Fig 1.
Aphid probing during host and non-host interactions.
(A) Autofluorescence around aphid probe sites, indicated by white arrows, was visualized using laser confocal microscopy. Scale bars 100μm. (B) Aphid colonization of Arabidopsis by Myzus persicae, M. cerasi and Rhopalosiphum padi. Graph shows the mean number of nymphs produced after two weeks on wild type Col-0 plants. Error bars indicate standard error. A Student’s t-test was used for statistical analysis of M. persicae versus M. cerasi progeny (*** indicates p-value < 0.001). Three independent biological replicates were carried out, with 10 plants per treatment per replicate.
Fig 2.
Clustering of 874 differentially expressed Arabidopsis genes during host and non-host interactions with aphids.
Using one-way ANOVA in GeneSpring (Bonferroni correction, p-value ≤ 0.05), we identified 874 genes that display significant differential expression across different aphid treatments and timepoints. Hierarchical gene tree cluster analysis of these 874 genes using GeneSpring software identified three main clusters of genes (A, B and C) according to their expression profiles across all treatments and timepoints. Low gene expression levels are indicated by blue colour and high gene expression levels are indicated by red colour. Mc indicates Myzus cerasi, Mp indicates M. persicae and Rp indicates Rhopalosiphum padi.
Fig 3.
Overlap of Arabidopsis differentially expressed genes across different aphid interactions.
Venn diagram analyses of 874 differentially expressed as determined by one-way ANOVA (Bonferroni correction, p-value ≤ 0.05) across different aphid interactions and timepoints. (A) Venn diagrams showing the numbers of genes that are down-regulated during different aphid interactions at 3h, 6h and 24h post aphid exposure. (B) Venn diagrams showing the numbers of genes that are up-regulated during different aphid interactions at 3h, 6h and 24h post aphid exposure. Mc indicates Myzus cerasi, Mp indicates M. persicae and Rp indicates Rhopalosiphum padi.
Fig 4.
Expression profile of Arabidopsis genes showing opposite gene expression changes during aphid host and non-host interactions.
Using volcano plot analyses (fold change ≥ 2.0, p-value ≤ 0.05) in GeneSpring, we identified 11 genes with statistically significant opposite gene expression changes during different aphid interactions. Hierarchical gene tree cluster analysis in GeneSpring generated an overview of the expression changes of these 11 genes 3h, 6h and 24h after aphid exposure. Low gene expression levels are indicated by blue color and high gene expression levels are indicated by red color.
Fig 5.
Arabidopsis knock-out mutants show altered susceptibility to Myzus persicae and Myzus cerasi.
Four-week old plants were exposed to two adult aphids and nymph production was counted after 10 days. Average nymph production was calculated from three independent replicated experiments, with 10 plants per replicate per treatment. Error bars indicate standard error. The two-tailed Student's t-test was used for statistical analyses (*** indicates p-value < 0.001, ** indicates p-value < 0.01, * indicates p-value < 0.05). Mc indicates Myzus cerasi, Mp indicates M. persicae.
Fig 6.
Arabidopsis knock-out mutants show altered susceptibility to Myzus persicae, Myzus cerasi and Rhopalosiphum padi.
(A) Graph showing R. padi aphid survival on the control (Col-0) and a vsp1 mutant line over 6 days. Five adult aphids were placed on four-week old plants and survival was monitored the following 6 days. Three independent biological replicates were carried out, with 10 plants per replicate. Error bars indicate standard error. (B) Graph shows the percentage of R. padi survival between day 3 and 4 on a vsp1 mutant line and Col-0 wild-type plants. Data is from the same experiment as described in (A). The two-tailed Student's t-test was used for statistical analyses (*** indicates p-value<0.001). Error bars indicate standard error. (C) M. persicae and M. cerasi performance on an Arabidopsis vsp1 knock-out line and Col-0 wild-type plants. Four-week old plants were exposed to two adult aphids and nymph production was counted after 10 days. Average nymph was calculated from three independent replicated experiments, with 10 plants per replicate per treatment. The two-tailed Student's t-test was used for statistical analyses (* indicates p-value<0.05). (D) Graph showing R. padi aphid survival on the control (Col-0) and a mutant line for an Arabidopsis gene predicted to encode an ABA-responsive protein over 6 days. Same experimental set-up and analyses as described in A. Error bars indicate standard error. (E) Graph showing the percentage of R. padi survival between day 3 and 4 on a mutant line for an Arabidopsis gene predicted to encode an ABA-responsive protein and Col-0 wild-type plants. Data are from the same experiment as described in D. The two-tailed Student's t-test was used for statistical analyses (*** indicates p<0.001). Error bars indicate standard error. (F) M. persicae and M. cerasi performance on a mutant line for an Arabidopsis gene predicted to encode an ABA-responsive protein and Col-0 wild-type plants. Same experimental set-up as described in (C). The two-tailed Student's t-test was used for statistical analyses (* indicates p-value < 0.05).
Fig 7.
ROS levels during host and non-host interactions with aphids.
Leaves exposed to different aphid species were incubated with the dye DCFH-DA (dichlorodihydro-fluorescein diacetate) to compare reactive oxygen species (ROS) levels during host and non-host interactions. Images were taken 3, 6, 12 and 24 hours after aphid exposure using a laser confocal microscope and processed in ImageJ to generate graph bars representing relative fluorescence to the control treatment (no aphids). Graph indicates fluorescence ratios of leaf samples exposed to aphids compared to a no-aphid control (C). Average relative ratios are based on 5 different leaf samples per treatment. Three independent replicated experiments, with 5 leaf samples per treatment per replicate, were performed with similar results. Graph shows results of one of the two replicates. The additional replicate is shown in S9 Fig.
Fig 8.
Arabidopsis atrbohD-3 and atrbohF-3 knock-out mutants show reduced non-host resistance and enhanced susceptibility to aphids.
(A) Myzus persicae (Mp) and M. cerasi (Mc) performance on Arabidopsis atrbohD-3 and atrbohF-3 knock-out lines. Four-week old plants were exposed to two adult aphids and nymph production was counted after 10 days. Average nymph production for M. persicae and M. cerasi was calculated from three independent replicated experiments, with 10 plants per replicate per treatment. (B) Graph shows R. padi aphid survival on control (Col-0) and Arabidopsis atrbohD-3 and atrbohF-3 knock-out lines over 6 days. Three independent biological replicates were carried out, with 10 plants per replicate. Error bars indicate standard error. (C) Rhopalosiphum padi (Rp) survival on Arabidopsis atrbohD-3 and atrbohF-3 mutants and Col-0 wild-type plants between 3 and 4 days of the experiment. The two-tailed Student's t-test was used for statistical analyses (*** indicates p-value<0.001). Error bars indicate standard error. (D) ROS levels in atrbohD-3 and atrbohF-3 mutants during host and non-host interactions with aphids. Images were taken 3 and 24 hours after aphid exposure using a laser confocal microscope and processed in ImageJ to generate graph bars representing relative fluorescence to the control treatment (no aphids). Graph indicates fluorescence the relative fluorescence of leaf samples exposed to aphids compared to a no aphid control (indicated by C). Average relative ratios are based on 5 different leaf samples per treatment. Three independent replicated experiments, with 5 leaf samples per treatment per replicate, were performed with similar results. Graph shows results of one of the three replicates. Additional replicates are shown in S10 Fig.