The Arabidopsis thaliana Brassinosteroid Receptor (AtBRI1) Contains a Domain that Functions as a Guanylyl Cyclase In Vitro

BACKGROUND: Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) from guanosine 5′-triphosphate (GTP). Cyclic GMP has been implicated in an increasing number of plant processes, including responses to abiotic stresses such as dehydration and salt, as well as hormones. PRINCIPLE FINDINGS: Here we used a rational search strategy based on conserved and functionally assigned residues in the catalytic centre of annotated GCs to identify candidate GCs in Arabidopsis thaliana and show that one of the candidates is the brassinosteroid receptor AtBR1, a leucine rich repeat receptor like kinase. We have cloned and expressed a 114 amino acid recombinant protein (AtBR1-GC) that harbours the putative catalytic domain, and demonstrate that this molecule can convert GTP to cGMP in vitro. CONCLUSIONS: Our results suggest that AtBR1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors, NPR1 and NPR2. The findings also suggest that cGMP may have a role as a second messenger in brassinosteroid signalling. In addition, it is conceivable that other proteins containing the extended GC search motif may also have catalytic activity, thus implying that a significant number of GCs, both in plants and animals, remain to be discovered, and this is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains over 90 annotated putative CGs

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

An Arabidopsis Clathrin Assembly Protein with a Predicted Role in Plant Defense Can Function as an Adenylate Cyclase

Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP261–379) can produce cyclic adenosine 3′,5′ monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization

Calcium is the switch in the moonlighting dual function of the ligand–activated receptor kinase phytosulfokine receptor 1

Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca²⁺ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca²⁺ in a concentration-dependent manner. Conversely, increasing Ca²⁺ levels inhibits kinase activity up to 500-fold at 100 nM Ca²⁺. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases

An Arabidopsis Clathrin Assembly Protein with a Predicted Role in Plant Defense Can Function as an Adenylate Cyclase

Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP261–379) can produce cyclic adenosine 3′,5′ monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization

Calcium is the molecular switch shifting the phytosulfokine receptor 1 (PSKR1) from kinase to guanylate cyclase activity

Conference presentationMany plant responses are mediated by interactions between intracellular calcium and the second messenger cGMP formed by guanylate cyclases (GCs). Previously we identified a novel class of receptor-GCs containing the GC catalytic center embedded within the kinase domain and showed that the recombinant cytoplasmic domain of phytosulfokine receptor AtPSKR1 has both guanylate cyclase and kinase activity in vitro (Kwezi et al. 2011 J Biol Chem 286: 22580-8). We now show that physiological increases in calcium levels enhance GC activity of AtPSKR1 whereas these calcium levels reversibly inhibit kinase activity. In addition PSKR1 kinase activity is reduced in the presence of the GC product cGMP. Recombinant AtPSKR1 can undergo in vitro autophosphorylation and we have confirmed it has 14 phosphorylation sites in its cytoplasmic domain including 8 serine, 3 threonine and 3 tyrosine residues. Three phospho-serine residues at the juxta-membrane position were mutated to either mimic phosphorylation on or off states. Kinase activity was enhanced in the on mutant and suppressed in the off mutant while GC activity was unaffected suggesting calcium acts as a molecular switch of PSKR1- mediated signalling that can be modulated by the phosphorylation state. The challenge now lies in understanding how molecular interactions between the GC and kinase domains are capitalized on in the plant

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens