Auto-acetylation on K289 is not essential for HopZ1a-mediated plant defense suppression

Abstract

The Pseudomonas syringae type III-secreted effector HopZ1a is a member of the HopZ/YopJ superfamily of effectors that triggers immunity in Arabidopsis. We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2. HopZ1a has been shown to possess acetyltransferase activity, and this activity is essential to trigger immunity in Arabidopsis. HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue. In this paper we analyze the relevance of autoacetylation of lysine residue 289 in HopZ1a ability to suppress plant defenses, and on the light of the results obtained, we also revise its relevance for HopZ1a avirulence activity. Our results indicate that, while the HopZ1aK289R mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1aC216A, since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant. We also present evidence that the HopZ1aK289R mutant still displays virulence activities, partially suppressing both ETI and SAR.

How Microbes Twist Jasmonate Signaling around Their Little Fingers

“…Indeed, post-translational modifications, including acetylation, have been shown to induce or repress proteasomal degradation [ 64 ]. Recently, the threonine residue T346 was identified as the main auto-acetylation site of HopZ1a by mass spectrometry, whereas two additional neighboring serine residues (S349 and S351) are required for the acetyltransferase activity of HopZ1a in vitro and indispensable for the virulence function of HopZ1a in planta [ 65 , 66 ]. These serine residues are important for the enzymatic activity of HopZ1a and are indispensable for inositol hexakisphosphate (IP6)-induced activation of the effector in eukaryotic hosts [ 65 ].…”

Suppression of HopZ Effector-Triggered Plant Immunity in a Natural Pathosystem

“…The first to be characterized, HopZ1a, suppresses several layers of the plant defense response, including pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI), as well as systemic acquired resistance (SAR) ( Macho et al, 2009 , 2010 ; Lewis et al, 2014 ; Rufián et al, 2015 ). To date, HopZ1a has been shown to function as an acetyltransferase ( Lee A.H. et al, 2012 ; Jiang et al, 2013 ; Lewis et al, 2013 ; Ma et al, 2015 ; Rufián et al, 2015 ), and an assortment of host proteins have been proposed as targets of its virulence function ( Zhou et al, 2011 ; Jiang et al, 2013 ). However, the exact nature of its relevant virulence target(s) within the plant is still under discussion.…”

The bacterial effector HopZ1a acetylates MKK7 to suppress plant immunity

“…For comparing more than two mean values 233 statistical significance was tested using one-way ANOVA (α=0.05) with Tukey's 234 multiple comparisons test. 235 Transient expression assays in N. benthamiana were performed as previously described 236 (Rufián et al, 2015). Briefly, 5-week old plants were infiltrated with an Agrobacterium 237 C58C1 solution at OD 600 0.5 in 10 mM MgCl 2 , 10 mM MES (SIGMA, USA), 200 µM 238 3′,5′-Dimethoxy-4′-hydroxyacetophenone (acetosyringone) (SIGMA, USA) carrying the 239 corresponding binary plasmids ( Table 1).…”

“…AvrRps4 and AvrRpm1 (Macho et al, 2010;Rufián et al, 2015); and (iii) SAR 64 triggered by either virulent or avirulent bacteria (Macho et al, 2010;Rufián et al, 65 2015). On the other hand, HopZ1a triggers ETI in Arabidopsis upon recognition by the 66 NLR ZAR1 (HOPZ-ACTIVATED RESISTANCE 1) (Lewis et al, 2010), a defense 67 response that is independent of salicylic acid (SA) and EDS1 (Lewis et al, 2010;68 Macho et al, 2010).…”

“…HopZ1a acetyltransferase activity is completely dependent on the 77 integrity of the catalytic triad cysteine (C216), since a HopZ1a C216A mutant behaves as a 78 catalytically inactive mutant (Lee et al, 2012). Likewise, residue C216 is essential for 79 all described HopZ1a virulence and avirulence functions in planta (Ma et al, 2006;80 Lewis et al, 2008;Macho et al, 2010;Lewis et al, 2014;Rufián et al, 2015). HopZ1a 81 has also been described to auto-acetylate in two serine residues that are essential for 82…”