The phytoplasma SAP54 effector acts as a molecular matchmaker for leafhopper vectors by targeting plant MADS-box factor SVP
- John Innes Centre, Norwich Research Park, United Kingdom
Figures
Figure 1 with 3 supplements
M. quadrilineatus leafhopper preference to reproduce and feed on SAP54 versus GFP plants is dependent on leaf exposure to leafhopper males.
(A) Experimental design of 6 choice tests (treatments) with 10 male and/or 10 female insects, as indicated, on 6 weeks old A. thaliana rosettes. Dashed circles indicate clip-cages and the arrow indicates the removal of males before the start of the choice test. Each choice test (treatment) is placed in a separate cage. (B) Percentages (%) of nymphs found on SAP54 versus GFP plants. (C) Percentages (%) of leafhopper honeydew secretions in choice tests where insects are allowed to feed on either SAP54 or GFP plants. Horizontal bars in (B, C) indicate the mean ± 1 SEM. *p<0.05, ***p<0.001. The entire series of choice tests 1–6 were performed in parallel and repeated independently three times for progeny count and two times for honeydew quantification - data presented in (B) and (C) include the pooled results of the independent choice test series.
Figure 1—figure supplement 1
Data distributions of independent repeats that were used to generate graphs displayed in Figure 1B and C.
Experimental design for choice tests (treatments) 1–6 represented in Figure 1 of the main text is shown in (A). Each choice test was performed in a separate choice cage (arena) but simultaneously with the other choice tests in the same room. Boxplots show variation among repeated experiments for progeny (B) and feeding (honeydew excretion) data (C). Each data point in (B) represents oviposition choice of 10 female insects. Each data point in (C) corresponds to feeding choice of 20 adult insects within a single choice arena. No preference for either test or control plant in a choice cage is represented by the 50% reference line. Survived progeny or feeding preference for SAP54 plants is characterized by the skewed distribution above the 50% reference line. Each reproduction choice experiment consisted of six choice arenas and was performed independently three times (Repeat1, Repeat2, Repeat3 with corresponding bar colors), involving a total of 540 insects. Each feeding choice experiment consisted of six choice arenas and was performed independently two times (Repeat1 and Repeat2 with corresponding bar colors), involving a total of 480 insects. Paired t-test statistics for combined repeated experiments are summarized in the table below the boxplots.
Figure 1—figure supplement 2
Macrosteles quadrilineatus female leafhoppers show no preference for A. thaliana Col-0 wild-type plants exposed to conspecific male leafhoppers.
(A) In each choice arena 10 female leafhoppers were allowed to choose to lay eggs between insect-free A. thaliana Col-0 plants (with an empty clip-cage) and Col-0 plants with 10 male insects confined in clip-cages. Eggs laid by females were counted over the entire plant. Bars are 1 standard error of the mean. (B) This experiment was independently repeated three times with combined total of 180 female insects. Paired t-test was performed on combined dataset considering all repeated experiments (t=1.09; p=0.325).
Figure 1—figure supplement 3
Female M. quadrilineatus preference for male-exposed SAP54 plants are unlikely to involve long-distance cues.
Choice tests were performed in separate cages, inside which two test plants were placed in non-transparent black plastic boxes that permit plant volatiles to escape but conceal the plants inside. Transparent or green sticky landing platforms were placed over each box (horizontal dashed bars). Twenty (20) female insects were released in a choice arena and females sticking to the transparent or sticky landing platforms were recorded after 1 hr. Bars indicate the percentage of recaptured females on each trap type. Boxplots show data distribution for recaptured females on SAP54 plants. (A) Each choice test contained four choice arenas (cages) corresponding to a single datapoint in the boxplot. Each test was repeated independently two times. Females preferred the green over transparent platforms regardless of plant identity in within the trap. Figure shows the results of combined repeated choice tests. Choice test 1, t7=1.521, p=0.172; Choice test 2, t7=0.226, p=0.828; Choice test 3, t7=21.826, p<0.001; Choice test 4, t7=21.104, p<0.001. (B) Females do not show preference for platforms of cages that contain male-exposed plants over insect free plants. Figure shows the results of single choice test with four choice arenas. Choice test 1, t3=0.245, p=0.822; Choice test 2, t3=0.322, p=0.769; Choice test 3, t3=0.302, p=0.783; Choice test 4; t3=0.555, p=0.617.
