Redox proteomics of tomato in response to Pseudomonas syringae infection

Abstract

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation-reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

Figure 1

Reverse cysTMT-labeling workflow. Control and Pst-treated samples were prepared at the same time. Blocking reduced thiols with IAM followed by reduction of the oxidized thiols with TCEP allows the previously oxidized thiols due to Pst treatment to be labeled with the isobaric cysTMT reagents.

Figure 2

Comparison of redox proteomes and identified redox proteins in different tomato genotypes. (a) Venn diagram showing the number of shared proteins among three different biological replicates. (b) The number of proteins underwent reduction (gray) or oxidation (dark) in the susceptible prf3 and resistant PtoR genotypes at early and late stage of Pst infection. PtoR clearly showed more oxidized proteins at both 4 hai and 24 hai, compared to prf3.

Figure 3

Comparative ontology analysis of the redox proteins after Pst infection between susceptible and resistant genotypes at early and late stages of infection. Relevant biological process GO terms are shown on the y-axis. Numbers of proteins significantly redox-regulated in each genotype and time point after infection are shown on the x-axis.

Figure 4

Alignment of tomato KAT2 sequence with its Arabidopsis ortholog. The shaded cysteine residue with a star underneath was identified as being redox-regulated upon Pst infection in tomato. This cysteine is localized in the active site of the enzyme and known to form a disulfide bond with the other cysteine at position 192 in Arabidopsis (highlighted in gray).

Figure 5

The role of redox regulation in plant defense response. (a) Role of redox regulation of primary metabolic enzymes in plant defense response at early stage of infection. Redox regulation may serve as an activity switch to turn on or off the different connections between primary metabolism and defense response, playing a role in energy as well as signaling to directly or indirectly trigger defense responses. (b) Role of cysteine synthase oxidation and GS in plant defense response. Cysteine is the sulfur amino acid precursor of GSH, which plays a crucial role in maintaining cellular redox homeostasis. Oxidation of cysteine synthase may cause an increase in enzyme activity, leading to synthesis of more cysteines and further increase in the amount of GSH. Glutamate is a precursor of GSH and glutamine. Oxidation of GS decreases its activity, leading to less glutamine. This may cause an increase in the availability of glutamate for GSH production. (c) Role of redox regulation of enzymes belonging to carbohydrate metabolism, protein synthesis, and chaperone activity in plant defense response. It is likely that a synchronized interaction between sugar and hormonal signaling pathways leads to effective immune responses. Redox regulation of proteins of carbohydrate metabolism might affect apoplastic sugar levels, through which SAR is regulated. Redox regulation of proteins involved in protein synthesis may regulate enzyme activity in protein synthesis, which is an energy-consuming process, and therefore it is considered an important regulation step in stress responses. Oxidative stress is known to enhance misfolded proteins in the endoplasmic reticulum (ER), causing ER stress or unfolded protein response where proteins with chaperone activity play a crucial role. (d) Role of oxidation of KAT2 in the antagonistic interaction between SA and JA. Oxidation of KAT2, which is one of the three core enzymes that catalyze β-oxidation of JA synthesis, may lead to inactivation of KAT2.