Role of nitric oxide and reactive oxide species in disease resistance to necrotrophic pathogens

Nitric oxide (NO) and reactive oxygen species (ROS) are important signaling molecules in plant immunity. However, roles of NO and ROS in disease resistance to necrotrophic pathogens are not fully understood. We have recently demonstrated that NO plays a pivotal role in basal defense against Botrytis cinerea and the expression of the salicylic acid (SA)-responsive gene PR-1 in Nicotiana benthamiana. By contrast, ROS function negatively in resistance or positively in expansion of disease lesions during B. cinerea-N. benthamiana interaction. Here, analysis in NahG-transgenic N. benthamiana showed that SA signaling is not involved in resistance to B. cinerea in N. benthamiana. We discuss how NO and ROS participate in disease resistance to necrotrophic pathogens on the basis of recent reports.Key words: NO burst, oxidative burst, necrotrophic pathogen, salicylic acid, plant immunity, MAPKNecrotrophs are pathogens that kill host cells by means of toxic molecules and lytic enzymes, and they feed on the remains for their own growth. If the toxic molecule shows differential activity to one or a few plant species, the pathogen has a limited host range and the metabolite is referred to as a host-selective toxin (HST).¹ Several well-studied necrotrophs, in particular Cochliobolus and Alternaria spp., produce HSTs required for the pathogenicity. There are also necrotrophic fungal pathogens with a broad host range, particularly those in the order of Helotiales, including Sclerotinia sclerotiorum and Botrytis cinerea.Rapid production of nitric oxide (NO) and reactive oxygen species (ROS), called NO burst and oxidative burst, respectively, is one of the earliest responses of plants to pathogen attacks. Our recent study showed that NO and oxidative bursts accompanied by activation of the mitogen-activated protein kinase (MAPK)² are induced after inoculation with B. cinerea, and that NO plays a key role, but ROS have an opposite effect in basal defense against B. cinerea in Nicotiana benthamiana.³ NO and ROS are believed to play key roles independently or coordinately in plant innate immunity.^4,5 NO signaling comprises complex processes including increases in cytosolic Ca²⁺ concentration, cyclic GMP (cGMP), cyclic ADP ribose and activation of protein kinases. NO also modulates protein activities directly by cysteine S-nitrosylation.⁶ In addition, NO appears to act as an antioxidant of ROS, because NO can react quickly with superoxide (O₂⁻) to form peroxynitrite (ONOO⁻) and then, reduces the amount of endogenous ROS. Actually, treatment with a mammalian NO synthase inhibitor and silencing NbNOA1 decreased endogenous NO levels and increased the levels of ROS after inoculation with B. cinerea.³ The suppression of NO burst induced high susceptibility to B. cinerea, and depletion of oxidative burst by an NADPH oxidase inhibitor or silencing NbRBOHB led to reduction in disease lesions by B. cinerea,³ suggesting that the growth of B. cinerea might be determined by endogenous levels of ROS which is an important component of virulence.⁷ However, depletion of both NO and oxidative bursts by double silencing NbNOA1/NbRBOHB resulted in expansion of disease lesions compared with reduction of oxidative burst alone by silencing NbRBOHB.³ Similarly, our most recent study showed that silencing NbRibA which compromises production of both NO and ROS do not affect basal resistance against B. cinerea.⁸ These findings suggest that NO positively functions in resistance to necrotrophic pathogens in the manner other than as an antioxidant of ROS.The relationship between NO and salicylic acid (SA) has been studied.⁹ SA signaling-deficient mutants of Arabidopsis thaliana show high susceptibility to B. cinerea.^10,11 We have suggested that reduced basal defense against B. cinerea in N. benthamiana resulting from compromised endogenous NO production may be due to depletion of SA signaling, because NbNOA1-silenced plants showed suppression of the SA-responsive gene NbPR-1 expression induced by inoculation with B. cinerea.³ To confirm the possibility, we used N. benthamiana expressing NahG that converts all SA to catechol. NahG and non-NahG (WT) leaves were inoculated with B. cinerea. NahG plants showed similar susceptibility to B. cinerea compared with WT plants (Fig. 1). We also evaluated effects of silencing NbNOA1 and NbRBOHB in NahG plants on susceptibility to B. cinerea. Like NbNOA1-silenced WT plants shown previusly,³ NbNOA1-silenced NahG leaves showed high susceptibility to B. cinerea. On the other hand, NbRBOHB-silenced NahG leaves showed marked reduction of disease lesions compared with silencing-control NahG leaves. NbNOA1/NbRBOHB-silenced NahG leaves showed expansion of disease lesions compared with NbRBOHB-silenced NahG leaves (Fig. 2). These results suggest that NO-mediated basal defense against B. cinerea is not due to SA signaling, and effects of ROS on disease lesions may not depend on SA in N. benthamiana.Open in a separate windowFigure 1Effects of NahG transgene on susceptibility to B. cinerea. NahG and non-NahG (WT) leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 days postinoculation (dpi). (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from fourteen experiments.Open in a separate windowFigure 2Effects of silencing NbNOA1 (N), NbRBOHB (B) or NbNOA1/NbRBOHB (N/B) in NahG plants on susceptibility to B. cinerea. Silenced NahG leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 dpi. (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from four experiments. Data were subjected to Student''s t-test. *p < 0.05 versus silencing-control plants (TRV). **p < 0.05 versus NbRBOHB-silenced plants.Recently, it has been reported that NO and ROS are involved in HSTs responses.^12–15 Victorin, an HST produced by Cochliobolus victoriae, elicits generation of NO and ROS in victorin-sensitive oat leaves.¹² Cell death induced by victorin is suppressed by treatment with ROS scavengers.¹³ Similarly, treatment with ToxA, an HST produced by Pyrenophora triticirepentis, induces oxidative burst, and scavenging ROS compromises ToxA-inducible cell death in ToxA-sensitive wheatleaves.^14,15 SA-induced MAPK, which regulates both NO and ROS production,² is activated by AAL-toxin produced by Alternaria alternata f. sp. lycopersici in AAL-toxin-sensitive tobacco (Mizuno et al. unpublished data). These findings indicate requirement of ROS for the HST-inducible cell death and participation of NO in HST responses.In conclusion, NO and ROS appear to play a contrasting role in disease resistance to necrotrophic pathogens as shown in Figure 3. However, how NO signaling participates in defense responses against necrotrophic pathogens has yet to be elucidated. Recently, several targets of protein S-nitrosylation during hypersensitive response have been characterized in A. thaliana.¹⁶ Evidence is also accumulating for cGMP as an important component of NO-related signal transduction.¹⁷ Further investigations of NO signaling will lead to our understanding of interactions between plants and necrotrophic pathogens.Open in a separate windowFigure 3Model showing role of NO and oxidative bursts in disease resistance to necrotrophic pathogens. After recognition of necrotrophs, plants immediately provoke activation of MAPK which could regulate production of both NO and ROS,² and then NO and oxidative bursts. NO burst plays an important role in disease resistance to necrotrophic pathogens, whereas oxidative burst has a negative role in resistance or has a positive role in expansion of disease lesions by necrotrophs.