Erica J. Washington, M. Shahid Mukhtar, Omri M. Finkel, Li Wan, Mark J. Banfield, Joseph J. Kieber, and Jeffery L. Dangl

Significance

Pseudomonas syringae is a Gram-negative bacterium that uses a type III secretion system to inject type III effector (T3E) proteins into the host to cause disease in plants. Multiple P. syringae T3Es promote virulence by targeting immune system signaling pathways using diverse biochemical mechanisms. We provide evidence for a molecular function of the P. syringae T3E HopAF1. We demonstrate that the C-terminal region of HopAF1 has structural homology to deamidases. We demonstrate that an enzyme important for production of the gaseous signaling hormone ethylene is a target for HopAF1 and show that HopAF1 targets methylthioadenosine nucleosidase proteins MTN1 and MTN2 to dampen ethylene production during bacterial infection.

 Abstract

HopAF1 is a type III effector protein of unknown function encoded in the genomes of several strains of Pseudomonas syringae and other plant pathogens. Structural modeling predicted that HopAF1 is closely related to deamidase proteins. Deamidation is the irreversible substitution of an amide group with a carboxylate group. Several bacterial virulence factors are deamidases that manipulate the activity of specific host protein substrates. We identified Arabidopsis methylthioadenosine nucleosidase proteins MTN1 and MTN2 as putative targets of HopAF1 deamidation. MTNs are enzymes in the Yang cycle, which is essential for the high levels of ethylene biosynthesis in Arabidopsis. We hypothesized that HopAF1 inhibits the host defense response by manipulating MTN activity and consequently ethylene levels. We determined that bacterially delivered HopAF1 inhibits ethylene biosynthesis induced by pathogen-associated molecular patterns and that Arabidopsis mtn1 mtn2 mutant plants phenocopy the effect of HopAF1. Furthermore, we identified two conserved asparagines in MTN1 and MTN2 from Arabidopsis that confer loss of function phenotypes when deamidated via site-specific mutation. These residues are potential targets of HopAF1 deamidation. HopAF1-mediated manipulation of Yang cycle MTN proteins is likely an evolutionarily conserved mechanism whereby HopAF1 orthologs from multiple plant pathogens contribute to disease in a large variety of plant hosts.

See http://www.pnas.org/content/113/25/E3577.full

PNAS June 21 2016; vol.113; no.25: E3577–E3586

Fig. 1. HopAF1 putative catalytic residues in HopAF1 transgenic lines are required for an increase in susceptibility to a disarmed pathogen and inhibition of flg22-induced defense. (A) Homology model of the HopAF1 reference allele (blue) from Pto DC3000 residues 130–284 aligned with CheD (PDB ID code 2F9Z) (silver). The side chains of the putative catalytic residues of HopAF1 D154, C159, and H186 are shown in green. The side chains of the CheD catalytic residues C27, H44, and T21 are shown in silver. The N and C termini of the proteins are labeled, as are the key structural elements (α helix 1, α helix 2, β sheet 3, and β sheet 5). PyMOL was used to generate the figure. (B) Bacterial growth of Pto DC3000D28E in Col-0 plants and two independent transgenic lines expressing either estradiol-inducible HopAF1-cerulean-HA or estradiol-inducible HopAF1_C159AH186A-cerulean-HA was measured 0 and 3 d after hand-inoculation with bacteria (1 × 10⁵ cfu/mL). Transgene expression was induced with 20 μM estradiol 24 h before bacterial inoculation. Error bars represent SEM. An ANOVA was performed among the day 3 samples, followed by Tukey’s post hoc analysis (P < 0.05). The different letters indicate groups that differ significantly. (C) Col-0, estradiol-inducible HopAF1-cerulean-HA, and estradiol-inducible HopAF1_C159AH186A-cerulean-HA plants were sprayed with 20 μM estradiol for 12 h and then were infiltrated with 1 μM flg22 or water for 24 h before infiltration with 1 × 10⁵ cfu/mL Pto DC3000. Bacterial growth was determined 0 and 3 d after bacterial inoculation. Error bars represent SEM. An ANOVA was performed among the day 3 samples, followed by Tukey’s post hoc analysis (P < 0.05). The different letters indicate groups that differ significantly.