The anticipated potential nuclear localization sequence of ‘Candidatus Phytoplasma mali’ SAP11-like protein is required for TCP binding but not for transport into the nucleus

The plant pathogen ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of ‘Ca. P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenic A. thaliana.


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The delivery of effector proteins and small molecules into the plant host is a common strategy of 25 plant pathogens, including bacteria, fungi, oomycetes and nematodes, to enhance the hosts´ 26 susceptibility and benefit their infectiousness (1). The function of these effectors reaches from 27 suppression of the plant immune system to alteration of plant behavior and development (1). Thus,

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identifying targets of plant pathogen effectors and revealing plant-microbe interactions enable to 29 better understand the infectious mechanisms and consequently to control phytoplasma diseases.  In 'Candidatus Phytoplasma mali' ('Ca. P. mali') strain AT, a putative pathogenic-related effector 49 protein ATP_00189 (GenBank: CAP18376.1) was identified which shares 41% homology on 50 amino acid level with AY-WB_SAP11 and was therefore called SAP11-like protein (8). 'Ca.' P.

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mali is the cause of apple proliferation (AP) causing symptoms such as witches' broom, enlarged 52 stipules, tasteless and dwarf fruits and thus leading to massive yield losses and economic damage 53 in apple production. AY-WB_SAP11 and the AP_SAP11-like protein share a signal-peptide motif 54 of the phytoplasma-specific sequence-variable mosaic (SVM) protein signal sequence (Pfam 55 entry: PF12113), linking these proteins to a rapid evolution (9). Both AY-WB_SAP11 and 56 AP_SAP11-like protein are found to be expressed in infected plants (8,5).  amino acids 40 to 56 (Fig.1b), which correspond to the predicted NLS of AY-WB_Sap11 sequence 138 alignment (Fig. 1a), and fused the protein to GFP, resulting in AP_SAP11-like_PM19 Δ40-56 -GFP.

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The result of the protoplasts isolated from the infiltrated leaves shows that AP_SAP11-  144 Different constructs were fused to GFP or RFP and transiently expressed in N. benthamiana.

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Protoplasts of Agrobacterium-infiltrated leaves were analyzed by confocal microscopy using GFP 146 and RFP filters. a. AP_SAP11-like_PM19 and AP_SAP11-like_PM19 Δ40-56 localize mainly in the 147 plant nucleus but also partly in the cytoplasm. Co-localization with biNLS-RFP is indicated by 148 yellow coloring in the merge. b. Investigation of nuclear leading activity of AP_SAP11_PM19.

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Fusion of AP_SAP11-like_PM19 to Gus-GFP localizes mainly in the cytoplasm, whereas fusion 150 with biNLS leads to a mainly nuclear localization. c. Fusion of AP_SAP11-like-PM19-GFP with a 151 nuclear export sequence (NES) leads to cytoplasmic localization. Scale bar = 10 µm. Ten

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protoplasts expressing the proteins in questions were analyzed and all showed the same results.

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Nuclear localization cannot only be achieved by active nuclear import, but also via passive 156 transport. Even though it was long assumed that the maximum size for protein diffusion through

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The result shows that Gus-GFP localized exclusively in cytoplasm while the biNLS-Gus-GFP is 165 detected mainly in the nucleus (Fig.2b, upper and middle panel). As suspected, AP_SAP11-166 like_PM19 could not target the Gus-GFP into the nucleus. (Fig.2b, lower panel). These results

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suggest that the nuclear localization of AP_SAP11-like_PM19 is rather due to the size than the 168 function of previously predicted NLS sequence (amino acids 40 to 56).

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The expression of AY-WB_SAP11 in A. thaliana induces stem proliferation, leading to witches' 173 broom symptoms, and alteration of leaf and silique shape (6) while the expression of AP_SAP11-174 like protein of Ca. P. mali in N. benthamiana also leads to morphological changes with stunted 175 growth and crinkled leaves (13).

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To analyze the effects of AP_SAP11-like_PM19 protein in A. thaliana, we produced transgenic  Table S2.

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The obtained values were normalized to the arithmetic average of AP_SAP11-like_PM19 for better indicates that AP_SAP11-like_PM19 protein does not only exclusively interact with class II but 269 also with some of class I AtTCPs.

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In the next step, we repeated Y2H screens of all candidate preys that showed interaction with   Figure S1). n.a.

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Class I neg. n.a.   to locate into the nucleus when its size is increased (fused with Gus-GFP), it cannot be ruled out 366 that the protein could enter the nucleus via passive transport or other unknown mechanisms.

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It was reported that NLS of AY-WB_SAP11 protein is not required for binding some AtTCPs (7).

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It was discussed that the AY-WB_SAP11 enters the nucleus to interact with and destabilize 382 AtTCPs, however a cytoplasmic function of the protein was not completely excluded (7). Since our      Table S2 and S3 respectively.

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The relative expression levels were calculated using the ddCt method (41)

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To test for interaction between bait and prey, the respective expression plasmids were co- 506 Acceptor photobleaching FRET

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The genes of the two potential interaction partners fused to GFP (donor) and RFP (acceptor) 508 respectively, were cloned into the same pPZP200 binary vector (38), to ensure coexpression in 509 the same plant cell. The proteins were then transiently expressed in N. benthamiana and 510 protoplasts were isolated, as described before.

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For fluorescence resonance energy transfer (FRET) analysis the signal of the donor GFP was 512 observed using a Zeiss Observer Z1 with LSM510 confocal laser-scanning head before and after

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of these Y2H experiments are also summarized in Table 1.