Figure 2 with 3 supplements
SAP54 plants display a dramatically altered leaf response to male leafhoppers by transcriptionally downregulating the majority of biotic stress and plant defence related processes.
(A-B) Euler-Venn diagrams illustrating DEGs in leaves of GFP plants (A) and SAP54 plants (B) exposed to female leafhoppers compared to male leafhoppers, versus leaves of plants in the control group (cage-only, non-exposed plants). DEG analysis was performed on 17,153 leaf-expressed genes available in Supplementary file 1. DEG IDs listed within each Venn diagram are provided in Supplementary file 2. (C-D). MapMan diagrams of A. thaliana DEGs involved in biotic stress from female (red insect) or male (blue insect) exposed GFP (C) or SAP54 plants (D). Biotic stress related pathways were significantly enriched with DEGs from male exposed SAP54 plants compared to other functions listed in Supplementary file 3. Names of functional bins (e.g. respiratory burst or MAPK) are listed next to the corresponding color boxes and fully listed in Supplementary file 4 along with individual transcript names and their fold changes. Red color boxes indicate upregulated, but green – downregulated DEGs based on log2(fold change).
Figure 2—figure supplement 1
Experimental design and selection of transcripts for downstream analysis.
(A) Five male or five female M. quadrilineatus individuals were placed within a clip-cage onto a single rosette leaf of 35 S:GFP-SAP54 or 35 S:GFP plants. Empty clip-cages without insects served as controls. (B). Mapped SAP54 reads plotted against GFP reads and color coded for treatments (m=male; f=female; n=no insect) on GFP and SAP54 plants. (C) Multi-dimensional analysis (MDA) plot demonstrates grouping of cDNA libraries according to treatment. (D) Transcripts with normalized read count (FRKM) ≥1 in any of the sequenced libraries (10,196) and significantly differentially expressed transcripts (DEGs) from any of the treatment pairwise comparisons (6947) were considered for downstream analysis (total = 17,153 transcripts).(E). Median of all transcript FRKM plotted against GFP reads and color coded for treatments (m=male; f=female; n=no insect) on GFP and SAP54 plants.
Figure 2—figure supplement 2
Biological variation and role of outliers in separation of treatments and identification of differentially expressed genes.
Euler-Venn diagrams illustrating DEGs that differentiate SAP54 and GFP plants exposed to female or male or no leafhoppers when outliers are retained (A) or removed (B). Principal component analysis (PCA) plots illustrate variation within and among treatments when outliers are retained (C) or removed (D). Arrows in (C) indicate the outliers (SAP54_male, GFP_male, SAP54_female) that were removed in panel (D).
Figure 2—figure supplement 3
The cage-only SAP54 vs cage-only GFP treatments show a limited number of biotic stress DEGs.
(A) Euler-Venn diagrams illustrating DEGs in SAP54 plants exposed to female or male or no leafhoppers compared to no insect exposed (empty cage-only) GFP plants. (B) Mapman diagram of DEGs involved in biotic stress in the cage-only SAP54 vs cage-only GFP plants. Pathways are indicated and each square is a gene with red versus green shades illustrating the level of up- or downregulation.
Figure 3 with 1 supplement
Genes involved in A. thaliana defence responses are predominantly down regulated in male-exposed leaves of SAP54 plants.
MapMan diagrams with the manually curated bins for plant cell-surface receptors, NLRs, RLCKs, MAPKs and hormone biosynthesis and signaling proteins involved in plant responses to biotic stress. Full list of transcripts assigned to each bin can be retrieved from Supplementary file 5. DEGs are indicated as boxes above or adjacent to each protein category. Red color boxes indicate upregulated, but green – downregulated DEGs based on log2(fold change). Female (red insect) or male (blue insect) exposed GFP or SAP54 plants are always compared to insect free plants for DEG analysis and indicated by the insect image above each panel. Individual transcript names and their fold changes within each panel are listed in Supplementary file 6. The manually drawn defence signaling background image can be downloaded as Figure 3—figure supplement 1.
