Fig 1.
CRISPR-Cas 9 stylin-01 edited aphid lines (Sty01-KO and Sty01-Cter) and wild-type non edited line (WT).
(A) Schematic representation of the domains of the mature proteins and their respective 3D structure predicted by AlphaFold2. The N-terminal (Nter), C-terminal (Cter) and RR-1 chitin-binding domains are indicated in green, red and blue respectively. Mutations in the amino acid sequences (Mut) are indicated in purple. Credit images Yu Fu. (B) Detection of Stylin-01 proteins in A. pisum lines by western blot analysis. Whole-body crude extract proteins from WT, Sty01-KO, and Sty01-Cter aphids (lanes 1, 2 and 3, respectively) were analyzed by SDS-PAGE and stained with Coomassie blue to compare the protein amount loaded on the gel, or transferred onto a nitrocellulose membrane prior to immunolabeling with antibodies anti-1-09 or anti-1-L14Cter antibodies (S2A and B, S1 Table). (C) Detection of Stylin-01 Cter mutant protein in dissected stylets of the 3 aphid lines using immunofluorescence labeling and confocal microscopy Alexafluor-488 conjugated secondary antibodies were used to label anti-1-L14Cter antibody. The mutant protein is detected as green dots at the tip of maxillary stylets of Sty01-Cter aphids (indicated with a white arrow). Stylets of the wild-type or Sty01-KO aphids are not labeled by this antibody. Bars: 10 µm.
Fig 2.
Impact of Stylin-01 mutations on CaMV transmission efficiency.
The values are presented as the means ± SE of all independent replicates. Asterisks indicate significant differences according to binomial generalized linear model followed by a post-hoc Tukey’s honestly significant difference (HSD) test (GLM, *P < 0.05; **P < 0.01; ***P < 0.001).
Fig 3.
In vitro interaction assays between dissected aphid stylets and the viral protein CaMV P2 fused to Green Fluorescent Protein (P2-GFP).
(A) Representative images of the detection of P2-GFP on the acrostyle of dissected stylets of WT, Sty01-KO and Sty01-Cter adults. Stylets were scored (positive, partial, negative) according to the detection and localization of the green fluorescence labeling as shown in the schematic diagram on the left panel. Scale bars represent 10 µm. (B) Proportion of maxillary stylets labeled for each category out of the total number of maxillary stylets observed. The number of positive stylets was significantly lower in the two mutant lines Sty01-KO and Sty01-Cter than in the wild-type aphid line (P-values from a binomial GLM), “N” indicates the total number of maxillary stylets observed for each treatment from three (Sty01-KO, Sty01-Cter) or four (WT) biological replicates.
Fig 4.
Observation of the stylet ultrastructure in wild-type and Stylin-01 mutant aphids using transmission electron microscopy (TEM).
(A) Schematic representation of the approximative position of the cross sections of the apical part of aphid stylets observed under TEM in (B). The stylet bundle visible on the electron micrograph shows the two mandibular stylets (md) surrounding the two maxillary stylets (mx). (B) Electron micrographs of cross sections of the maxillary stylets of each aphid line (upper panel), observed at higher magnification in the bottom panel. The acrostyle, visible as an electron-dense area in the maxillary stylets, is indicated with a red arrowhead in (A) and (B, upper panel). Bars: 500 nm in (A), 200 nm in (B, upper panel), 50-100 nm in (B, bottom panel).
Fig 5.
Distribution of RR-1 stylins in wild-type and Stylin-01 mutant aphids.
Representative images of labeling of stylin peptides observed in maxillary stylets of WT, Sty01-KO and Sty01-Cter aphids. The primary antibodies used (Ab anti-1-11, Ab anti-1-15, Ab anti-1-16) and the stylins they theoretically target (Stylin-01, -02, -03, -04/-04bis) is indicated above the images (S2 Fig and S1 Table). The stylins detected in aphid stylets are indicated in green at the bottom of each image. The main difference observed at the acrostyle surface is outlined in red. Bars: 10 µm.
Fig 6.
Stylin-02 is upregulated in the Sty01-KO mutant during the synthesis of adult stylets.
Relative expression levels of stylins at N4 instar 46 h post-molt were measured in WT and mutant lines. The gene encoding NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS88) was used as internal control to compare the expression of all RR-1 stylins in each aphid line. The values are presented as the means ± SE (n = 7-8 biological replicates from two independent experiments, different letters indicate significant differences according to one-way ANOVA followed by the least significant difference (LSD) test.
Fig 7.
Feeding behavior of wild-type (WT) and mutant A. pisum lines related to CaMV acquisition and inoculation.
(A, C) Schematic diagrams illustrating the waveforms related to CaMV acquisition and inoculation, respectively. The duration of first C to first pd (time from the beginning of the first probe to the first intracellular puncture) during CaMV acquisition (B) and inoculation (D) show no difference between all aphid lines (n = 21-22, Kruskal-Wallis test, ns = not significant at the 5% level). Below the dotted line are most of the aphids that completed the sequence from the first C to the first pd. The duration of the sub-phase II-3 in the first pd related to ingestion of cell content (B) or the duration of subphase II-2 in the first pd related to CaMV inoculation (D) are similar in all aphid lines (n = 21-22, Kruskal-Wallis test, ns = not significant at the 5% level).