Salicylic acid binding protein 2 (SABP2) that converts methyl salicylate to salicylic acid (SA) plays an obligatory role in the SA-mediated disease resistance pathway in plants. SABP2 interacts with SBIP24 in a yeast two-hybrid screening. SBIP24 belongs to the stearoyl-acyl carrier protein-desaturase protein family. To biochemically characterize the SBIP24, it was cloned from tobacco leaves using RT-PCR and expressed in E. coli. Recombinant SBIP24 was affinity purified using Ni-NTA chromatography. RT-PCR was performed to determine the role of SABP2 in modulating the expression SBIP24. TMV infected transgenic C3 (control tobacco plant containing empty silencing vector) and 1-2 (SABP2-silenced) transgenic tobacco plants were used. Preliminary results indicate that SABP2 may regulate the expression of SBIP24 in tobacco plants. Further studies are needed to confirm these preliminary results.

Silent Information Regulator 2 (SIR2) have a phylogenetically conserved catalytic domain from bacteria to humans. It catalyzes NAD+ dependent deacetylase activity post-translationally on acetylated lysine residues present in the protein. Because SIR2 are NAD+ dependent, its activity gets influenced by the change in the level of NAD+. SIR2 is responsible for calorie restriction and increased replicative yeast lifespan. It breakdown high energy bond in nicotinamide adenine dinucleotide (NAD), and the synthesis of O-acetyl-ADP-ribose which is a novel product. Lysine de/acetylation of histone molecule plays a significant role in chromatin dynamics in eukaryotes, but little is known in term of non-histone molecule modification by SIR2 enzyme especially in the case of the plant. SIP-428 is one of the SABP2 interacting protein (SIP) that exhibit SIR2 deacetylase activity. SABP2 is one of the essential components of salicylic acid (SA) signaling pathway that converts inactive methyl salicylate (MeSA) to active SA to induce local as well as SAR. AtSRT2, an Arabidopsis homolog of SIP-428 negatively regulate the basal resistance. Although catalytic domain is conserved, functional divergence has been reported in the case of SIR2 homologs. Presence of acetylated lysine residue in many cellular and organellar proteins implicated the possible physiological and metabolic role of SIP-428. Our result demonstrated SIP-428 exhibited NAD+ dependent deacetylase activity, but its lysine residue found to be acetylated, which raises the possibility of a post-translation regulatory mechanism that modulates the activity of SIP-428. SIP-428 have non-histone substrate, the negative regulator of basal resistance, and SAR. To understand better about the role of SIP-428 in plant physiology how it plays a vital role in SABP2 signaling pathway we will be using transgenic tobacco plant with altered expression of SIP-428 (Silence and inducible overexpression). Verified T3 generation of silence line and T2 generation of overexpression were created. These transgenic plant will be used to answer the possible link between SIP-428 and SABP2 in response to pathogen infection.

Plants are resilient organisms that are continually evolving and continue to withstand an adverse and dynamic world. SABP2-interacting protein (SIP)-470 is a non-specific lipid transfer protein (nsLTP) that was identified in tobacco. SIP470 was discovered during a yeast two-hybrid screening with SABP2, which is an important methyl esterase enzyme which catalyzes the conversion of immobile MeSA into active salicylic acid (SA) during pathogenic challenge. SA activation and mobility allows for immunity to be carried to other, non-infected parts of the plant. This induced responses is called systemic acquired resistance (SAR) and it is a broad spectrum defense. Like many nsLTPs, SIP470 is small and has a predicted characteristic hydrophobic cavity. nsLTPs are found in higher plants and have repeatedly demonstrated protection in biotic stress including disease resistance, and greater resistance to both bacterial and fungal pathogens in overexpressed transgenic lines. This diverse class is also significantly involved in plant adaptation to environmental changes, namely drought, salinity, and freezing, but also in osmotic stress and wounding. Furthermore, nsLTPs are involved in wax metabolism and seed development. Subcellular localization of nsLTPs varies considerably during in vitro and in recent in vivo studies. SIP470 was originally identified in tobacco plants, and therefore, it is important to study its role directly in tobacco plants. SIP470 and eGFP fusion construct has been generated to study the subcellular localization of SIP470 in tobacco cells. SIP470 localization has shown a discontinuous, punctate arrangement around the membrane periphery which is being further verified by subcellular fractionation. Transgenic tobacco lines that are silenced in SIP470 via RNAi have been generated, and these plants are being screened. Overexpressor transgenic lines of SIP470 have been generated and are under the control of an estradiol-inducible promoter. These transgenic lines will be tested for their response in basal resistance and SAR.

Glucosyltransferases catalyze the transfer of glucose molecules from an active donor to acceptor molecules and are involved in many plant processes. SIP68, a tobacco glucosyltransferase protein, is a SABP2-interacting protein. It was identified in a yeast two-hybrid screen using SABP2 as bait and tobacco proteins as prey. SABP2, converts methyl salicylate to salicylic acid (SA) as a part of the signal transduction pathways in SA-mediated defense signaling. Subcellular localization is a crucial aspect of protein functional analysis to assess its biological function. The recombinant SIP68 tagged with eGFP was expressed transiently in Nicotiana benthamiana and observed under confocal microscopy. Fluorescent signals were observed in the epidermal cells. Subcellular fractionation of the tobacco leaves transiently expressing SIP68-+eGFP confirmed that SIP68 is localized in the cytosol. To study the role of SIP68 in plant stress signaling, transgenic lines with altered SIP68 expression were generated using RNAi and CRISPR Cas9 and analyzed.

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant known to accumulate less SA. Taken together expression of SBIP-428 is negatively correlated to the levels of SA in plants. The AtSRT2 plants exhibit no altered growth phenotype but exhibit a higher pathogen resistance against bacterial pathogen. Our results indicate that SBIP-428 is an important regulator in plant defense pathway.

