The major focus of our research is to understand, at the biochemical, molecular and cellular levels, how plants protect themselves against microbial pathogens. The interaction of tobacco and Arabidopsis with their viral pathogens Tobacco Mosaic Virus (TMV) and Turnip Crinkle Virus (TCV), respectively, are the two principal (but not exclusive) model systems used. Our main goal is to decipher the signal transduction pathway(s) that leads to induction of plant defenses.
During the past two decades we have identified salicylic acid (SA) and nitric oxide (NO) as two important defense signals involved in the activation of defense responses to pathogens. Current studies are directed at defining components in the SA- and NO-mediated signalingpathways and determining their mechanisms of action using a combination of biochemical, pharmacological, molecular and genetic approaches. The TCV-Arabidopsis pathosystem is being utilized to help understand the early, as well as late, steps in activation of defense responses.
One of the major projects during the past several years is the continued characterization of tobacco SA-binding proteins (SABPs) as part of our effort to decipher the SA-mediated signal transduction network. Much of our recent effort has been focused on SABP2 since it was a likely receptor for SA, given its very high affinity and specificity for SA and extremely low abundance. Silencing of SABP2 using RNAi technology showed that it is involved in both basal resistance and resistance (R) gene-mediated resistance, as well as in systemic acquired resistance (SAR; Kumar and Klessig, 2003). Biochemical and structural studies of SABP2 revealed i) its methyl salicylate (MeSA) esterase activity, ii) its 3-D structure, and iii) feedback inhibition of its esterase activity by binding of SA in its active site (Forouhar et al., 2005). To assess the specificity of RNAi-mediated silencing and to facilitate structure-function analyses of SABP2, a novel approach, termed synthetic gene complementation, was developed (Kumar et al., 2006).
Characterization of SABP2’s role in SAR revealed that its MeSA esterase activity is required in the systemic tissue to perceive/process the mobile SAR signal and this esterase activity must be inhibited by SA binding in the primary infected tissue to generate the SAR signal. These results together with quantification of MeSA in the various tissue, including phloem exudate, demonstrated that MeSA is a long-sought, phloem-mobile signal for SAR and SABP2’s function is to cleave MeSA to release active SA, which then activates/primes defenses in the systemic tissue leading to SAR (Park et al., 2007). The involvement of MeSA esterase and MeSA in SAR has been demonstrated in Arabidopsis (Vlot el al., 2008) and is currently being studied in potato. We have recently identified a large number of new putative SABPs. Their characterization is a major focus of our current and future research.
Identification of defense signaling components using genetics
During the 1990s and early 2000s we developed the Arabidopsis-TCV pathosystem. This led to identification of HRT, which encodes a CC (coiled coil)-NBS-LRR type R protein required for resistance to TCV, and also RRT, a yet to be cloned gene that regulates resistance to TCV. Further analyses of this pathosystem suggest that the reason why HRT, while necessary, is not sufficient for resistance is that SA levels are not sufficiently elevated after infection to enhance HRT expression and thus facilitate stable resistance. The recessive rrt allele, which together with HRT is sufficient for resistance, may correct this SA deficiency (Chandra-Shekara et al., 2004). A genetic screen has uncovered a novel ATPase that is required for resistance to TCV and other pathogens; it physically interacts with HRT and other R proteins (Kang et al., 2008). Its role in defense signaling is under investigation.
Cyclic Nucleotide-Gated Ion Channel (CNGCs) mutants:
A genetic screen of a T-DNA insertion mutant Arabidopsis library identified a mutant which constitutively expresses defense-associated PR genes (cpr22), has elevated levels of SA and displays enhanced resistance (Yoshioka et al., 2001). Further characterization of cpr22 revealed that it contains a deletion which results in loss of two CNGCs, CNGC11 and CNGC12, and formation of a chimeric CNGC11/12 with new signaling properties. This study also demonstrated that the defense signaling components NDR1, EDS1 and PAD4 mediate other resistance signaling functions in addition to SA and PR protein accumulation (Yoshioka et al., 2006).
