The Effect of the Anticipated Nuclear Localization Sequence of ' Candidatus Phytoplasma mali' SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor

Abstract

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of 'Candidatus Phytoplasma mali' ('Ca. P. mali') strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches' broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

Keywords: SAP11; TCP transcription factors; phytoplasmas; plant-pathogen interaction; ‘Candidatus Phytoplasma mali’.

Figure 1

Sequence alignment and schematic representation of the AP_SAP11-like_PM19 amino acid sequence used in this study. (a) Multiple sequence alignment of AP_SAP11-like protein of strains PM19, STAA and AT, and AY-WB_SAP11. Annotations: single black line represents sequence-variable mosaic protein signal peptide (SVM-SP), double black line represents nuclear leader sequence (NLS), and the dotted line represents TCP-binding area of AY-WB_SAP11. (b) Schematic representation of deletions and additions to AP_SAP11-like_PM19 in comparison to AY-WB_SAP11. The black squares represent the SVM-SP, the striated squares represent the NLS, and the dotted squares represent the TCP-binding area. The deletion of amino acids 40 to 56 in AP_SAP11-like_PM19Δ40–56 is indicated by a line connecting the squares.

Figure 2

Localization studies of AP_SAP11-like_PM19 protein in infiltrated N. benthamiana leaves. AP_SAP11-like_PM19 and AP_SAP11-like_PM19Δ40–56 were fused to GFP and biNLS with RFP to decorate the nucleus. The localization of expressed proteins in infiltrated leaves was analyzed by confocal microscopy using GFP and RFP filters. AP_SAP11-like_PM19 (SAP11-GFP) and AP_SAP11-like_PM19Δ40–56 (ΔNLS-SAP11-GFP) localize not only in the plant nucleus but also in the cytoplasm. Co-localization with biNLS-RFP is indicated by yellow coloring in the merge. Scale bar = 100 µm.

Figure 3

Localization studies of AP_SAP11-like_PM19 protein in protoplasts of infiltrated N. benthamiana leaves. AP_SAP11-like_PM19 and AP_SAP11-like_PM19Δ40–56 were fused to GFP and biNLS with RFP to decorate the nucleus. The localization of expressed proteins was analyzed by confocal microscopy using GFP and RFP filters. AP_SAP11-like_PM19 (SAP11-GFP) and AP_SAP11-like_PM19Δ40–56 (ΔNLS-SAP11-GFP) localize not only in the plant nucleus but also in the cytoplasm. Co-localization with biNLS-RFP is indicated by yellow coloring in the merge. Scale bar = 10 µm.

Figure 4

Amino acids 40 to 56 of AP_SAP11-like_PM19 protein are required for symptom development in A. thaliana. (a) Transgenic A. thaliana lines expressing AP_SAP11-like_PM19 (SAP11-like) and AP_SAP11-like_PM19Δ40–56 (SAP11-like-Δ40–56) under the control of the Cauliflower mosaic virus 35S promoter compared to the wt Arabidopsis Col-0 plant. All plants were grown for 8 weeks in long-day (16 h/8 h light/dark) conditions. For each of the three constructs, at least 3 lines were examined, all showing similar phenotypic characteristics. (b) Leaves of transgenic plant lines shown in (a). Scale bar = 1 cm. (c) Siliques of transgenic lines shown in (a).

Figure 5

(a) Number of primary stems developed by transgenic A. thaliana lines compared to the wt Col-0 plant. (b) Relative expression levels of transgenes of transgenic lines compared to the geometric average of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2 (PP2A). All values show a p-value lower than 0.05 in ANOVA.

Figure 6

Example for Y2H results of AP_SAP11-like_PM19 and AP_SAP11-like_PM19Δ40–56 with different AtTCPs. Y2H screens were performed using the binding domain fused to AP_SAP11-like_PM19 (BD-SAP11-like) or AP_SAP11-like_PM19Δ40–56 (BD-SAP11-like-Δ40–56) and the activation domain (AD) fused to different AtTCPs. For the negative control, only BD was used. Co-transformed yeast cells were patched on double drop-out medium (DDO) to select for presence of both expression plasmids and DDO media containing Aureobasidin A and X-α-Gal (DDOXA) to select for protein interaction. n.a. = not applicable. A figure showing all Y2H results can be found in the Supplementary Materials (Figure S1).

Figure 7

TCP destabilization assay. The relative quantities of AtTCPs (3, 4, 6, 12, and 19) were monitored in the presence of AP_SAP11-like_PM19 and AP_SAP11-like_PM19Δ40–56 when they were transiently expressed from the same vector in N. benthamiana. The amounts of AtTCPs and SAP11 were examined using a monoclonal anti-HA for detecting HA-AtTCPs (middle panel) and anti-GFP for SAP11-GFP (low panel) in Western blotting analysis. For controlling the total protein loading amount, the large subunit of Rubisco visualized with Coomassie Brillant Blue staining is indicated by an arrowhead (upper panel).