5-17.
Original images for blots in Supplementary Figure 20-25.
Identification of RDP and SP101 homologs in insect species
