Extracellular pyridine nucleotides as immune elicitors in arabidopsis

ABSTRACT

The pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are coenzymes that function in both metabolic reactions and intracellular signaling. Emerging evidence from animal research indicates that NAD(P) also acts in the extracellular space (ECS). We have shown in the model plant Arabidopsis that (1) exogenous NAD(P) induces immune responses, (2) pathogen infection causes leakage of intracellular NAD(P) into the extracellular fluid at concentrations sufficient to induce immune responses, and (3) removal of extracellular NAD(P) [eNAD(P)] by expressing the human NAD(P)-metabolizing ectoenzyme CD38 partially compromises systemic acquired resistance. Based on these results, we hypothesize that eNAD(P) is a novel damage-associated molecular pattern (DAMP) in plants; during plant-microbe interaction, intracellular NAD(P) is released from dead or dying cells into the ECS where it interacts with the adjacent healthy cells' surface receptors/targets, which in turn activate downstream specific immune signaling pathways. Our recent identification of LecRK-I.8, a lectin receptor kinase, as the first cell surface NAD⁺-binding receptor has provided compelling evidence for this hypothesis. Further identification of cell surface eNAD(P) receptors/targets and their downstream signaling components in Arabidopsis as well as determination of the generality of eNAD(P) signaling in crops will help establish eNAD(P) as a conserved DAMP in plants.

The pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are universal coenzymes, which not only participate in metabolic reactions and intracellular signaling,^1–7 but also functions in the extracellular space (ECS).⁸ A number of NAD(P)-metabolizing ectoenzymes such as CD38, CD157 and mono(ADP-ribosyl)transferases (ARTs) have been identified in animal cells.^9–12 CD38 combines ADP-ribosyl cyclase, cyclic ADP-ribose (cADPR)-hydrolase and NAD-hydrolase activities,^13,14 which uses extracellular NAD(P) [eNAD(P)] as the substrate to produce cADPR and nicotinic acid adenine dinucleotide phosphate (NAADP).¹⁵ NAADP may function in the ECS to trigger purinoceptors, resulting in elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i).^16,17 Both cADPR and NAADP can move across the plasma membrane to reach their intracellular targets and raise [Ca²⁺]_i.^18,19 ARTs are glycosylphosphatidylinositol (GPI)-anchored or secreted ectoenzymes that utilize NAD to ADP-ribosylate plasma membrane signaling proteins, leading to changes in their signaling activities.^20–23

eNAD(P) may directly bind to plasma membrane receptors/targets to trigger signal transduction. NAD⁺ has been shown to bind to rat brain synaptic membranes,²⁴ and several purinergic P2X and P2Y receptors have been reported to function in eNAD(P)-induced biological responses including activation of adenylate cyclase and production of cAMP, activation of CD38 and production of cADPR, as well as increase of [Ca²⁺]_i via Ca²⁺ influx.^25–28 However, direct binding of NAD(P) to these receptors has not been reported. To the best of my knowledge, no cell surface receptor has been shown to bind NAD(P). Thus, the nature and identity of eNAD(P)-binding receptors have been elusive till we recently identified the first eNAD⁺-binding receptor in the model plant Arabidopsis.²⁹

The existence of eNAD(P) and its role in plants were fortuitously discovered. We previously showed that the plant immune transcription coactivator NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is regulated by cellular redox potential changes.³⁰ Since NADPH is the reducing power for most anabolic reactions, we thought that exogenous NADPH might activate NPR1 and immune responses in plants. Indeed, exogenous addition of NADPH induces strong immune responses in Arabidopsis.³¹ Surprisingly, other pyridine nucleotides, including NADP⁺, NADH, and NAD⁺, all similarly induce immune responses in Arabidopsis,³¹ indicating that the immunity-inducing activity of NADPH is not owing to its reducing power. Furthermore, exogenously added NAD(P) appears not to perturb intracellular NAD(P) homeostasis,³¹ suggesting that it may act in the ECS to trigger immune signaling in plants.³² Importantly, during plant-microbe interaction, NAD(P) leaks out into the ECS at concentrations sufficient to induce immune responses,³¹ and removal of extracellular NAD(P) [eNAD(P)] by expressing the human NAD(P)-metabolizing ectoenzyme CD38 partially compromises systemic acquired resistance (SAR).³³ Thus, we hypothesize that eNAD(P) is a novel damage-associated molecular pattern (DAMP) in plants; during plant-microbe interaction, intracellular NAD(P) is released from dead or dying cells into the ECS where it interacts with the adjacent healthy cells' surface receptors/targets, which in turn activate downstream specific immune signaling pathways.

