Exogenous Nicotinamide Adenine Dinucleotide Induces Resistance to Citrus Canker in Citrus

Abstract

Nicotinamide adenine dinucleotide (NAD) is a universal electron carrier that participates in important intracellular metabolic reactions and signaling events. Interestingly, emerging evidence in animals indicates that cellular NAD can be actively or passively released into the extracellular space, where it is processed or perceived by ectoenzymes or cell-surface receptors. We have recently shown in Arabidopsis thaliana that exogenous NAD induces defense responses, that pathogen infection leads to release of NAD into the extracellular space at concentrations sufficient for defense activation, and that depletion of extracellular NAD (eNAD) by transgenic expression of the human NAD-hydrolyzing ectoenzyme CD38 inhibits plant immunity. We therefore hypothesize that, during plant-microbe interactions, NAD is released from dead or dying cells into the extracellular space where it interacts with adjacent naïve cells' surface receptors, which in turn activate downstream immune signaling. However, it is currently unknown whether eNAD signaling is unique to Arabidopsis or the Brassicaceae family. In this study, we treated citrus plants with exogenous NAD⁺ and tested NAD⁺-induced transcriptional changes and disease resistance. Our results show that NAD⁺ induces profound transcriptome changes and strong resistance to citrus canker, a serious citrus disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). Furthermore, NAD⁺-induced resistance persists in new flushes emerging after removal of the tissues previously treated with NAD⁺. Finally, NAD⁺ treatment primes citrus tissues, resulting in a faster and stronger induction of multiple salicylic acid pathway genes upon subsequent Xcc infection. Taken together, these results indicate that exogenous NAD⁺ is able to induce immune responses in citrus and suggest that eNAD may also be an elicitor in this woody plant species.

Keywords: Citrus sinensis; Xanthomonas citri subsp. citri; citrus canker; defense gene; disease resistance; extracellular NAD; transcriptional changes.

FIGURE 1

Exogenous NAD⁺-induced resistance to citrus canker in citrus. (A) Phenotypes of the citrus canker lesions on leaves of citrus plants treated with soil drenches of water, 1 mM NAD⁺, or Actigard. Photos were taken 14 days after Xcc inoculation. (B) Phenotypes of the citrus canker lesions on leaves pre-infiltrated with water or 1 mM NAD⁺. Photos were taken 14 days after Xcc inoculation. (C) Numbers of the citrus canker lesions on leaves of citrus plants treated with soil drenches of water, Actigard, or different concentrations (1, 5, and 10 mM) of NAD⁺. Data represent means of five biological replicates with standard error (SE). Different letters above the bars indicate significant differences (Newman–Keuls test, p < 0.05). (D) Numbers of the citrus canker lesions on leaves pre-infiltrated with water or various concentrations (0.25, 0.5, 0.75, 1, 5, and 10 mM) of NAD⁺. Data represent means of five biological replicates with SE. Different letters above the bars indicate significant differences (Newman–Keuls test, p < 0.05).

FIGURE 2

The effectiveness of NAD⁺ treatment over time. (A) Numbers of the citrus canker lesions on leaves of new shoots emerged on the citrus plants that were previously treated with soil drenches of water, Actigard, or different concentrations (1, 5, and 10 mM) of NAD⁺. The plants were pruned below the inoculation point 6 weeks after NAD⁺ treatment. Data represent means of five biological replicates with SE. Different letters above the bars indicate significant differences (Newman–Keuls test, p < 0.05). (B) Numbers of the citrus canker lesions on leaves of new shoots emerged on the citrus plants with leaves previously infiltrated with water or various concentrations (0.25, 0.5, 0.75, 1, 5, and 10 mM) of NAD⁺ and inoculated with Xcc. The plants were pruned below the inoculation point 6 weeks after NAD⁺ treatment. Data represent means of five biological replicates with SE. Different letters above the bars indicate significant differences (Newman–Keuls test, p < 0.05).

FIGURE 3

NAD⁺-mediated priming of several SA-related defense genes. Expression of CsCM2, CsCM1, CsICS, CsPAL, CsNPR1, and CsPR5 at the indicated time points in citrus leaf tissues first infiltrated with 5 mM NAD⁺ and then inoculated with Xcc. The arrows indicate the time point when the leaf tissues were inoculated with Xcc after NAD⁺ treatment. Expression of the target genes was normalized against the constitutively expressed Cs18S. The y-axis values represent the relative expression levels of the indicated genes, which were calculated using the formula 2^{[Ct(Cs18S)-Ct(targetgene)]}. Data represent means of three biological replicates with SE.