The effect of nitrosative stress on histone H3 and H4 acetylation in Phytophthora infestans life cycle.

The oomycete Phytophthora infestans is one of the most destructive phytopathogens globally. It has a proven ability to adapt to changing environments rapidly; however, molecular mechanisms responsible for host invasion and adaptation to new environmental conditions still need to be explored. The study aims to understand the epigenetic mechanisms exploited by P. infestans in response to nitrosative stress conditions created by the (micro)environment and the host plant. To characterize reactive nitrogen species (RNS)-dependent acetylation profiles in avirulent/virulent (avr/vr) P. infestans, a transient gene expression, ChIP and immunoblot analyses, and nitric oxide (NO) emission by chemiluminescence were used in combination with the pharmacological approach. Nitrosative stress increased total H3/H4 acetylation and some histone acetylation marks, mainly in sporulating hyphae of diverse (avr/vr) isolates and during potato colonization. These results correlated with transcriptional up-regulation of acetyltransferases PifHAC3 and PifHAM1, catalyzing H3K56 and H4K16 acetylation, respectively. NO or peroxynitrite-mediated changes were also associated with H3K56 and H4K16 mark deposition on the critical pathogenicity-related gene promoters (CesA1, CesA2, CesA3, sPLD-like1, Hmp1, and Avr3a) elevating their expression. Our study highlights RNS-dependent transcriptional reprogramming via histone acetylation of essential gene expression in the sporulating and biotrophic phases of plant colonization by P. infestans as a tool promoting its evolutionary plasticity.

Histone (de)acetylation in epigenetic regulation of Phytophthora pathobiology.

Phytophthora species are oomycetes that have evolved a broad spectrum of biological processes and improved strategies to cope with host and environmental challenges. A growing body of evidence indicates that the high pathogen plasticity is based on epigenetic regulation of gene expression linked to Phytophthora's rapid adjustment to endogenous cues and various stresses. As 5mC DNA methylation has not yet been identified in Phytophthora, the reversible processes of acetylation/deacetylation of histone proteins seem to play a pivotal role in the epigenetic control of gene expression in oomycetes. To explore this issue, we review the structure, diversity, and phylogeny of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in six plant-damaging Phytophthora species: P. capsici, P. cinnamomi, P. infestans, P. parasitica, P. ramorum, and P. sojae. To further integrate and improve our understanding of the phylogenetic classification, evolutionary relationship, and functional characteristics, we supplement this review with a comprehensive view of HATs and HDACs using recent genome- and proteome-level databases. Finally, the potential functional role of transcriptional reprogramming mediated by epigenetic changes during Phytophthora species saprophytic and parasitic phases under nitro-oxidative stress is also briefly discussed.

Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.

Endogenous plant nitroxyl, a new component of nitric oxide biology.

Nitroxyl (HNO), a one-electron reduced and protonated congener of nitric oxide (•NO), was recently discovered in Arabidopsis thaliana. Due to its distinct chemical properties, we believe HNO must be further studied to determine how many physiological processes it impacts.

Comparative genomic analysis of the aldehyde dehydrogenase gene superfamily in Arabidopsis thaliana - searching for the functional key to hypoxia tolerance.

Flooding entails different stressful conditions leading to low oxygen availability for respiration and as a result plants experience hypoxia. Stress imposed by hypoxia affects cellular metabolism, including the formation of toxic metabolites that dramatically reduce crop productivity. Aldehyde dehydrogenases (ALDHs) are a group of enzymes participating in various aspects of plant growth, development and stress responses. Although we have knowledge concerning the multiple functionalities of ALDHs in tolerance to various stresses, the engagement of ALDH in plant metabolism adjustment to hypoxia is poorly recognized. Therefore, we explored the ALDH gene superfamily in the model plant Arabidopsis thaliana. Genome-wide analyses revealed that 16 AtALDH genes are organized into ten families and distributed irregularly across Arabidopsis 5 chromosomes. According to evolutionary relationship studies from different plant species, the ALDH gene superfamily is highly conserved. AtALDH2 and ALDH3 are the most numerous families in plants, while ALDH18 was found to be the most distantly related. The analysis of cis-acting elements in promoters of AtALDHs indicated that AtALDHs participate in responses to light, phytohormones and abiotic stresses. Expression profile analysis derived from qRT-PCR showed the AtALDH2B7, AtALDH3H1 and AtALDH5F1 genes as the most responsive to hypoxia stress. In addition, the expression of AtALDH18B1, AtALDH18B2, AtALDH2B4, and AtALDH10A8 was highly altered during the post-hypoxia-reoxygenation phase. Taken together, we provide comprehensive functional information on the ALDH gene superfamily in Arabidopsis during hypoxia stress and highlight ALDHs as a functional element of hypoxic systemic responses. These findings might help develop a framework for application in the genetic improvement of crop plants.

