Post-translational cleavage and self-interaction of the phytoplasma effector SAP11

Abstract

Phytoplasmas are insect-transmitted intracellular plant bacterial pathogens that secrete effector molecules into host cells that interfere with the host’s developmental or metabolic processes. Recently, the secreted Aster Yellows phytoplasma strain Witches’ Broom protein11 (SAP11) has been shown to act as a virulence factor that alters the development, hormone biosynthesis, phosphate (Pi) homeostasis, and defense responses in the affected plants. We found that SAP11 undergoes proteolytic processing in planta and self-interaction in vitro. These biochemical studies provide foundational insights necessary for the functional characterization of SAP11; however, the biological relevance of post-translational cleavage and self-interaction of SAP11 to its role as a virulence factor warrants further investigation.

Phytoplasmas are intracellular plant bacterial pathogens that are responsible for significant agricultural losses worldwide.¹ Phytoplasmas are known to infect more than 700 plant species, eliciting various symptoms such as phyllody (conversion of floral organs into leafy structures), virescence (greening of petals), witches' broom (clustering of branches), purple top (purple coloration of leaves), apical dwarfing, and yellowing.² Although phytoplasmas cannot be cultured in vitro, the availability of its complete genomic sequence facilitates the exploration of its interactions with host plants, toward a better molecular understanding of the symptoms associated with phytoplasma infections.³

The identification of phytoplasma-secreted proteins has revealed that phytoplasma effectors are virulence factors responsible for inducing morphological changes in infected plants.^4,5 For example, SAP11 secreted by the Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) and TENGU secreted by the Onion Yellows phytoplasma strain M (OY-M) induce the proliferation of branches similar to the witches’ broom symptom.^6,7 SAP54 of AY-WB was reported to induce green leaf-like flowers symptomatic of phyllody and virescence.⁸ In addition to altering the plant morphology, SAP11 enhanced insect reproduction through the suppression of jasmonate (JA) synthesis and modulated phosphate (Pi) homeostasis by triggering Pi starvation responses.^7,9 While these findings provide novel insights into understanding the pathogenesis of phytoplasmas, the molecular mechanisms underlying the role of phytoplasma effectors remain largely unknown.

A recent study showed that TENGU, a 38-amino acid long protein, is modified through post-translational cleavage by an unidentified host protease.¹⁰ The protein was found to be processed into small functional peptides, including an 11-amino acid sequence at the N-terminus that is necessary to induce branch proliferation. Previously, SAP11 was found to bind to and destabilize CIN-TCPs, which inhibits LOX2 expression and reduces JA biosynthesis.^7,11 In the present study, we showed that SAP11 undergoes proteolytic processing in planta and self-interaction in vitro. These biochemical characterizations of SAP11 are essential for providing a better understanding of the molecular basis of phytoplasma pathogenesis.

Using a specific antibody against SAP11 of AY-WB, we found two SAP11-related bands with a molecular weight (MW) between 11 kDa and 17 kDa among the total cell extracts of 35S::SAP11 transgenic plants (Fig. 1A). To further examine the sizes of the two SAP11-related bands, the SAP11 recombinant protein was constructed. A PCR product encoding SAP11 was subcloned into the SUMO-pET vector. The N-terminal His-SUMO fusion SAP11 (23.7 kDa) was induced in Escherichia coli BL21 (DE3) cells and purified by Ni²⁺-NTA affinity chromatography. The His-SUMO tag (12.6 kDa) was then cleaved off by a ubiquitin-like specific protease1, and the reaction mixture was further loaded onto a size-exclusion chromatography column (HiPrep 16/60 Sephacryl S-200 HR column); thus the SAP11 protein (11.1 kDa) was obtained. Using the recombinant SAP11 as an MW standard, we found that the upper band of SAP11-related proteins that appeared in the immunoblotting experiments had a protein size similar to the recombinant protein whereas the lower band did not (Fig. 1B). This result indicates that the lower band of SAP11-related proteins in 35S::SAP11 transgenic plants is a protein processed through post-translational cleavage.