Suppressors of SA insensitive mutants:
In the late 1990’s we isolated several Arabidopsis mutants termed suppressor of the SA insensitive (ssi1-ssi4) phenotype of npr1/sai1; NPR1/SAI1 is a key positive regulator of SA-mediated signaling. Characterization of ssi4 showed that it is a constitutively active R protein, which confers constitutive activation of SA-mediated defenses, enhanced disease resistance, elevated levels of reactive oxygen species, activation of the defense-associated MAP kinases MPK3 and MPK6, and induction of various defense genes (Shirano et al., 2002; Zhou et al., 2004). The ssi4 mutation also causes morphological alterations which differentially require SGT1b, whereas activation of defenses requires RAR1 (Zhou et al., 2008). Similarly, the ssi2 mutation leads to activation of SA-mediated defense signaling but suppression of jasmonic acid (JA)-mediated defense signaling. The mutation is in a fatty acid (stearoyl) desaturase resulting in reduced levels of oleic acid (18:1) which is responsible for both constitutive SA-mediated signaling and the resulting enhanced resistance to biotrophic pathogens like Pseudomonas syringae, and for compromised JA-mediated signaling and the resulting reduced resistance to necrotrophic pathogens such as Botrytis cinerea (P. Kachroo et al., 2001; P. Kachroo et al., 2003; A. Kachroo et al., 2003; Nandi et al., 2005).
Mitogen-activated protein kinases (MAP kinases)
In the late 1990s we purified the first plant MAP kinase (SA-induced protein kinase, SIPK) and demonstrated its involvement in protection against pathogens. In 2004, Arabidopsis’ homolog of SIPK, MPK6, was shown to be required for disease resistance (Menke et al., 2004) and in 2005, one of the first substrates of a plant MAP kinase was identified. SIPK was found to phosphorylate the defense-associated transcription factor NtWRKY1 (Menke et al., 2005).
NO and plant defense
In the late 1990s, we and the Chris Lamb/Rick Dixon group demonstrated the involvement of NO in plant immunity. Several direct targets of NO were subsequently identified, including aconitase, catalase, and ascorbate peroxidase. Further studies demonstrated that plant aconitases are bifunctional, like their mammalian counterparts. In the presence of NO, aconitases lose their iron-sulfur cluster in the catalytic center, and hence their enzymatic activity, but gain RNA binding activity. The Arabidopsis aconitase binds the 5’-UTR of the chloroplastic Cu/Zn superoxide dismutase mRNA and appears to have a role in resisting oxidative stress and activating cell death (Moeder et al., 2007).
Most of our research on NO signaling over the past decade has focused on identifying the NO synthase (NOS) responsible for NO synthesis during infection. Unfortunately, its identity is still unknown. Our work on the putative NOS activity of the P protein could not be reproduced. Moreover, our subsequent studies of AtNOS1 (identified by Nigel Crawford’s group) indicate that it is not an NOS, but is a GTPase (Moreau et al., 2008).
The major focus of our research is to understand, at the biochemical, molecular and cellular levels, how plants protect themselves against microbial pathogens. The interaction of tobacco and Arabidopsis with their viral pathogens tobacco mosaic virus (TMV) and turnip crinkle virus (TCV), respectively, are the two principal (but not exclusive) model systems used. Our main goal is to decipher the signal transduction pathway(s) that leads to induction of defense genes, such as the pathogenesis-related (PR) genes which appear to underlie disease resistance.
During the past decade we have identified salicylic acid (SA) and nitric oxide (NO) as two important secondary signals involved in the activation of defense responses to pathogens. Current studies are directed at defining components in the SA- and NO-mediated signaling pathways and determining their mechanisms of action using a combination of biochemical, pharmacological, molecular and genetic approaches. The TCV-Arabidopsis pathosystem is being utilized to help understand the early, as well as late, steps in the defense reaction, including the initial recognition of the pathogen via direct or indirect interaction with the product of the cognate host resistance gene.