Studies On Autoimmune Response, Redox - Sensitive Protein And MART Activity Of Arabidopsis Thaliana

Autoimmunity in the Arabidopsis nudt6-2nudt7-1 Double MutantThe plant innate immune system is bipartite. One branch of this system is based on the detection of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) in plants. Well characterized examples include the perception of bacterial flagellin through the receptor kinase FLS2 and the recognition of bacterial elongation factor Tu (EF-Tu) by the EF-Tu receptor (EFR). The recognition of PAMPs elicits downstream immune responses called PAMP-triggered immunity (PTI). To dampen the host PTI, pathogens deliver a repertoire of effectors through the type III secretion system into host cells. In turn, plants have evolved another layer of surveillance system to specifically recognize the presence of effectors either directly or indirectly through resistance (R) proteins, culminating in a strong defense response termed effector-triggered immunity (ETI), often characterized by a hypersensitive response (HR) at the site of infection.The onset and amplitude of plant innate immunity need to be tightly controlled to ensure optimal growth and development, especially for ETI, which is quite potent and might lead to constitutive defense responses if left unleashed. R genes determine the signal input of ETI, thus it’s not surprising that several mutants identified in recent years exhibit constitutive defense response resulting from improper activation of R genes. The transcription of R genes needs to be strictly controlled, as overexpression of SNC1 or RPS4, two TIR-NB-LRR type R genes, leads to autoimmunity. The autoimmune response could also result from the loss of function of negative regulators of defense response, such as cprl, which encodes an F-box containing protein negatively regulating the accumulation of SNC1 and RPS2 protein. Another negative regulator of plant defense response is AtNUDT7. Plants without a functional AtNUDT7 constitutively overexpress PR genes, accumulate high levels of SA without pathogen perception and exhibit spontaneous microscopic cell death. They are also more resistant to several virulent and avirulent pathogens.Previous reports have shown that AtNUDT7 negatively regulate the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) pathway. EDS1 is essential for basal resistance to some virulent biotrophic and hemibiotrophic pathogens and also for ETI mediated by Toll/Interleukin-1 receptor-nucleotide binding-leucine rich repeat (TIR-NB-LRR) type R proteins. It has long been established that EDS1 functions downstream of activated TIR-NB-LRR receptors as a signal transducer. In the Arabidopsis genome, AtNUDT6 encodes a protein with the highest similarity to AtNUDT7. The nudt6-2nudt7-1 double mutant shows phenotypes characterized of autoimmunity, including extreme dwarfism, constitutive defense related gene overexpression and spontaneous cell death. By studying the nudt6-2nudt7-1 double mutant, we determined that AtNUDT6 and AtNUDT7 both negatively regulate the EDS1 pathway, by suppressing the activity of SNC1 and probably other TIR-NB-LRR R genes. We also found that the phenotype of the nudt6-2nudt7-1 double mutant could be suppressed by growth at 28℃, which was suggested to target R genes, and also by a low nitrate/ammonium ratio in the growth medium, at least partly through a reduction of the level of nitric oxide (NO) in vivo. Our finding would help better understanding of negative regulators of plant defense responses and the interaction of the EDS1 pathway with NO.Proteomic Analysis of Redox-Sensitive ProteinsWhen challenged with abiotic or biotic stresses, one important physiological response of plants is the induction of reactive oxygen species (ROS). Accumulating evidence has proved that ROS is not merely a toxic by-product of metabolism, but also plays a pivotal role in signal transduction, which is mainly mediated by oxidative modification of its cellular substrates (for example, redox-sensitive proteins). Recent advances have revealed that regulation of protein function through oxidative modification is an important molecular mechanism modulating various biological processes. Here, we report a proteomic study of redox-sensitive proteins in Arabidopsis suspension cells subjected to H2O2 treatment. Four gel-based approaches were employed, leading to the identification of four partially overlapping sets of proteins whose thiols underwent oxidative modification in the H2O2-treated cells. Using a method based on differential labeling of thiols followed by immunoprecipitation and Western blot analysis, several selected putative redox-sensitive proteins were confirmed to undergo oxidative modification following the oxidant treatment in Arabidopsis leaves. Another method, which is based on differential labeling of thiols coupled with protein electrophoretic mobility shift assay, was used to reveal that one of the H2O2-sensitive proteins, a homologue of human cytokine-induced apoptosis inhibitor 1 (AtCIAPIN1), also underwent oxidative modification in Arabidopsis leaves after treatments with salicylic acid (SA) or the peptide elicitor flg22, two inducers of plant defense signaling. The redox-sensitive proteins identified from the proteomic study are involved in various biological processes, such as metabolism, the antioxidant system, protein biosynthesis and processing, and cytoskeleton organization. The identification of novel redox-sensitive proteins will be helpful toward better understanding of cellular components or pathways previously unknown to be redox-regulated.Protein mono-ADP-ribosylation Catalyzed by Endogenous mART Activity in Arabidopsis thalianaProtein mono-ADP-ribosylation post-translationally transfers an ADP-ribose moiety from a P-NAD+ donor onto various protein acceptors. Such a reaction is catalyzed by the mono-ADP-ribosyltransferase (mART). This type of modification has been widely characterized in animals, yeast and prokaryotes, and shown to regulate protein functions, but has never been reported in plants. In this study, using [32P]NAD+ as the substrate, ADP-ribosylated proteins in Arabidopsis were discovered and investigated. In adult rosette leaves, one protein substrate with an apparent molecular weight of 32 kDa was found to be radio-labeled. Heat treatment, protease sensitivity assay and nucleotide derivative competition assays suggested a covalent reaction of NAD+ with the 32 kDa protein. [carbonyl-14C]NAD+ could not label the 32 kDa protein, confirming that the modification was ADP-ribosylation. Poly (ADP-ribose) polymerase inhibitor failed to suppress the reaction, but chemicals that destroy mono-ADP-ribosylation on specific amino acid residues could break the linkage, suggesting that the reaction was not a poly-ADP-ribosylation but rather a mono-ADP-ribosylation. This modification mainly exists in leaves and was enhanced by oxidative stresses. In young seedlings, two more protein substrates of 45 kDa and over 130 kDa, respectively, were observed in addition to the 32 kDa protein, indicating that different proteins were modified at different developmental stages. Although the substrate proteins remain to be identified, this is the first report on the discovery and characterization of endogenously mono-ADP-ribosylated proteins in plants.