Biology subjects

Clay, N. K.

Publications and source records attributed to Clay, N. K..

4 recordsLinked to original sources

bioRxiv2019

Discovery of novel Defense Regulated WD40-repeat proteins DRW1/2 and their roles in plant immunity

Plant heterotrimeric G proteins transduce extracellular signals that activate plant immunity. Plants encode canonical and non-canonical G and G{gamma} subunits, but only a single canonical G{beta} subunit is known. The existence of only one G{beta} subunit limits the number of heterotrimeric G protein combinations able to transduce different signals. It remains unknown whether non-canonical G{beta} subunits exist. Here, we identify two WD40-repeat genes that negatively regulate plant immunity. The proteins encoded by these two genes, DEFENSE REGULATED WD40-REPEAT 1 and 2 (DRW1/2), are structurally similar to AGB1. DRW2 localizes to the plasma membrane and interacts with the canonical G and G{gamma} subunits. Reduced levels of DRW in the drw1 and drw2 single mutants resulted in greater MAPK activation in response to flagellin treatment and conferred increased resistance to the bacterial pathogen Pseudomonas syringae. Furthermore, the drw1 drw2 double-mutant also displayed increased MAPK activation upon flagellin treatment and broad-spectrum resistance against bacterial and fungal pathogen infection. The function of DRW1 and DRW2 is opposite of AGB1, which promotes immune signaling, suggesting that the function of these potential non-canonical G{beta} subunits are not conserved with the canonical G{beta} subunit. Our study identifies additional heterotrimeric G protein components, greatly increasing the number of heterotrimeric G protein complexes that participate in signal transduction.

plant biology↗

bioRxiv2018

Heterotrimeric G-proteins in unfolded protein response mediate plant growth-defense tradeoffs upstream of steroid and immune signaling

FLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

plant biology↗

bioRxiv2018

Catalytic promiscuity potentiated the divergence of new cytochrome P450 enzyme functions in cyanogenic defense metabolism

Cytochrome P450 monooxygenases (P450s) constitute the largest metabolic enzyme family in plants, responsible for synthesizing hundreds of thousands of specialized metabolites with essential roles in chemical defenses against herbivores and pathogens (Banks et al., 2011; Nelson and Werck-Reichhart, 2011; Wurtzel and Kutchan, 2016). Substrate promiscuity has been documented to play a central role in the evolution of plant specialized metabolic enzymes (Weng et al., 2012; Leong and Last, 2017), however most plant P450s are highly substrate-specific (Verpoorte, 2013). Here, we show the rapid inversion of primary and weak secondary (promiscuous) catalytic activities between two distinct yet evolutionarily linked multifunctional P450s, CYP71A12 and CYP71A13, based on intramolecular epistasis of two amino acid residues under positive selection in CYP71A12. Furthermore, we uncover previously undocumented catalytic activity during the inversion as well as naturally occurring amino acid substitution patterns that could have been present in evolutionary intermediates between the two enzymes. Comparative expression profiling and homology modeling reveal that natural selection acted on the promoter of CYP71A13 and the substrate-recognition elements of CYP71A12 to improve the efficiencies of their promiscuous reactions. The rise in catalytic promiscuity potentiated the divergence of new P450 enzyme functions in cyanogenic defense metabolism. Directed evolution of promiscuous reactions is one of the core technologies underpinning the field of synthetic biology. Our results provide a more complete understanding of how natural selection uses promiscuous reactions to generate new enzymes in nature and chemical diversity in pathogen defense, as well as demonstrate a novel strategy for identifying their molecular origins in highly divergent, related enzymes.

evolutionary biology↗

bioRxiv2018

Expansion of a core regulon in specialized metabolism by mobile genetic elements promotes chemical diversity in Arabidopsis thaliana

Plants synthesize hundreds of thousands of ecologically specialized, lineage-specific metabolites through biosynthetic gene duplication and functional specialization. However, the rewiring of duplicated genes into existing regulatory networks remains unclear. We show that the duplicated gene CYP82C2 was recruited into the WRKY33 regulon and indole-3-carbonylnitrile (ICN) biosynthetic pathway through exaptation of a retroduplicated LINE retrotransposon (EPCOT3) into a novel enhancer. The stepwise development of a chromatin-accessible WRKY33-binding site on EPCOT3 potentiated the regulatory neofunctionalization of CYP82C2 and the evolution of inducible defense metabolite 4-hydroxy-ICN in Arabidopsis thaliana. Transposable elements (TEs) have long been recognized to have the potential to rewire regulatory networks; these results establish a more complete understanding of how duplicated genes and TEs contribute in concert to chemical diversity and pathogen defense.

evolutionary biology↗