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 Ca2+ and H2O2 burst in SlGC-silenced plants. Additionally, silencing of these genes altered the expression of a set of Ca2+ 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.
Highlights
Guanylate cyclases (GCs) are enzymes that catalyze the reaction to produce cyclic GMP, a key signaling molecule in eukaryotes
Alighment of the GC catalytic center (GC-CC) motifs predicted using GCPred with the highest confidence together with the one for sequence #41 predicted from alignment analysis revealed that all these motifs contained [SK]1, [GSC]3 and [KR]14, the three amino acid (AA) associated with catalysis function in the plant GC-specific GC-CC motif, and the sequences were recognized as tomato GC candidates (Fig. 1)
Pst DC3000 bacterial growth in SlGC17 and SlGC18 co-silenced plants was signigicantly increased by approximately one order of magnitude, while that in SlGC17 and SlGC18 individually silenced plants was increased by about 0.6 order of magnitude, compared with that in the eGFP control plants at 3 d post inoculation (Fig. 5B)
Summary
Guanylate cyclases (GCs) are enzymes that catalyze the reaction to produce cyclic GMP (cGMP), a key signaling molecule in eukaryotes. Searching analyses with a motif deduced from representatives of annotated catalytic domains of GCs from prokaryotes and eukaryotes ([RKS] [YFW] [CTGH] [VIL] [FV] G [DNA] x [VIL] x{4} [KR]) demonstrated that sequences carry the motif of functional amino acid (AA) residues of the GC catalytic center (GC-CC) do exist in plants[35,36,37,38] In this motif, the AA at position 1 forms the hydrogen bond with the guanine, the residue at position 3 confers substrate specificity for GTP, the AA at position 14 stabilizes the transition state from GTP to cGMP, while two or three AAs away from the C-terminal end of the motif is the residue that interacts with the Mg2+/Mn2+ ions[35,36]. Our results provide first insights into the geonome-wide composition and function of GCs in crop species
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