Figure 3—figure supplement 1
Manually drawn MapMan image for defence signaling pathway visualization.
Figure 4
Biotic stress response genes are predominantly downregulated in male-exposed SAP54 versus GFP plants.
Plant biotic stress is among the most enriched bins with male-specific responses in SAP54 leaves.
Figure 5 with 1 supplement
SAP54 interacts with multiple MADS-box transcription factors and mediates their degradation.
(A) Western blots showing degradation of MTFs in the presence of SAP54 in A. thaliana protoplasts. Assays were repeated twice with similar results and available in Figure 5—figure supplement 1 and the associated source data. (B) Yeast two-hybrids assays with GAL4-activation (AD) and GAL4-DNA binding (BD) domains fused to the test proteins. -L-W-H-A denote auxotrophic SD media lacking leucine, tryptophan, histidine or adenine, conditionally supplemented with 3-Amino-1,2,4-triazole (3AT). EV, empty vector control.
Figure 5—figure supplement 1
SAP54 mediates degradation of multiple MADS-box transcription factors.
Two independent transient expression experiments (assays) showing degradation of MTFs in the presence of SAP54 in A. thaliana protoplasts. The destabilization efficiency was calculated as the HA peak intensity divided by the RuBisCo large subunit (rbcL) peak intensity from the same sample using ImageJ. MTF levels in presence of SAP54 expressed as ratio relative to MTF intensity in GFP lanes which was normalized to 1.
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Figure 5—figure supplement 1—source data 1
Gel/blot source data.
- https://cdn.elifesciences.org/articles/98992/elife-98992-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Gel/blot source data.
- https://cdn.elifesciences.org/articles/98992/elife-98992-fig5-figsupp1-data2-v1.zip
Figure 6 with 2 supplements
The MADS-box transcription factor SVP is required for female preference to reproduce on male-exposed SAP54 plants.
(A-C) Choice test with equal numbers of 10 males and 10 females on wild type and MTF null mutant A. thaliana rosettes (A), 35 S:GFP-SAP54 A. thaliana rosettes (panel B) and AY-WB phytoplasma-infected plants (C). (D). Choice tests with 10 females on A. thaliana svp and maf5 null mutants without males. The entire series of choice tests depicted in panels (A–D) were conducted in parallel and repeated independently two times. ** p<0.01, *** p<0.001. Bars are ± 1 SEM. (E). Expression levels of SVP and MAF5 in leaves among treatments showing that SVP and MAF5 are upregulated in male-exposed SAP54 plants.
Figure 6—figure supplement 1
SVP enhances female egg-laying preference for male exposed plants in phytoplasma and SAP54-dependent manner.
MTF mutants maf5 and svp display preferential leafhopper reproduction over wild-type plants in mixed-sex choice tests (A). Preferential leafhopper reproduction on SAP54 plants is abolished in svp but not maf5 mutant (B). Preferential leafhopper reproduction on svp and maf5 mutants is abolished in AY-WB infected plants (C). svp displays preferential leafhopper reproduction over wild-type plants in presence of males but not when males are removed (D). Each datapoint represents reproductive choice of 10 female and 10 male insects within single choice arena. Experiment consists of six choice arenas. Each experiment was repeated independently two times. No preference for either test or control plant in a choice cage is represented by the 50% reference line. Oviposition preference for test or control plant is characterized by significant deviation from the 50% reference line. All pairwise comparisons done with paired t-tests by combining all datapoints from the two repeated experiments for each choice test. Test statistics summarized in table (E).
Figure 6—figure supplement 2
Comparison between male and female colonized svp plants reveal SVP-dependent insect regulated biotic stress genes.
