Nitric oxide and salicylic acid signaling in plant defense

Abstract

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H(2)O(2)-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IkappaBalpha and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.

Figure 1

The effect of a NOS inhibitor on TMV induction of PR-1 gene expression. Xanthi nc (NN) plants were mock inoculated (M) or inoculated with the U1 strain of TMV (1 μg/ml) and then were maintained for 48 h at 32°C in a growth chamber. One hour before shifting plants from 32°C to 22°C, the intercellular spaces of the inoculated leaves were infiltrated with either H₂O, the active form (5 mM N^G-monomethyl-l-arginine monoacetate, l-NMMA), or inactive form (5 mM d-NMMA) of an NOS inhibitor. Samples were taken for RNA preparation at 0, 5, 7 h after the shift to 22°C. A portion (10 μg) of total RNA was fractionated on a formaldehyde-agarose gel and was subjected to Northern blot analysis using standard protocols (74). The tobacco acidic PR-1a cDNA clone was used as a probe.

Figure 2

Induction of PR-1 gene expression with various concentrations of NOS. Five- to eight-week-old Xanthi nc (NN) plants were injected with 40 mM Hepes (pH 7.4) containing all of the cofactors and substrate of NOS (22) and different concentrations of recombinant rat neuronal NOS (0–5 units per ml; Alexis Biochem, San Diego). Samples were taken for RNA preparation immediately after (0 h) or 24 h after injection; the RNA was subjected to Northern blot analysis as described in Fig. 1, with the addition that β-tubulin was used as an internal control for gel loading and transfer.

Figure 3

Proposed SA- and NO-mediated pathway for activation of certain defense genes and elevation of intracellular free Fe²⁺. Key signaling molecules in the cascade include NO, cGMP, cADPR, Ca²⁺, and SA. Important enzymes are NOS, GC, cGMP-dependent protein kinase, and ADPRC. PAL and PR-1 are two important defense genes activated by pathogens. Activation of NOS by TMV infection increases NO levels, which activate GC and lead to elevated cGMP levels. cGMP activates ADPRC, via a cGMP-dependent protein kinase, which results in rising cADPR levels. cADPR activates ruthenium red (RR)-sensitive Ca²⁺ ion channels, which leads to higher cytosolic Ca²⁺ levels. Ca²⁺ induces SA biosynthesis, perhaps by activation of PAL gene expression. The SA-induced MAP kinase, SIPK, may play a role in SA signaling through NPR1. NPR1 transmits the SA signal to the PR-1 gene via its interaction with members of the TGA/OBF family of transcription factors, which bind the SA-responsive TGACG element of the PR-1 promoter. In a separate branch of the pathway, NO inhibits cytosolic aconitase and may convert it into a iron regulatory protein (IRP), thereby facilitating an increase in intracellular free Fe²⁺. GSNO and SNAP are NO donors whereas PTIO is an NO scavenger.

Figure 4

A cADPR antagonist suppresses the NO induction of PR-1 gene expression. Five- to eight-week-old Xanthi nc (NN) plants were injected with 40 mM Hepes (pH 7.4) containing all of the cofactors and substrate of NOS (22) either without NOS as a negative control (C) or with NOS (1 unit/ml) as a positive control (N). A second set of plants were co-injected with NOS, cofactors and substrate plus 200 μM of the cADPR antagonist 8-bromo-cADPR. Samples were taken for RNA preparation at 0, 16, and 20 h after injection; the RNA was subjected to Northern blot analysis as described in Fig. 2.