Plant homocysteine, a methionine precursor and plant's hallmark of metabolic disorders.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid, which arises from redox-sensitive methionine metabolism. In plants, Hcy synthesis involves both cystathionine ß-lyase and S-adenosylhomocysteine hydrolase activities. Thus, Hcy itself is crucial for de novo methionine synthesis and S-adenosylmethionine recycling, influencing the formation of ethylene, polyamines, and nicotianamine. Research on mammalian cells has shown biotoxicity of this amino acid, as Hcy accumulation triggers oxidative stress and the associated lipid peroxidation process. In addition, the presence of highly reactive groups induces Hcy and Hcy derivatives to modify proteins by changing their structure and function. Currently, Hcy is recognized as a critical, independent hallmark of many degenerative metabolic diseases. Research results indicate that an enhanced Hcy level is also toxic to yeast and bacteria cells. In contrast, in the case of plants the metabolic status of Hcy remains poorly examined and understood. However, the presence of the toxic Hcy metabolites and Hcy over-accumulation during the development of an infectious disease seem to suggest harmful effects of this amino acid also in plant cells. The review highlights potential implications of Hcy metabolism in plant physiological disorders caused by environmental stresses. Moreover, recent research advances emphasize that recognizing the Hcy mode of action in various plant systems facilitates verification of the potential status of Hcy metabolites as bioindicators of metabolism disorders and thus may constitute an element of broadly understood biomonitoring.

Dynamics of nitration during dark-induced leaf senescence in Arabidopsis reveals proteins modified by tryptophan nitration.

Nitric oxide (NO) is a critical molecule that links plant development with stress responses. Herein, new insights into the role of NO metabolism during leaf senescence in Arabidopsis are presented. A gradual decrease in NO emission accompanied dark-induced leaf senescence (DILS), and a transient wave of peroxynitrite (ONOO-) formation was detected by day 3 of DILS. The boosted ONOO- did not promote tryptophan (Trp) nitration, while the pool of 6-nitroTrp-containing proteins was depleted as senescence progressed. Immunoprecipitation combined with mass spectrometry was used to identify 63 and 4 characteristic 6-nitroTrp-containing proteins in control and individually darkened leaves, respectively. The potential in vivo targets of Trp nitration were mainly related to protein biosynthesis and carbohydrate metabolism. In contrast, nitration of tyrosine-containing proteins was intensified 2-fold on day 3 of DILS. Also, nitrative modification of RNA and DNA increased significantly on days 3 and 7 of DILS, respectively. Taken together, ONOO- can be considered a novel pro-senescence regulator that fine-tunes the redox environment for selective bio-target nitration. Thus, DILS-triggered nitrative changes at RNA and protein levels promote developmental shifts during the plant's lifespan and temporal adjustment in plant metabolism under suboptimal environmental conditions.

Fungal and oomycete pathogens and heavy metals: an inglorious couple in the environment.

Heavy metal (HM) contamination of the environment is a major problem worldwide. The rate of global deposition of HMs in soil has dramatically increased over the past two centuries and there of facilitated their rapid accumulation also in living systems. Although the effects of HMs on plants, animals and humans have been extensively studied, yet little is known about their effects on the (patho)biology of the microorganisms belonging to a unique group of filamentous eukaryotic pathogens, i.e., fungi and oomycetes. Much of the literature concerning mainly model species has revealed that HM stress affects their hyphal growth, morphology, and sporulation. Toxicity at cellular level leads to disturbance of redox homeostasis manifested by the formation of nitro-oxidative intermediates and to the induction of antioxidant machinery. Despite such adverse effects, published data is indicative of the fact that fungal and oomycete pathogens have a relatively high tolerance to HMs in comparison to other groups of microbes such as bacteria. Likely, these pathogens may harbor a network of detoxification mechanisms that ensure their survival in a highly HM-polluted (micro)habitat. Such a network may include extracellular HMs immobilization, biosorption to cell wall, and/or their intracellular sequestration to proteins or other ligands. HMs may also induce a hormesis-like phenomenon allowing the pathogens to maintain or even increase fitness against chemical challenges. Different scenarios linking HMs stress and modification of the microorganisms pathogenicity are disscused in this review.

