Dhirendra Kumar

High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity

Salicylic acid (SA) is a critical hormone for signaling innate immunity in plants. Here we present the purification and characterization of SA-binding protein 2 (SABP2), a tobacco protein that is present in low abundance and specifically binds SA with high affinity. Sequence analysis predicted that SABP2 is a lipase belonging to the α/β fold hydrolase super family. Confirming this prediction, recombinant SABP2 exhibited lipase activity against several synthetic substrates. Moreover, this lipase activity was stimulated by SA binding and may generate a lipid-derived signal. Silencing of SABP2 expression suppressed local resistance to tobacco mosaic virus, induction of pathogenesis-related 1 (PR-1) gene expression by SA, and development of systemic acquired resistance. Together, these results suggest that SABP2 is an SA receptor that is required for the plant immune response. We further propose that SABP2 belongs to a large class of ligand-stimulated hydrolases involved in stress hormone-mediated signal transduction.

Differential induction of tobacco MAP kinases by the defense signals nitric oxide, salicylic acid, ethylene, and jasmonic acid.

In tobacco, two mitogen-activated protein (MAP) kinases, designated salicylic acid (SA)-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) are activated in a disease resistance-specific manner following pathogen infection or elicitor treatment. To investigate whether nitric oxide (NO), SA, ethylene, or jasmonic acid (JA) are involved in this phenomenon, the ability of these defense signals to activate these kinases was assessed. Both NO and SA activated SIPK; however, they did not activate WIPK. Additional analyses with transgenic NahG tobacco revealed that SA is required for the NO-mediated induction of SIPK. Neither JA nor ethylene activated SIPK or WIPK. Thus, SIPK may function downstream of SA in the NO signaling pathway for defense responses, while the signals responsible for resistance-associated WIPK activation have yet to be determined.

Identification of likely orthologs of tobacco salicylic acid‐binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana

Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2s methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active alpha/beta fold hydrolases that share 32-57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.

Tobacco transcription factor WRKY1 is phosphorylated by the MAP kinase SIPK and mediates HR-like cell death in tobacco.

The salicylic acid-induced protein kinase (SIPK) of tobacco, which is a mitogen-activated protein kinase (MAPK), is activated by various biotic and abiotic treatments. Overexpression of SIPK has been shown to trigger cell death. In this study, a targeted yeast two-hybrid approach identified the tobacco transcription factor WRKY1 as a potential substrate. SIPK phosphorylated WRKY1, which resulted in enhanced DNA-binding activity of WRKY1 to its cognate binding site, a W box sequence from the tobacco chitinase gene CHN50. SIPK-mediated enhancement of WRKY1 DNA-binding activity was inhibited by staurosporine, a general kinase inhibitor. Co-expression of SIPK and WRKY1 in Nicotiana benthamiana led to more rapid cell death than expression of SIPK alone, suggesting that WRKY1 is involved in the formation of hypersensitive response-like cell death and may be a component of the signaling cascade downstream of SIPK.

Interactive effects of lead, copper, nickel and zinc on growth, metal uptake and antioxidative metabolism of Sesbania drummondii.

Sesbania drummondii seedlings were grown in a medium to which lead (Pb), copper (Cu), nickel (Ni) and zinc (Zn) were added singly and in combinations in order to assess the effects of metal interactions on seedling growth, metal accumulation and anti-oxidative system. S. drummondii growth was significantly inhibited with metal treatments. S. drummondii accumulated substantially higher concentrations of metals in the roots than shoots. The uptake of metals followed the order Pb>Cu>Zn>Ni in roots and Pb>Zn>Cu>Ni in shoots. In addition, uptake of a single metal by S. drummondii was affected by the presence of a second metal, suggesting an antagonistic effect or competition between metals at the plant uptake site. A significant increase in both enzymatic [superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR)] and non-enzymatic (glutathione) antioxidant was observed in the S. drummondii seedlings exposed to different metal treatments. The enhancement in enzyme activities followed the order of Cu>Ni>Pb>Zn. However, compared to the effect of individual metal, metals in combination increased the enzyme activities to varying degrees.

Salicylic acid signaling in disease resistance

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.

SABP2, a methyl salicylate esterase is required for the systemic acquired resistance induced by acibenzolar-S-methyl in plants

Tobacco SABP2, a 29 kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar‐S‐methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2‐silenced tobacco plants when treated with ASM, fail to express PR‐1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2‐silenced plants. These results show that functional SABP2 is required for ASM‐mediated induction of resistance.

CLONING AND CHARACTERISATION OF A GENE ENCODING AN ANTIVIRAL PROTEIN FROM CLERODENDRUM ACULEATUM L.

The Clerodendrum aculeatum-systemic resistence inducing (CA-SRI) protein, a 34 kDa basic protein, plays a key role in inducing strong systemic resistance in susceptible plants against various plant viruses [22]. We have cloned the cDNA encoding the CA-SRI from C. aculeatum leaves using antibodies raised against the purified protein and degenerate oligonucleotide probes derived from microsequencing of the CA-SRI protein. The full-length cDNA consisted of 1218 nucleotides with an open reading frame of 906 bp. The deduced amino acid sequence of CA-SRI protein showed varying homology (ranging from 11 to 54%) to the ribosome inactivating proteins (RIPs) from other plant species. CA-SRI inhibited in vitro protein synthesis both in rabbit reticulocyte lysate and wheat germ lysate but not in Escherichia coli in vitro translation system. The CA-SRI open reading frame was expressed in an E. coli expression vector and the purified recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate. Southern blot analysis indicated that the CA-SRI gene may be present in low copy number.

The search for the salicylic acid receptor led to discovery of the SAR signal receptor.

Systemic acquired resistance (SAR) is a state of heightened defense which is induced throughout a plant by an initial infection; it provides long-lasting, broad-spectrum resistance to subsequent pathogen challenge. Recently we identified a phloem-mobile signal for SAR which has been elusive for almost 30 years. It is methyl salicylate (MeSA), an inactive derivative of the defense hormone, salicylic acid (SA). This discovery resulted from extensive characterization of SA-binding protein 2 (SABP2), a protein whose high affinity for SA and extremely low abundance suggested that it might be the SA receptor. Instead we discovered that SABP2 is a MeSA esterase whose function is to convert biologically inactive MeSA in the systemic tissue to active SA. The accumulated SA then activates or primes defenses leading to SAR. SABP2’s esterase activity is inhibited in the initially/primary infected tissue by SA binding in its active site; this facilitates accumulation of MeSA, which is then translocated through the phloem to systemic tissue for perception and processing by SABP2 to SA. Thus, while SABP2 is not the SA receptor, it can be considered the receptor for the SAR signal. This study of SABPs not only illustrates the unexpected nature of scientific discoveries, but also underscores the need to use biochemical approaches in addition to genetics to address complex biological processes, such as disease resistance. Addendum to: Park S-W, Kaimoyo E, Kumar D, Mosher S, Klessig DF. Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 2007; 318:113-6.

Salicylic Acid- And Nitric Oxide-Mediated Signal Transduction In Disease Resistance

Salicylic acid (SA) has been shown to play a critical role in signaling the activation of plant defense responses following pathogen infection. These responses include the induction of cell death and disease resistance in the infected leaf, as well as the activation of systemic disease resistance in the uninfected portions of the plant. SA has been shown to induce these phenomena through a variety of mechanisms that may include altering the activity or synthesis of certain enzymes, increasing the expression of various defense genes and/or potentiating the activation of certain defense responses.