A hydrophobic anchor mechanism defines a deacetylase family that suppresses host response against YopJ effectors

Abstract

Several Pseudomonas and Xanthomonas species are plant pathogens that infect the model organism Arabidopsis thaliana and important crops such as Brassica. Resistant plants contain the infection by rapid cell death of the infected area through the hypersensitive response (HR). A family of highly related α/β hydrolases is involved in diverse processes in all domains of life. Functional details of their catalytic machinery, however, remained unclear. We report the crystal structures of α/β hydrolases representing two different clades of the family, including the protein SOBER1, which suppresses AvrBsT-incited HR in Arabidopsis. Our results reveal a unique hydrophobic anchor mechanism that defines a previously unknown family of protein deacetylases. Furthermore, this study identifies a lid-loop as general feature for substrate turnover in acyl-protein thioesterases and the described family of deacetylases. Furthermore, we found that SOBER1's biological function is not restricted to Arabidopsis thaliana and not limited to suppress HR induced by AvrBsT.

Fig. 1

Phylogenetic and structural analysis of plant hydrolases reveals two major protein clades. a Phylogenetic tree of related plant hydrolases. b Multiple sequence alignment highlighting conserved sequence blocks within the clades. c Superimposition of AtSOBER1 (gray) and ZmB6T1C9 (black) showing the structural elements encoded by those blocks in the same colors

Fig. 2

ZmB6T1C9 has an open substrate tunnel while the tunnel of SOBER1 is blocked. a Tunnel architecture of ZmB6T1C9 with docked pNP-palmitate, displaying the continuous tunnel through the protein and the position of catalytic serine S126. b K _M values of different para-nitrophenyl esters from pNP acetate (C2) through pNP-palmitate (C16) to ZmB6T1C9/ZmAPT2. Data were measured in triplicates and error bars indicate standard deviation. c Transient co-expression of AtSOBER1 (wild type and enzymatic dead mutant), AtTIPSY1 and AT5G20060 (closest Arabidopsis homolog of ZmB6T1C9) with avrBsT in N. benthamiana. d Open tunnel architecture in ZmB6T1C9. e Blocked tunnel architecture in AtSOBER1 and location of tunnel blocking residues L63 and F65

Fig. 3

Altering SOBER1 protein surface changes substrate specificity and biological function. a K _M values of AtSOBER1 wt and L63A to pNP acetate (C2), pNP butyrate (C4), and pNP-valerate (C5). Data were measured in triplicates and error bars indicate standard deviation. b Docked pNP acetate into the catalytic site of wild type AtSOBER1. c Docked pNP-valerate into the catalytic site of AtSOBER1 L63A. d Transient co-expression of AtSOBER1 (wild type and mutant variants) with avrBsT in N. benthamiana

Fig. 4

Suppression of HR is not determined by the protein termini of SOBER1 and TIPSY1. a Terminal sequences of both Arabidopsis proteins. b, c Localization of YFP-tagged AtSOBER1 (b) and AtTIPSY1 (c) in epidermal root cells of transgenic Arabidopsis seedlings. YFP signals are depicted in green, while propidium iodide, which was used for counterstaining, is shown in red. d Transient co-expression of AtSOBER, AtTIPSY1 and their truncated mutant variants with avrBsT in N. benthamiana. Scale bar = 5 µm

Fig. 5

Enzymatic and biological properties are conserved between AtSOBER1 and BnSOBER1. a–e Lid-loop temperature factors of wild type AtSOBER1, AtSOBER1 F65L and of the acyl-protein thioesterases ZmB6T1C9 and human APT1. f Catalytic efficiencies of wild type and mutant SOBER1/TIPSY1 from Arabidopsis thaliana and Brassica napus on different para-nitrophenyl esters. Data were measured in triplicates and error bars indicate standard deviation. g Transient co-expression of SOBER1/TIPSY1 from Arabidopsis thaliana and B. napus with avrBsT in N. benthamiana

Fig. 6

SOBER1 is a protein deacetylase. a HA:AvrBsT variants (wild type and catalytic dead mutant C222A) were in vitro translated, affinity enriched and incubated with [¹⁴C]-acetyl coenzyme A and inositol hexakisphosphate. Subsequent addition of wild type AtSOBER1 deacetylated AvrBsT, while mutant variants (mut1: H192A, mut2: S106A H192A) were inactive. Autoradiography was used to visualize acetylation. (see also Supplementary Fig. 6). b FLAG:ACIP1 was in vitro translated, affinity enriched and incubated with in vitro translated HA:AvrBsT, [¹⁴C]-acetyl coenzyme A and inositol hexakisphosphate. Subsequent addition of wild type AtSOBER1 deacetylated ACIP1, while the mutant variant H192A was inactive. Autoradiography was used to visualize acetylation. c Acyl-protein thioesterases feature a long accessible tunnel for accommodation of long-chain fatty acids and release the product by use of a flexible loop that constitutes the tunnel lid. Deacetylases use a hydrophobic residue as anchor to fix the lid and another residue to close the tunnel entrance. This causes an alteration in protein surface and loop rigidity changing the catalytic preference to deacetylation