APhelipanche ramosaKAI2 Protein Perceives enzymatically Strigolactones and Isothiocyanates

Abstract

22Phelipanche ramosa is an obligate root-parasitic weed threatening major crops in central 23Europe. For its germination, it has to perceive various structurally diverging host-exuded 24 signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors 25 involved are still uncharacterized. Here, we identified five putative SL receptors in P. ramosa, 26 of which PrKAI2d3 is involved in seed germination stimulation. We established the high 27 plasticity of PrKAI2d3, allowing interaction with different chemicals, including ITCs. The SL 28 perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic 29 plants. We provide evidence that the PrKAI2d3 enzymatic activity confers hypersensitivity to 30SLs. Additionally, we demonstrated that methylbutenolide-OH binds PrKAI2d3 and 31 stimulates P. ramosa germination with a bioactivity comparable to that of ITCs. This study 32 highlights that P. ramosa has extended its signal perception system during evolution, a fact to 33 be considered in the development of specific and efficient biocontrol methods. 34Besides their involvement in germination, SLs have a strong stimulating activity on 54 arbuscular mycorrhizal fungi (Glomeromycotina) by promoting mitochondrial metabolism 55 and hyphal branching 7 , thereby mediating the establishment of the oldest mutualistic 56 interaction of land plants 8 . In addition to their role in rhizosphere signaling, SLs act as 57 hormones in planta with pervasive roles throughout the plant development ,9 and, as such, are 58 perceived in vascular plants by the α/β-hydrolase DWARF14 (D14) 10,11 . Biochemical 59 analyses with recombinant D14 proteins by means of the synthetic SL analog GR24 revealed 60 that the SL signal transduction requires GR24 cleavage 11,12 . One of the cleavage products, the 61 D ring, may remain covalently attached to the receptor 13,14 , thereby probably allowing the 62 recruitment of partners for downstream processes 15,16 . The required SL cleavage for signal 63 transduction is still under debate 15,16 . 64 In Arabidopsis thaliana, D14 belongs to a small gene family, including KARRIKIN 65 INSENSITIVE2/HYPOSENSITIVE TO LIGHT (AtKAI2/AtHTL) that shares the α/β-66 hydrolase catalytic triad. However, AtKAI2 regulates AtD14-independent processes, such as 67 seed germination, and preferentially perceives (−)-GR24 that mimics non-natural SLs, 68 karrikins 17 , and supposedly a still unknown endogenous ligand 18 . Interestingly, the KAI2 gene 69 family has expanded during the evolution of obligate parasitic-plant genomes 19 . For example, 70Phelipanche aegyptiaca and Striga hermonthica possess five and eleven KAI2/HTL genes, 71 respectively. Two KAI2 paralogs play a role in SL response in P. aegyptiaca 19 and six S. 72 hermonthica KAI2/HTL proteins are hypersensitive to SLs 19,20 , with ShHTL7 exhibiting the 73 same perception mechanism as D14 21 . The remarkable expansion of the KAI2 gene family in 74Orobanchaceae along with the capacity of P. ramosa to perceive ITCs let us...

Desmethyl butenolides are optimal ligands for karrikin receptor proteins

“…In addition, the affinity of the ligand–receptor relationship can be inferred using ITF measurements, in which ligand binding alters the tryptophan microenvironment and therefore the overall fluorescence properties of the protein (Ghisaidoobe & Chung, 2014). ITF measurements are only appropriate for ligands that do not interfere with a fluorescence signal from tryptophan, and ITF has been used elsewhere to infer a ligand‐binding relationship between strigolactone analogues such as GR24 and various D14 and KAI2 homologues (de Saint Germain et al ., 2016; de Saint Germain et al ., 2020). ITF data have also been documented as evidence for binding of KAR 1 by KAI2 (Toh et al ., 2014), but we contend that karrikins are incompatible with ITF assays because they absorb strongly at the same wavelengths (280–295 nm) that excite tryptophan (Fig.…”

“…It is conceivable that there exists an endogenous enzyme with strigolactone demethylase activity to generate KL. However, the preferred 2′( S ) stereochemistry of KAI2 ligands is opposite to that of natural strigolactones, and natural strigolactone‐like compounds with a desmethyl D‐ring have yet to be reported, even though such compounds are likely to be discovered by seed germination bioassays (de Saint Germain et al ., 2020). Furthermore, there has been evolutionary loss of key enzymes in strigolactone biosynthesis (e.g.…”

ThePhyscomitrium (Physcomitrella) patensPpKAI2L receptors for strigolactones and related compounds highlight MAX2 dependent and independent pathways

“…The copyright holder for this preprint this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.395954 doi: bioRxiv preprint Bythell-Douglas et al 2017), the expansion of the PpKAI2L family in P. patens (and not in other bryophytes such as Marchantia polymorpha, that contains two MpKAI2 genes), as in parasitic angiosperms Toh et al 2015), may have allowed the emergence of SL sensitivity (de Saint Germain et al 2020). Neofunctionalization of additional KAI2 copies in P. patens ancestry towards SL perception is therefore a possible explanation for our findings in this moss and would reveal a convergent evolution process, relative to the emergence of D14 in seed plants.…”

Structural and Functional Analyses Explain Pea KAI2 Receptor Diversity and Reveal Stereoselective Catalysis During Signal Perception

“…The ageold question in receptor diversity has been the evolutionary purpose and functional significance of KAI2 duplication events. It has been shown that D14 and KAI2 are not able to complement each other functions in planta [37][38][39][40][41] . To this end, within the ligand binding site of KAI2 receptors the substitution of a few amino acids can alter ligand specificity between KAI2 duplicated copies 39,42 .…”

“…Thus far, several crystal structures of KAI2/D14 receptors have been reported and have led to a greater understanding of receptor-ligand perception and the hydrolytic activity of the receptor towards certain ligands 11,12,19,31,36,38,[44][45][46][47][48][49][50][51] . The divergence between duplications of KAI2 receptors to confer altered ligand specificity has been partially addressed at the physiological and biochemical level for only few plant species, and a structural examination has been limited [38][39][40]42 .…”