Fig 2: Insect-directed engineering of At RPN10 confers resistance to SAP05 action(A) Schematic of domain organizations of At and M. quadrilineatus RPN10 proteins and alignment of the first 70 residues of the vWA domains. Highly divergent residues are highlighted below the alignment. Alignments of full-length RPN10 homologs are shown in Figure S5D.(B) Specific residues within the At RPN10 vWA domain are required for SAP05 interaction in Y2H assays. See the legends of Figures 2 and 5A for abbreviations. Yeast growth on -L-W medium is shown in Figure S6C.(C) Specific residues within the At RPN10 vWA domain are required for SAP05 degradation of plant GATA and SPL in At protoplasts. See the legend of Figure 2E.(D–J) Specific residues within the At RPN10 vWA domain are required for leaf and stem proliferation of At plants in the presence of constitutively expressed SAP05 (D and E) and during AY-WB phytoplasma infection (F–J). At plants included in these experiments were rpn10-2 null mutants complemented with wild-type AtRPN10 (cRPN10) or AtRPN10 m1 (eRPN10). Scale bars, 1 cm. Symptomatic leaves in (F) and lateral shoots in (I) are circled. Phenotypes were analyzed statistically for number of leaves of 4-week-old plants (E), number of newly produced rosette leaves after AY-WB infection (G), plant survival time after AY-WB inoculation (H), and numbers of lateral shoots in control and infected plants (J). Data are mean ± SD from 2 independent experiments. ∗p < 0.05, two-tailed Student’s t test.

Fig 3: Phenotypic analysis of transgenic plants or plants infected with AY-WB phytoplasma, related to Figures 4 and 5(A) Different SAP05 homologs induce distinct leaf morphologies in A. thaliana reminiscent of either MIR156 overexpression or a GATA mutant. Arrowheads indicate leaf serrations. The A. thaliana han mutant (han-2) produces rosettes with a smooth margin while the overexpression of a Cucumis sativus L. GATA18 homolog (CsHAN) under the control of the 35S promoter in the A. thaliana han-2 background leads to leaves with more severe serrations. Scale bar, 1 cm.(B) An engineered RPN10 allele rescues the developmental defects of the rpn10-2 mutant. The rpn10-2 mutant was complemented by either a wild-type AtRPN10 allele (cRPN10) or an engineered RPN10 allele (AtRPN10 m1, eRPN10) under the control of the native promoter. At least two independent lines for each complementation were obtained, with consistent plant phenotype. Scale bar, 1 cm.(C-E) Statistical analysis of the flowering time (C), branching (D) and seed weight (E) of cRPN10 plants and eRPN10 plants. The number of lateral shoots was scored both at 6 weeks after germination and 8 weeks after germination. Data are mean ± SD; n.s., no significant difference, two-tailed unpaired Student’s t tests.(F) Rosette leaf morphology on healthy and AY-WB phytoplasma-infected plants. All plants were kept in short-day conditions throughout the experiment. Circled areas correspond to rosette leaves that emerged during phytoplasma infection. Scale bar, 1 cm.(G) eRPN10 plants show phyllody symptoms during AY-WB infection. Typical flower morphology on healthy plants or infected plants is shown. Asterisks indicate leaf-like flowers on infected plants. Scale bar, 1 cm.

Fig 4: SAP05 destabilizes plant TFs of the SPL and GATA families via interaction with the ubiquitin receptor AtRPN10(A and B) SAP05 interacts with most members of the A. thaliana GATA (A) and SPL (B) TFs families in Y2H assays. The phylogenies show SAP05 interactors in red and those that were not tested or had autoactivation activities in gray. Conserved zinc-finger (ZnF) domains are shown in the top left corners, with red and yellow dots indicating cysteine and histidine residues, respectively. SBP, SQUAMOSA promoter-binding protein; ∗, regulated by miR156.(C) SAP05 interacts with the ZnF domains of GATAs and SPLs in Y2H assays. EV, empty vector control. AD, GAL4-activation domain. BD, GAL4-DNA binding domain.(D) SAP05 interacts with AtRPN10 in Y2H assays. Top panel: graphical representation of AtRPN10 domains, with locations indicated in amino acids underneath. See Figure S6A for yeast growth on -L-W (lacking leucine and tryptophan) medium.(E) Western blot analysis of SAP05-mediated degradation of GATA and SPL proteins in protoplasts from wild-type A. thaliana. GFP, control; HA, hemagglutinin; rSPL, miR156-resistant form. Numbers on the left indicate molecular weight markers in kilodaltons. Red dots on the left of the blots indicate the expected sizes of the transiently expressed proteins. Protein loading was visualized using amido black staining.(F) Western blot analysis of SAP05 degradation assays in rpn10-2 protoplasts.(G and H) GUS staining produced by the GUS fusions of the miR156-resistant forms of SPL11 (rSPL11) and SPL13 (rSPL13) in the transgenic A. thaliana is reduced in AY-WB-infected plants on the right compared with non-infected plants on the left. Bar graphs show the percentages of GUS-stained areas of leaves, indicated by red dots. Data are mean ± SD; ∗p < 0.05, two-tailed Student’s t-tests.

Fig 5: SAP05 can mediate substrate degradation in different systems, related to Figure 3(A and B) A SAP05-GBP fusion protein mediates the destabilization of GFP in A. thaliana protoplast. (A) Proposed model for SAP05-GBP-mediated GFP degradation.(B) western blot analysis of GFP abundance in the presence of SAP05-GBP or an empty vector control when transiently expressed in A. thaliana protoplasts. The GBP (GFP-binding protein) derived from a single-chain antibody domain specifically recognizing GFP was fused to SAP05 at its C-ter via a Glycine-rich linker. ‘tail’ represents an unstructured region that serves as an initiation site for proteasomal degradation.(C and D) Purified human 26S proteasomes degrade His-SPL5 in the presence of SAP05 and A. thaliana vWA. (C) Repeat 1; (D) Repeat 2. Western blots shown are from protein extracts of recombinant human 26S proteasome preparations (BostonBiochem) in the presence of purified His-SPL5 and SAP05 with or without A. thaliana RPN10 vWA (AtvWA) or proteasome inhibitor MG132 probed with antibodies to HA, GFP and SAP05 as shown at left. Red dots at left of the blots indicate the expected sizes of TFs. Protein loading was visualized using Amido black staining.