Structural insights into the interaction between phytoplasmal effector causing phyllody 1 and MADS transcription factors

Abstract

Summary Phytoplasmas are bacterial plant pathogens which can induce severe symptoms including dwarfism, phyllody and virescence in an infected plant. Because phytoplasmas infect many important crops such as peanut and papaya they have caused serious agricultural losses. The phytoplasmal effector causing phyllody 1 (PHYL1) is an important phytoplasmal pathogenic factor which affects the biological function of MADS transcription factors by interacting with their K (keratin‐like) domain, thus resulting in abnormal plant developments such as phyllody. Until now, lack of information on the structure of PHYL1 has prevented a detailed understanding of the binding mechanism between PHYL1 and the MADS transcription factors. Here, we present the crystal structure of PHYL1 from peanut witches’‐broom phytoplasma (PHYL1PnWB). This protein was found to fold into a unique α‐helical hairpin with exposed hydrophobic residues on its surface that may play an important role in its biological function. Using proteomics approaches, we propose a binding mode of PHYL1PnWB with the K domain of the MADS transcription factor SEPALLATA3 (SEP3_K) and identify the residues of PHYL1PnWB that are important for this interaction. Furthermore, using surface plasmon resonance we measure the binding strength of PHYL1PnWB proteins to SEP3_K. Lastly, based on confocal images, we found that α‐helix 2 of PHYL1PnWB plays an important role in PHYL1‐mediated degradation of SEP3. Taken together, these results provide a structural understanding of the specific binding mechanism between PHYL1PnWB and SEP3_K.

Structural requirements of the phytoplasma effector protein SAP54 for causing homeotic transformation of floral organs

“…The conditions during analytical ultracentrifugation thus may have promoted the dissociation of protein complexes. In line with this view, dimers seemed to be present in the crystals assessed by Liao et al (2019). In fact, employing dynamic light scattering, we found complexes with an apparent molecular mass of ~20 kDa, suggesting that SAP54 predominantly formed homodimers under our experimental conditions (Figure 5a, Table 2).…”

“…Taken all three studies together, and considering the high sequence similarity also to other homologous proteins (Figure 1), we have now a pretty good impression about the structure of SAP54-like proteins: it comprises about 90 amino acids that fold into two α-helices connected by a small, unstructured loop. SAP54-like proteins may not have a rigid structure, however; to a certain degree it may depend on environmental conditions, and it may undergo conformational changes upon binding to and release from K-domain (Liao et al 2019). Also the functional relevance of the structure for interaction with target proteins and for causing disease phenotypes is now well documented (this study; Iwabuchi et al, 2019).…”

“…While insertions in either the first or second helix abolished the interaction with some MIKC-type MTFs (SEP1 -SEP4), the degradation of SEP3, and the ability to induce disease symptoms (phyllody), insertion in the linker region had no such effects. Likewise, Liao et al (2019) reported that some double amino acid substitutions in both either the first or second helix of PHYL1PnWB abolished the interaction with the K-domain of SEP3 in Y2H experiments. These findings, summarized in Figure 6 strongly corroborate all structure-function relationships involving SAP54 outlined above.…”

“…While this manuscript was in preparation, Iwabuchi et al (2019) PHYL1OY shares about 87 % sequence identity with SAP54 ( Figure 1a) and generates similar symptoms in planta, suggesting that both proteins work in very similar ways (Maejima et al, 2014). In contrast, PHYL1PnWB shares only about 53% sequence identity with SAP54 and is hence probably more distantly related than PHYL1OY (Figure 1a; Liao et al, 2019).…”

“…It is well known, however, that Y2H experiments are biased against the detection of homodimer formation (Newman et al, 2000;Smirnova et al, 1999), so this conclusion does not appear to be cogent. Liao et al (2019) concluded from sedimentation rates during analytical ultracentrifugation that native PHYL1PnWB exist as a monomer. However, in their kind of experiment they also found monomers (and tetramers) of K-domain fragments of SEP3, whereas in other experiments only dimers and tetramers had been detected (Puranik et al, 2014).…”