Pairwise comparisons are schematically depicted and abbreviated in panel A and correspond to the Venn diagrams and MapMan biotic stress graphs in panels B-E. The two Venn diagrams in panel B display the differences in the magnitude of response to male and female leafhopper exposure of SAP54 plants and svp mutants compared to insect-exposed GFP and wild-type controls, respectively. Panel C represents log2 fold change of biotic stress DEGs in male-and female-exposed svp mutants according to the MapMan annotation. 8 vs 6 includes male 347+460 DEGs, 7 vs 5 includes female 464+460 DEGs, and male specific 8 vs 6 includes male 347DEGs. Panel D displays the overlap between 49 male-specific (not regulated in female) DEGs of SAP54 plants and svp mutants while the panel E shows 155 DEGs that are shared by male-exposed SAP54 and male-exposed svp plants regardless of their regulation in female-exposed SAP54 or svp plants.
Figure 7
Model illustrating how the phytoplasma effector SAP54 facilitates leaf colonization by leafhopper vectors, which are essential for phytoplasma transmission and spread.
During phytoplasma infection, the SAP54 effector is secreted and promotes the degradation of MADS-box transcription factors. This degradation of MADS-box transcription factor short vegetative phase (SVP) specifically leads to the downregulation of biotic stress responses to male leafhoppers, thereby attracting female leafhoppers to the leaves. The females then lay eggs, and their offspring acquire phytoplasmas during feeding. Thus, SAP54 likely enhances phytoplasma spread by increasing female insect vector reproduction in a process that requires SVP and the presence of males. An additional implication of this model is that male leafhoppers might benefit from phytoplasma infection by attracting more mates. This work corroborates the previous findings that both the SAP54-mediated degradation of MADS-box transcription factors and the leafhopper attraction phenotype are dependent on the 26 S proteasome shuttle factor RAD23 (MacLean et al., 2014) and that leafhoppers are specifically attracted to leaves and not the leaf-like flowers that are induced by SAP54 actions (Orlovskis and Hogenhout, 2016).
Additional files
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Supplementary file 1
FPKM and differential expression values of 17'153 genes included in the response analyses of plants to SAP54 vs GFP and male vs female leafhopper exposure.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp1-v1.xlsx
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Supplementary file 2
IDs and log2-fold changes of differentially expressed genes (DEGs) of male and female M. quadrilineatus leafhopper-exposed GFP and SAP54 plants compared to insect free GFP plants.
Tab A: GFP plants exposed to caged females versus cages alone. Tab B: GFP plants exposed to caged males versus cages alone. SAP54 plants exposed to caged females versus cages alone. Tab D: SAP54 plants exposed to caged males versus cages alone. Tab E: SAP54 versus GFP plants exposed to cages alone.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp2-v1.xlsx
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Supplementary file 3
MapMan build-in functional bins enriched for DEGs in male and female M. quadrilineatus leafhopper-exposed GFP and SAP54 plants compared to insect-free GFP plants.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp3-v1.xlsx
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Supplementary file 4
MapMan build-in biotic stress functional bins enriched for DEGs in male and female M. quadrilineatus leafhopper-exposed GFP and SAP54 plants compared to insect-free/cage-only GFP plants.
Tab A: MapMan build-in biotic stress functional bins enriched for DEGs. p-values based on Wilcoxon rank test with BH correction. Bins highlighted in bold are enriched for DEGs. Individual transcript identities within significantly enriched bins are provided in the tabs B, C, D, E. Tab B: list of DEGs within significantly enriched biotic stress bins for GFP plants exposed to caged females versus cages alone. list of DEGs within significantly enriched biotic stress bins for GFP plants exposed to caged males versus cages alone. Tab D: list of DEGs within significantly enriched biotic stress bins for SAP54 plants exposed to caged females versus cages alone. Tab E: list of DEGs within significantly enriched biotic stress bins for SAP54 plants exposed to caged males versus cages alone.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp4-v1.xlsx
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Supplementary file 5
Manually curated and assigned defence signalling bins for MapMan import.