Figure 1.SAP11 undergoes proteolytic processing in planta. (A) western blotting for the translated products of SAP11 in Arabidopsis. Black arrowhead indicates the SAP11-related bands detected by anti-SAP11 antibody (upper panel). Double asterisk indicates the tubulin band detected by anti-tubulin antibody (lower panel). Single asterisk indicates the cross-reacting band that appeared in all samples (upper panel). (B) Examination of the protein size of two SAP11-related bands. Black arrow indicates the recombinant SAP11 band detected by anti-SAP11 antibody (upper right panel, short exposure). White arrowhead indicates the translated product of SAP11ΔC in Arabidopsis (upper left panel, long exposure). Leaf morphologies of Arabidopsis WT plants and transgenic plants overexpressing SAP11 and SAP11ΔC (from left to right) are shown in the lower right panel. Bar = 8 mm.

SAP11 contains a bipartite nuclear localization signal in the N-terminal region and a coiled-coil domain in the C-terminal region, which are important for nuclear targeting and CIN-TCP destabilization.¹¹ In this study, we generated SAP11ΔC-overexpressing plants in which the conserved C-terminal motif (-GSSSKQPDDSKK-) of SAP11 was truncated. Although SAP11ΔC-overexpressing plants continue to exhibit a crinkling-leaf phenotype, only one protein band was detected by the anti-SAP11 antibody in the total cell extracts (Fig. 1B). This result suggests that the C-terminal motif of SAP11 is required for proteolytic processing in planta.

During the purification stage, we found that the lower MW fractions of SAP11 was eluted earlier than the higher MW fractions of His-SUMO tag in the size-exclusion chromatography experiment (Fig. 2A). This result suggests that SAP11 tends to preserve its self-associated state in vitro. As a control, we subcloned SAP54 into the SUMO-pET vector to obtain the N-terminal His-SUMO fusion SAP54. After cleavage by a ubiquitin-like specific protease1, the reaction mixture was loaded onto a size-exclusion chromatography column. As expected, we found that the higher MW fractions of His-SUMO tag was eluted earlier than the lower MW fractions of SAP54 (10.8 kDa) (Fig. 2B). However, different from SAP11, SAP54 was not able to be separated from the His-SUMO tag (Fig. 2B).

Figure 2.SAP11 interacts with itself in vitro. (A) The size-exclusion chromatography experiment of His-SUMO and SAP11. (B) The size-exclusion chromatography experiment of His-SUMO and SAP54. The upper panel indicates the UV trace (A: solid line; B: dashed line) of size-exclusion chromatography. The lower panel indicates the SDS-PAGE of column fractions.

SAP11 is known to cause crinkling-leaf and branching shoots phenotypes, reduce JA biosynthesis, and increase Pi content in plants.^7,9 Although the biological relevance of the proteolytic processing and self-interaction of SAP11 to these phenotypes requires further investigation, our findings provide fundamental molecular mechanisms that functionally characterize SAP11 and advance our understanding of morphological and physiological changes in plants that are altered by SAP11.

Materials and Methods

Generation of transgenic Arabidopsis plants

The 35S::SAP11 transgenic plants used in this study have been described previously.⁹ To construct 35S::SAP11ΔC, a codon-optimized version of SAP11 was used as template for PCR amplification and subcloned into pBA002 vector. After sequencing verification, the plasmid was introduced into Agrobacterium tumefaciens and transformed into Arabidopsis thaliana for generation of 35S::SAP11ΔC transgenic plants.

Immunoblotting analysis

The anti-SAP11 antibody described previously was used in the study.⁹ To detect SAP11 derivatives, 3-wk-old seedlings were ground, and total cell extracts were prepared by directly adding SDS sample buffer into grinding samples. Proteins were then separated by Tricine SDS-PAGE and blotted onto a PVDF membrane. The chemiluminescence signals were captured with Amersham ECL western-blotting reagents, using an ImageQuant LAS 4000 Mini (GE Healthcare)