Nitric Oxide Implication in Potato Immunity to Phytophthora infestans via Modifications of Histone H3/H4 Methylation Patterns on Defense Genes.

Nitric oxide (NO) is an essential redox-signaling molecule operating in many physiological and pathophysiological processes. However, evidence on putative NO engagement in plant immunity by affecting defense gene expressions, including histone modifications, is poorly recognized. Exploring the effect of biphasic NO generation regulated by S-nitrosoglutathione reductase (GNSOR) activity after avr Phytophthora infestans inoculation, we showed that the phase of NO decline at 6 h post-inoculation (hpi) was correlated with the rise of defense gene expressions enriched in the TrxG-mediated H3K4me3 active mark in their promoter regions. Here, we report that arginine methyltransferase PRMT5 catalyzing histone H4R3 symmetric dimethylation (H4R3sme2) is necessary to ensure potato resistance to avr P. infestans. Both the pathogen and S-nitrosoglutathione (GSNO) altered the methylation status of H4R3sme2 by transient reduction in the repressive mark in the promoter of defense genes, R3a and HSR203J (a resistance marker), thereby elevating their transcription. In turn, the PRMT5-selective inhibitor repressed R3a expression and attenuated the hypersensitive response to the pathogen. In conclusion, we postulate that lowering the NO level (at 6 hpi) might be decisive for facilitating the pathogen-induced upregulation of stress genes via histone lysine methylation and PRMT5 controlling potato immunity to late blight.

Insights into the expression of DNA (de)methylation genes responsive to nitric oxide signaling in potato resistance to late blight disease.

Our previous study concerning the pathogen-induced biphasic pattern of nitric oxide (NO) burst revealed that the decline phase and a low level of NO, due to S-nitrosoglutathione reductase (GSNOR) activity, might be decisive in the upregulation of stress-sensitive genes via histone H3/H4 methylation in potato leaves inoculated with avr P. infestans. The present study refers to the NO-related impact on genes regulating DNA (de)methylation, being in dialog with histone methylation. The excessive amounts of NO after the pathogen or GSNO treatment forced the transient upregulation of histone SUVH4 methylation and DNA hypermethylation. Then the diminished NO bioavailability reduced the SUVH4-mediated suppressive H3K9me2 mark on the R3a gene promoter and enhanced its transcription. However, we found that the R3a gene is likely to be controlled by the RdDM methylation pathway. The data revealed the time-dependent downregulation of the DCL3, AGO4, and miR482e genes, exerting upregulation of the targeted R3a gene correlated with ROS1 overexpression. Based on these results, we postulate that the biphasic waves of NO burst in response to the pathogen appear crucial in establishing potato resistance to late blight through the RdDM pathway controlling R gene expression.

Cadmium Stress Reprograms ROS/RNS Homeostasis in Phytophthora infestans (Mont.) de Bary.

Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.

Switchable Nitroproteome States of Phytophthora infestans Biology and Pathobiology.

The study demonstrates protein tyrosine nitration as a functional post-translational modification (PTM) in biology and pathobiology of the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato (Solanum tuberosum L.). Using two P. infestans isolates differing in their virulence toward potato cv. Sarpo Mira we found that the pathogen generates reactive nitrogen species (RNS) in hyphae and mature sporangia growing under in vitro and in planta conditions. However, acceleration of peroxynitrite formation and elevation of the nitrated protein pool within pathogen structures were observed mainly during the avr P. infestans MP 946-potato interaction. Importantly, the nitroproteome profiles varied for the pathogen virulence pattern and comparative analysis revealed that vr MP 977 P. infestans represented a much more diverse quality spectrum of nitrated proteins. Abundance profiles of nitrated proteins that were up- or downregulated were substantially different also between the analyzed growth phases. Briefly, in planta growth of avr and vr P. infestans was accompanied by exclusive nitration of proteins involved in energy metabolism, signal transduction and pathogenesis. Importantly, the P. infestans-potato interaction indicated cytosolic RXLRs and Crinklers effectors as potential sensors of RNS. Taken together, we explored the first plant pathogen nitroproteome. The results present new insights into RNS metabolism in P. infestans indicating protein nitration as an integral part of pathogen biology, dynamically modified during its offensive strategy. Thus, the nitroproteome should be considered as a flexible element of the oomycete developmental and adaptive mechanism to different micro-environments, including host cells.

