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 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. Horizontal bars in B, C indicate the mean ± 1 SEM. *p < 0.05, ***p<0,001. The entire series of choice tests 1 to 6 were performed in parallel and repeated independently 3 times for progeny count and 2 times for honeydew quantification - data presented in B and C include the pooled results of the independent choice test series.

The online version of this article includes the following source data and figure supplement(s) for figure 1:

Figure supplement 1: Data distributions of independent repeats that were used to generate graphs displayed in B and C.

Figure supplement 2: Macrosteles quadrilineatus female leafhoppers show no preference for A. thaliana Col-0 wild-type plants exposed to conspecific male leafhoppers.

Figure supplement 3: Female M. quadrilineatus preference for male-exposed SAP54 plants is unlikely to involve long-distance cues.

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). C, D. MapMan diagrams of A. thaliana DEGs involved in biotic stress. Pathways are indicated and each square is a gene with red versus green shades illustrating the level of up- or downregulation, respectively, of the gene in insect-exposed versus cage-only, non-exposed leaves of GFP (C) and SAP54 (D) plants.

The online version of this article includes the following source data and figure supplement(s) for figure 2:

Figure supplement 4. Experimental design and selection of transcripts for downstream analysis.

Figure supplement 5. Biological variation and role of outliers in separation of treatments and identification of differentially expressed genes.

Figure supplement 6. The cage-only SAP54 vs cage-only GFP treatments show few biotic stress DEGs.

Supplementary file 1. FPKM and differential expression values of 17’153 differentially expressed genes (DEGs) included in the response analyses of plants to SAP54 vs GFP and male vs female leafhopper exposure.

Supplementary file 2. IDs and log2-fold changes of DEGs of male and female M. quadrilineatus leafhopper-exposed GFP and SAP54 plants compared to insect-free GFP plants.

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.

Supplementary file 4. Source data for generating Fig. 2CD - Enrichment statistics of biotic stress bins and fold-change of DEGs in each bin.

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 signalling proteins involved in plant responses to biotic stress. The number of DEGs belonging to each entity are indicated as squares above or adjacent to each protein category with red versus green shades illustrating the level of up- or downregulation, respectively, of the gene in insect-exposed versus cage-only, non-exposed leaves as shown in the upper panel.

The online version of this article includes the following source data and figure supplement(s) for figure 3:

Figure supplement 7: Manually drawn MapMan image for defence signalling pathway visualisation.

Supplementary file 5: Manually curated and assigned defence signalling bins for MapMan import.

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.

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.

The online version of this article includes the following source data and figure supplement(s) for figure 4:

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.

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.

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. 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.

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 (panel A), 35S:GFP-SAP54 A. thaliana rosettes (panel B) and AY-WB phytoplasma-infected plants (panel 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 2 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.

The online version of this article includes the following source data and figure supplement(s) for figure 6:

Figure supplement 8: Data distributions of independent repeats that were used to generate graphs displayed A-D.

Supplemental file 9. Fold-expression changes of MADS-box transcription factor genes insect-exposed SAP54 vs GFP leaves.

Proposed model for SVP-dependent enhancement of insect vector colonisation of phytoplasma-infected host plants.

SAP54 recruits proteasome shuttle protein RAD23 to degrade MTFs in 26S proteasome dependent manner (MacLean et al., 2014). This destabilises SVP which is required for recruitment female leafhoppers to lay eggs on male-exposed leaves during vegetative growth phase. In addition, SAP54-induced perturbations in the functioning of MTF network leads to the generation of leaf-like flowers after the phase transition.

Data distributions of independent repeats that were used to generate graphs displayed in Fig. 1 B and C.

Experimental design for choice tests (treatments) 1 to 6 represented in Figure 1 of the main text is shown in panel 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 (panel B) and feeding (honeydew excretion) data (panel C). Each data point in panel B represents oviposition choice of 10 female insects. Each data point in panel 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 characterised by the skewed distribution above the 50% reference line. Each reproduction choice experiment consisted of 6 choice arenas and was performed independently 3 times (Repeat1, Repeat2, Repeat3 with corresponding bar colours), involving a total of 540 insects. Each feeding choice experiment consisted of 6 choice arenas and was performed independently 2 times (Repeat1 and Repeat2 with corresponding bar colours), involving a total of 480 insects). Paired t-test statistics for combined repeated experiments are summarised in the table below the boxplots.

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 3 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).

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 1h. 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 4 choice arenas (cages) corresponding to a single datapoint in the boxplot. Each test was repeated independently 2 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 4 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.

Experimental design and selection of transcripts for downstream analysis.

A. Five (5) male or 5 female M. quadrilineatus individuals were placed within a clip-cage onto a single rosette leaf of 35S:GFP-SAP54 or 35S:GFP plants. Empty clip-cages without insects served as controls. B. Mapped SAP54 reads plotted against GFP reads and colour 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 colour coded for treatments (m=male; f=female; n=no insect) on GFP and SAP54 plants.

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 panel C indicate the outliers (SAP54_male, GFP_male, SAP54_female) that were removed in panel D.

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.

Manually drawn MapMan image for defence signalling pathway visualisation.

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 6 choice arenas. Each experiment was repeated independently 2 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 characterised 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 summarised in table (E).

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.

Includes the following source data and figure supplement(s) for Figure supplement 9:

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.

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.

Supplementary file 12: GO-term and MapMan functional enrichment of shared 155 DEGs between ‘’male-exposed SAP54 vs male-exposed GFP’’ comparison and ‘’male-exposed svp vs male exposed wild type’’ comparison.