Characterization of tomato protein kinases embedding guanylate cyclase catalytic center motif

Abstract

Guanylate cyclases (GCs) are enzymes that catalyze the reaction to produce cyclic GMP (cGMP), a key signaling molecule in eukaryotes. Nevertheless, systemic identification and functional analysis of GCs in crop plant species have not yet been conducted. In this study, we systematically identified GC genes in the economically important crop tomato (Solanum lycopersicum L.) and analyzed function of two putative tomato GC genes in disease resistance. Ninety-nine candidate GCs containing GC catalytic center (GC-CC) motif were identified in tomato genome. Intriguingly, all of them were putative protein kinases embedding a GC-CC motif within the protein kinase domain, which was thus tentatively named as GC-kinases here. Two homologs of Arabidopsis PEPRs, SlGC17 and SlGC18 exhibited in vitro GC activity. Co-silencing of SlGC17 and SlGC18 genes significantly reduced resistance to tobacco rattle virus, fungus Sclerotinia sclerotiorum, and bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. Moreover, co-silencing of these two genes attenuated PAMP and DAMP-triggered immunity as shown by obvious decrease of flg22, chitin and AtPep1-elicited Ca 2+ and H 2 o 2 burst in SlGC-silenced plants. Additionally, silencing of these genes altered the expression of a set of Ca 2+ signaling genes. Furthermore, co-silencing of these GC-kinase genes exhibited stronger effects on all above regulations in comparison with individual silencing. Collectively, our results suggest that GC-kinases might widely exist in tomato and the two SlPEPR-GC genes redundantly play a positive role in resistance to diverse pathogens and PAMP/DAMP-triggered immunity in tomato. Our results provide insights into composition and functions of GC-kinases in tomato. Guanosine 3′,5′-cyclic monophosphate (cGMP) is a well known intracellular second messenger that regulates a broad range of physiological responses in animals, fungi and prokaryotes. However, the existence and function of cGMP in higher plants has been controversial for many years 1. The level of cGMP in higher plants is much lower than that in animals 2. Nevertheless, with the improvement of cGMP determination methods, it has been generally recognized that cGMP is present in plants 3-5. Furthermore, cGMP has been found to be involved in many molecular and cellular events such as protein phosphorylation 6,7 , transcription regulation 8,9 , hormone functioning 6,10,11 and cation flux regulation 8,12,13 , as well as a variety of developmental processes such as development of adventitious roots 14 , chloroplast development 15 , seed germination 16 , stomatal movement 17 , reorientation of pollen tube 18 , anthocyanin and flavonoid biosynthesis 9 , and photoperiodic flower induction 19. Additionally, cGMP is important in response to biotic stresses 20-24 and abiotic stresses 25,26. Moreover, cGMP appears to act on components such as cyclic nucleotide-gated ion channels (CNGCs), cGMP-dependent kinase G (PKG) and cyclic nucleotide phosphodiesterases (PDEs) to modulate plant responses 22,23,27-31. While cGMP ...

BdGUCD1 and Cyclic GMP Are Required for Responses of Brachypodium distachyon to Fusarium pseudograminearum in the Mechanism Involving Jasmonate

“…The available data in the literature indicate that cGMP and enzymes with in vitro confirmed GC activity are involved in the response to pathogen infection [ 4 , 5 , 6 , 7 , 27 , 28 ]. Active resistance is triggered by pathogens and involves biochemical and molecular responses.…”

Moonlighting Proteins Shine New Light on Molecular Signaling Niches

“…Several of the LRR RLK candidate guanylate cyclases (moonlighting kinases) identified in Arabidopsis [ 37 ] have since been shown to contain both kinase and guanylate cyclase activity at least in vitro. These include the brassinosteroid receptor, brassinosteroid insensitive 1 (AtBRI1; AT4G39400) [ 37 , 80 ], phytosulfokine receptor 1 (AtPSKR1; AT2G02220) [ 81 , 82 , 83 ], the DAMP receptor for Arabidopsis plant elicitor peptide 1 (AtPEP1), AtPEPR1 (AT1G73080), and its tomato homologs SlGC17 and SlGC18 [ 84 , 85 ]. BRI1 and PSKR1 are both important in plant growth, while PEPR1 is involved in plant immunity.…”

“…Both PEPR1/2 carry a guanylate cyclase catalytic center with conserved residues crucial for catalysis embedded in its kinase domain [ 84 , 120 ], but so far only the enzymatic activity of PEPR1 has been experimentally demonstrated [ 84 ]. Recombinant protein studies have demonstrated that AtPEPR1, H. hybridum HhPEPR1, and tomato homologs SlGC17 and SlGC18, can all generate cGMP [ 84 , 85 , 136 ]. Downstream signaling following PEP activation involves characteristic pattern induced responses including increased gene transcription that is enhanced in the presence of calcium possibly entering cells by cyclic nucleotide gated channels (CNGC) [ 84 , 125 ].…”

In Vitro Characterization of Guanylyl Cyclase BdPepR2 from Brachypodium distachyon Identified through a Motif-Based Approach

“…x(3) [VIL] x (4) [KR] is present in the catalytic center of guanylyl cyclases of several A. thaliana proteins, including brassinosteroid receptor AtBRI1 [8], wall-associated kinase like 10 AtWAKL10 [20], Pep1 receptor AtPepR1 [12], phytosulfokine receptor AtPSKR1 [9], nitric oxide-binding GC AtNOGC1 [21], plant natriuretic peptide receptor AtPNP-R1 [22], H. hybridum HpPepR1 [13], and 99 GC candidates in Solanum lycopersicum [15]. As presented in Figure 1B, the red amino acids at positions 1, 3 and 14 within the search motif are functionally assigned residues [16].…”