Abscisic acid (ABA) induces RD22, responsive to dehydration stress gene. Salicylic acid (SA) has been the focus of research due to its role against pathogens and abiotic stress. Interaction between ABA and SA signaling pathways is still poorly understood. SA-Binding Protein 2 (SABP2) converts methyl salicylate to SA. An attempt was made to identify proteins that interact with SABP2 using a yeast 2-hybrid screening. Several interactors were identified. One of them, SA-Binding Protein 2 Interacting Protein-355 (SBIP-355), showed high homology to RD22. Bioinformatic approaches showed that SBIP-355 contains a BURP domain. Phylogenetic analysis reveals that SBIP-355 clustered into the clade of RD22-like proteins. Thus, SBIP-355 gene might be a stress-inducible gene and encodes a dehydration-responsive protein, which is important for the stress tolerance of tobacco. The complementary DNA (cDNA) of tobacco SBIP-355 was cloned into pDEST-17 vector and then expressed in E. coli to detect the expression of SBIP-355 protein.

Plants are in a perpetual evolutionary arms race with a wide range of pathogens. Their sessile nature has led plants to evolve defense mechanisms that can quickly recognize a unique stressor and deploy a resistance tailored for a specific attack. The salicylic acid (SA) mediated defense pathway has been shown to be one of the major defense tactics plants can initiate to defend themselves against microbial pathogens. Following a pathogen attack high levels of methyl salicylate (MeSA) are produced that can be converted to SA by the enzyme salicylic acid binding protein 2 (SABP2). A yeast two-hybrid screening was performed to identify protein interactions with SABP2 to better understand the regulation of the enzyme on a cellular level. SBIP-436 is an interacting protein of tobacco SABP2 which showed high homology to phospholipase D-δ (PLD- δ). With an abundance of stimulators PLD- δ may be a lipid signaling modulator developed to perform various functions in different situations. PLD- δ may be a key player in a lipid signaling cascade in the SA mediated defense pathway. We present a novel Nicotiana tabacum PLD- δ putative gene construct. We demonstrate that the putative PLD- δ is subject to alternative splicing and its expression is differentially modulated under biotic and abiotic stress. Our results indicate that this putative PLD- δ may play a role in the SA mediated defense pathway.

Plant secondary metabolites are essential for normal growth and development in plants ultimately affecting crop yield. They play roles ranging from appearance of the plants to defending against pathogen attack and herbivory. They have been used by humans for medicinal and recreational purposes amongst others. Glycosyltransferases catalyze the transfer of sugars from donor substrates to acceptors. Glucosyltransferases are a specific type of glycosyltransferases known to transfer glucose molecules from a glucose donor to a glucose acceptor (aglycone) producing the corresponding glucose secondary metabolite or glycone, in this case glucosides. It was hypothesized that SBIP68, a tobacco putative glucosyltransferase-like protein glucosylated salicylic acid. Salicylic acid is an essential plant defense secondary metabolite. SBIP68 was cloned and heterologously expressed in both prokaryotic and eukaryotic systems. Results from activity screening suggest that SBIP68 is a UDP-glucose flavonoid glucosyltransferase with broad substrate specificity. Further studies are required to fully characterize SBIP68.

Biotic and abiotic stresses are signifi cant factors limiting the production of food and other supporting materials required to sustain increasing world population. Plant health is directly related to human health and is increasingly becoming signifi cant and demands more attention towards limiting the damages caused by biotic and abiotic stresses. Signifi cant progress has been made towards our understanding of the processes, which mediate both biotic and abiotic stress signaling in plants. Signifi cant role is played by various plant hormones, e.g., salicylic acid (SA) and jasmonic acid (JA) in biotic stress and abscisic acid (ABA) in abiotic stress (Annu Rev Cell Dev Biol 28:489-521, 2012). Other hormones with minor role include the cytokinins (CK), auxins (indole 3 acetic acid. IAA), and the brassinosteroids (BR) (Annu Rev Cell Dev Biol 28:489-521, 2012). Cross talk between these plant hormones is signifi cant and may result in either synergistic or antagonistic effect on stress responses (Annu Rev Cell Dev Biol 28:489-521, 2012). In recent years, extensive research carried out in various laboratories has implicated cross talk between the ABA and the SA in abiotic stress response. This is signifi cant in light of SA being key player in biotic stress responses in plants. This review will discuss the role of SA in biotic and abiotic stress signaling and its cross talk with other hormones in mediating abiotic stress signaling in plants.

Salicylic acid (SA) is a key plant hormone that mediates host responses against microbial pathogens. Identification and characterization of SA-interacting/binding proteins is a topic which has always excited scientists studying microbial defense response in plants. It is likely that discovery of a true receptor for SA may greatly advance understanding of this important signaling pathway. SABP2 with its high affinity for SA was previously considered to be a SA receptor. Despite a great deal work we may still not have true a receptor for SA. It is also entirely possible that there may be more than one receptor for SA. This scenario is more likely given the diverse role of SA in various physiological processes in plants including, modulation of opening and closing of stomatal aperture, flowering, seedling germination, thermotolerance, photosynthesis, and drought tolerance. Recent identification of NPR3, NPR4 and NPR1 as potential SA receptors and α-ketoglutarate dehydrogenase (KGDHE2), several glutathione S transferases (GSTF) such as SA binding proteins have generated more interest in this field. Some of these SA binding proteins may have direct/indirect role in plant processes other than pathogen defense signaling. Development and use of new techniques with higher specificity to identify SA-interacting proteins have shown great promise and have resulted in the identification of several new SA interactors. This review focuses on SA interaction/binding proteins identified so far and their likely role in mediating plant defenses.