A conserved glutamate residue in RPM1-INTERACTING PROTEIN4 is ADP-ribosylated by the Pseudomonas effector AvrRpm2 to activate RPM1-mediated plant resistance. The Plant cell Gram-negative bacterial plant pathogens inject effectors into their hosts to hijack and manipulate metabolism, eluding surveillance at the battle frontier on the cell surface. The effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola functions as an ADP-ribosyl transferase that modifies RESISTANCE TO P. SYRINGAE PV MACULICOLA1 (RPM1)-INTERACTING PROTEIN4 (RIN4), leading to the activation of Arabidopsis thaliana (Arabidopsis) resistance protein RPM1. Here we confirmed the ADP-ribosyl transferase activity of another bacterial effector, AvrRpm2Psa from P. syringae pv. actinidiae, via sequential inoculation of Pseudomonas strain Pto DC3000 harboring avrRpm2Psa following Agrobacterium-mediated transient expression of RIN4 in Nicotiana benthamiana. We conducted mutational analysis in combination with mass spectrometry to locate the target site in RIN4. A conserved glutamate residue (Glu156) is the most likely target for AvrRpm2Psa, as only Glu156 could be ADP-ribosylated to activate RPM1 among candidate target residues identified from the MS/MS fragmentation spectra. Soybean (Glycine max) and snap bean (Phaseolus vulgaris) RIN4 homologs without glutamate at the positions corresponding to Glu156 of Arabidopsis RIN4 are not ADP-ribosylated by bacterial AvrRpm2Psa. In contrast to the effector AvrB, AvrRpm2Psa does not require the phosphorylation of Thr166 in RIN4 to activate RPM1. Therefore, separate biochemical reactions by different pathogen effectors may trigger the activation of the same resistance protein via distinct modifications of RIN4. 10.1093/plcell/koac286

AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6. Plant physiology Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae, and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin-induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors. 10.1093/plphys/kiae186

Changes in the effective gravitational field strength affect the state of phosphorylation of stress-related proteins in callus cultures of Arabidopsis thaliana. Journal of experimental botany In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P <0.05) after 2 h of hypergravity (18 up-regulated, 10 down-regulated) could be identified. The corresponding proteins were largely involved in stress responses, including the detoxification of reactive oxygen species (ROS). In the present study, these investigations are extended to phosphorylated proteins. For this purpose, callus cell cultures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded the most reliable data. The first changes, however, were visible as early as 10 min after the start of treatment. In comparison to 1 g controls, exposure to hypergravity resulted in 18 protein spots, and random positioning in 25, respectively, with increased/decreased signal intensity by at least 2-fold (P <0.05). Only one spot (alanine aminotransferase) responded the same way under both treatments. After 30 min of RP, four spots appeared, which could not be detected in control samples. Among the protein spots altered in phosphorylation, it was possible to identify 24 from those responding to random positioning and 12 which responded to 8 g. These 12 proteins (8 g) are partly (5 out of 12) the same as those changed in expression after exposure to 2 h of hypergravity. The respective proteins are involved in scavenging and detoxification of ROS (32%), primary metabolism (20.5%), general signalling (14.7%), protein translation and proteolysis (14.7%), and ion homeostasis (8.8%). Together with our recent data on protein expression, it is assumed that changes in gravitational fields induce the production of ROS. Our data further indicate that responses toward RP are more by post-translational protein modulation (most changes in the degree of phosphorylation occur under RP-treatment) than by protein expression (hypergravity). 10.1093/jxb/ern324

Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. Rajjou Loïc,Belghazi Maya,Huguet Romain,Robin Caroline,Moreau Adrien,Job Claudette,Job Dominique Plant physiology The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor. 10.1104/pp.106.082057

Site-specific nitrosoproteomic identification of endogenously S-nitrosylated proteins in Arabidopsis. Hu Jiliang,Huang Xiahe,Chen Lichao,Sun Xuwu,Lu Congming,Zhang Lixin,Wang Yingchun,Zuo Jianru Plant physiology Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants. 10.1104/pp.15.00026