Tab A, Functional bin categories. Tab B, IDs of genes assigned to each of the functional bins listed in Tab A. ‘’Type T’’ means ‘’transcript’’, nomenclature used according to in-built MapMan pathway files.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp5-v1.xlsx
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Supplementary file 6
Functional bins for manually annotated defence genes enriched for DEGs in male and female M. quadrilineatus leafhopper-exposed GFP and SAP54 plants compared to insect free GFP plants.
Tab A: Functional bins for manually annotated defense genes enriched for DEGs. P-values based on Wilcoxon rank test with BH correction. Bins highlighted in bold are enriched for DEGs. Individual transcript identities within significantly enriched bins are provided in the tabs B, C, D, E. Tab B: list of DEGs within significantly enriched biotic stress bins for GFP plants exposed to caged females versus cages alone. list of DEGs within significantly enriched biotic stress bins for GFP plants exposed to caged males versus cages alone. Tab D: list of DEGs within significantly enriched biotic stress bins for SAP54 plants exposed to caged females versus cages alone. Tab E: list of DEGs within significantly enriched biotic stress bins for SAP54 plants exposed to caged males versus cages alone.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp6-v1.xlsx
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Supplementary file 7
MapMan build-in functional bins enriched for DEGs in SAP54 versus GFP plants with or without exposure to male and female M. quadrilineatus leafhoppers.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp7-v1.xlsx
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Supplementary file 8
Biotic stress bins from MapMan build-in and manually curated defence signalling pathway bins enriched for DEGs in GFP and SAP54 plants with or without exposure to male and female M. quadrilineatus leafhoppers.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp8-v1.xlsx
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Supplementary file 9
Fold-expression changes of MADS-box transcription factor genes insect-exposed SAP54 vs GFP leaves.
Tab A: List of 20 genes encoding MADS-box transcription factors that are expressed in leaves in the (insect-exposed) SAP54 and GFP plants. Tab B: List of all 107 genes annotated as MADS-box transcription factors in A. thaliana (de Folter et al., 2005).
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp9-v1.xlsx
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Supplementary file 10
DEG encoded function enrichment for all MapMan built-in bins in female- or male-exposed svp plants vs female- or male-exposed wild type plants. p-values based on Wilcoxon rank test and have BH correction.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp10-v1.xlsx
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Supplementary file 11
DEG encoded function enrichment for MapMan built-in built-in biotic stress and manually designed defence signalling pathways in female- or male-exposed svp plants vs female- or male-exposed wild type plants.
Tab A: biotic stress and defence signalling pathway bins enriched with female- or male-specific DEGs. p-value based on Wilcoxon rank test and BH correction. Tab B: list if DEGs within enriched bins for clip-caged females on svp plants vs clip-caged females on wild type plants. list if DEGs within enriched bins for clip-caged males on svp plants vs clip-caged males on wild type plants. Tab D: since no enriched bins found for male-specific DEGs in tab A, no transcripts highlighted here.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp11-v1.xlsx
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Supplementary file 12
GO-term and MapMan functional enrichment on shared 155 DEGs between ‘’male-exposed SAP54 vs male-exposed GFP’’ comparison and ‘’male-exposed svp vs male-exposed wild type’’ comparison.
Tab A: GO-term enrichment with 155 DEGs shared by male-exposed SAP54 plants (versus male-exposed GFP plants) and male-exposed svp plants (vs male-exposed wild type plants) and depicted in Figure 6—figure supplement 2D. Tab B: gene names, encoded function description and log2(fold) change for the 155 DEGs analysed in tab A and depicted in Figure 6—figure supplement 2D. Common functions identified between (1) MapMan function enrichment for 3816 DEGs on male-exposed SAP54 plants vs male-exposed GFP plants and (2) MapMan function enrichment for 807 DEGs on male-exposed svp plants vs male-exposed wild type plants; Venn diagram of the DEGs in Figure 6—figure supplement 2D.
- https://cdn.elifesciences.org/articles/98992/elife-98992-supp12-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/98992/elife-98992-mdarchecklist1-v1.docx
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The phytoplasma SAP54 effector acts as a molecular matchmaker for leafhopper vectors by targeting plant MADS-box factor SVP
eLife 13:RP98992.
https://doi.org/10.7554/eLife.98992.3
