Structural modelling and transcriptional responses highlight a clade of PpKAI2-LIKE genes as candidate receptors for strigolactones in Physcomitrella patens

Abstract

A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the α/β hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone receptors in moss.

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

“…The PEG-mediated transformation protocol was adapted to deliver a mix of the sgRNA plasmids along with the plasmid containing the S. pyogenes human codon-optimized Cas9 ( Sp -hCas9) into P. patens protoplasts (Figure S2). We initially selected five out of the 13 PpKAI2L genes (clade i) as targets, these genes being related most closely to the Arabidopsis KAI2/HTL gene (Lopez-Obando et al 2016). Two independent transformation experiments, I and II, used multiple gene-specific constructs targeting PpKAI2L-A to D and PpKAI2L-A to -E , respectively.…”

“…In order to test the robustness of this multiple gene knock-out strategy based on transient expression of the CRISPR-CAS9 module, we evaluated the efficiency of multiplex gene editing of the PpKAI2LF , PpKAI2LJ , and PpKAI2LK genes from the PpKAI2L clade i.i–iii (Lopez-Obando et al 2016), and of four genes ( Pp3c3_31490 , Pp3c3_31500 , Pp3c4_25130 , and Pp3c8_7340 ) belonging to the AP2/ERF transcription factor family (Mizoi et al 2012) (Table S7). After transient selection, 40 and 35 clones were evaluated for mutations in the three and four genes, respectively.…”

“…In order to test whether CRISPR-Cas9 technology could be used for efficient targeting of multiple genes in P. patens , we focused on targeting the P. patens KARRIKIN INSENSITIVE 2 LIKE ( PpKAI2L ) gene family. These genes are homologs of KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT ( KAI2/HTL ) and DWARF 14 ( D14 ) vascular plant genes, encoding receptors and candidate receptors of butenolide compounds such as strigolactones or karrikins (Lopez-Obando et al 2016). The 13 genes of this family are split into two clades, (i) and (i.i–iii), that were targeted separately.…”

From little things big things grow: karrikins and new directions in plant development

“…First, phylogenetic analysis strongly suggests that a KAI2-like protein was the evolutionary ancestor of D14, implying that the signalling function of KAI2 is fundamental and has been retained throughout the diversification of land plants (Delaux et al 2012;Waters et al 2012aWaters et al , 2015b. Given that D14 was an innovation of the seed plants but strigolactones are found in Charophyte algae, it is likely that KAI2 proteins in early land plants were receptors for strigolactones, and perhaps for other signalling compounds as well (Delaux et al 2012;Lopez-Obando et al 2016). Second, KAI2-dependent signalling requires an intact catalytic triad, and this requirement is evolutionarily conserved between lycophytes and angiosperms (Waters et al 2015a(Waters et al , 2015b.…”

Strigolactones, super hormones in the fight against Striga

“…SLs are produced and perceived in basal land plants extending back to the Charophyte lineage, but obvious D14 orthologs are not present [21, [40][41][42]. Thus it remains unclear whether basal land plants use a similar SL signaling system to angiosperms; one hypothesis is that KAI2-like proteins function as SL receptors [43]. Regardless, the discoveries made in model systems have been instrumental to understanding host perception in parasites.…”