To demonstrate this hypothesis, it is crucial to uncover the mechanisms perceiving eNAD(P) in plants. Unfortunately, although low activities of both ADP-ribosyl cyclase and cADPR hydrolase have been detected in Arabidopsis,³⁴ no protein with significant homology to animal CD38/CD157 or ARTs has been identified in any plant genome database.^12,34 Thus, plants most likely use proteins with very low homology to the animal eNAD(P)-processing proteins or novel mechanisms to perceive eNAD(P).³⁵

To uncover the mechanisms underlying eNAD(P) signaling in plants, we employed genetic approaches using Arabidopsis to identify signaling components of the eNAD(P) signaling pathway. In a forward genetic screen aimed at identifying mutants insensitive to exogenous NAD⁺ (ien) treatment, we found that the Mediator complex subunits MED14/STRUWWELPETER and MED16/SENSITIVE TO FREEZING6 / IEN1 as well as the Elongator complex function downstream of eNAD⁺.^36–38 However, no receptor protein was identified in the forward genetic screen. Since both Mediator and Elongator are general transcription regulators,^39,40 the forward genetic screen did not reveal any eNAD(P)-specific signaling component. On the other hand, a reverse genetic approach based on microarray analysis of exogenous NAD⁺-induced transcriptome changes in Arabidopsis identified a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD⁺ receptor.²⁹

Multiple lines of evidence support LecRK-I.8 as a potential eNAD⁺ receptor in Arabidopsis.²⁹ First, the LecRK-I.8 gene is NAD⁺ inducible. Second, LecRK-I.8 is localized in the plasma membrane and has kinase activity. Third, LecRK-I.8 specifically binds NAD⁺, but not NADP⁺, ATP, ADP, or AMP, and several other LecRKs, including DORN1 (DOES NOT RESPOND TO NUCLEOTIDES1, LecRK-I.9), LecRK-I.3, and LecRK-I.6, do not bind NAD⁺. Fourth, mutations in LecRK-I.8 particularly inhibit NAD⁺-induced, but not NADP⁺-, flg22 (a peptide corresponding to the 22 amino acids of the conserved N-terminal part of flagellin)-, and salicylic acid (SA)-induced, immune responses. Finally, mutations in LecRK-I.8 compromise resistance to bacterial infections.

However, a discrepancy between the ligand dissociation constant (Kd) of LecRK-I.8 and the concentration required for inducing measurable immune responses challenges the candidacy of LecRK-I.8 for an eNAD⁺ receptor. The concentration that is able to significantly induce early defense gene expression is about 10-fold higher than the Kd value of LecRK-I.8.²⁹ Although this result is difficult to reconcile with an eNAD⁺ sensor function of LecRK-I.8, it is not without precedent. Several ligands, including the plant hormone brassinolide, SA, and extracellular ATP, when used to trigger biological responses, the applied concentrations are generally also much higher than the Kd values of their corresponding receptors.^41–45 These results suggest that, in plants, exogenously applied ligands may not be able to efficiently reach the surface of their receptors.

LecRK-I.8 is not the sole eNAD⁺ perception mechanism and perhaps not the primary receptor of eNAD⁺ either. Mutations in LecRK-I.8 only inhibit low concentrations (0.4 mM or lower) of NAD⁺-induced immune responses, only partially compromise basal immunity, and have no effect on biological induction of SAR.²⁹ To firmly establish eNAD(P) as a conserved DAMP in plants, it is important to test the effect of largely blocking eNAD(P) signaling on plant immune responses by combining genetic mutations in most of the eNAD(P) perception mechanisms in Arabidopsis, to identify the downstream signaling components, and to test if eNAD(P) signaling exists in diverse plant species.

Acknowledgments

The work described in this addendum was supported by a grant from the Citrus Research and Development Foundation.