A physiological perspective on targets of nitration in NO-based signaling networks in plants.

Although peroxynitrite (ONOO-) has been well documented as a nitrating cognate of nitric oxide (NO) in plant cells, modifications of proteins, fatty acids, and nucleotides by nitration are relatively under-explored topics in plant NO research. As a result, they are seen mainly as hallmarks of redox processes or as markers of nitro-oxidative stress under unfavorable conditions, similar to those observed in human and other animal systems. Protein tyrosine nitration is the best-known nitrative modification in the plant system and can be promoted by the action of both ONOO- and related NO-derived oxidants within the cell environment. Recent progress in 'omics' and modeling tools have provided novel biochemical insights into the physiological and pathophysiological fate of nitrated proteins. The nitration process can be specifically involved in various cell regulatory mechanisms that control redox signaling via nitrated cGMP or nitrated fatty acids. In addition, there is evidence to suggest that nitrative modifications of nucleotides embedded in DNA and RNA can be considered as smart switches of gene expression that fine-tune adaptive cellular responses to stress. This review highlights recent advances in our understanding of the potential implications of biotargets in the regulation of intracellular traffic and plant biological processes.

BABA-Induced DNA Methylome Adjustment to Intergenerational Defense Priming in Potato to Phytophthora infestans.

We provide evidence that alterations in DNA methylation patterns contribute to the regulation of stress-responsive gene expression for an intergenerational resistance of ß-aminobutyric acid (BABA)-primed potato to Phytophthora infestans. Plants exposed to BABA rapidly modified their methylation capacity toward genome-wide DNA hypermethylation. De novo induced DNA methylation (5-mC) correlated with the up-regulation of Chromomethylase 3 (CMT3), Domains rearranged methyltransferase 2 (DRM2), and Repressor of silencing 1 (ROS1) genes in potato. BABA transiently activated DNA hypermethylation in the promoter region of the R3a resistance gene triggering its downregulation in the absence of the oomycete pathogen. However, in the successive stages of priming, an excessive DNA methylation state changed into demethylation with the active involvement of potato DNA glycosylases. Interestingly, the 5-mC-mediated changes were transmitted into the next generation in the form of intergenerational stress memory. Descendants of the primed potato, which derived from tubers or seeds carrying the less methylated R3a promoter, showed a higher transcription of R3a that associated with an augmented intergenerational resistance to virulent P. infestans when compared to the inoculated progeny of unprimed plants. Furthermore, our study revealed that enhanced transcription of some SA-dependent genes (NPR1, StWRKY1, and PR1) was not directly linked with DNA methylation changes in the promoter region of these genes, but was a consequence of methylation-dependent alterations in the transcriptional network.

BABA-Primed Histone Modifications in Potato for Intergenerational Resistance to Phytophthora infestans.

In this paper we analyzed ß-aminobutyric acid (BABA)-primed epigenetic adjustment of potato cv. "Sarpo Mira" to Phytophthora infestans. The first stress-free generation of the potato genotype obtained from BABA-primed parent plants via tubers and seeds showed pronounced resistance to the pathogen, which was tuned with the transcriptional memory of SA-responsive genes. During the early priming phase before the triggering stress, we found robust bistable deposition of histone marks (H3K4me2 and H3K27me3) on the NPR1 (Non-expressor of PR genes) and the SNI1 gene (Suppressor of NPR1, Inducible), in which transcription antagonized silencing. Switchable chromatin states of these adverse systemic acquired resistance (SAR) regulators probably reprogrammed responsiveness of the PR1 and PR2 genes and contributed to stress imprinting. The elevated levels of heritable H3K4me2 tag in the absence of transcription on SA-dependent genes in BABA-primed (F0) and its vegetative and generative progeny (F1) before pathogen challenge provided evidence for the epigenetic mark for intergenerational memory in potato. Moreover, our study revealed that histone acetylation was not critical for maintaining BABA-primed defense information until the plants were triggered with the virulent pathogen when rapid and boosted PRs gene expression probably required histone acetyltransferase (HAT) activity both in F0 and F1 progeny.

RNA and mRNA Nitration as a Novel Metabolic Link in Potato Immune Response to Phytophthora infestans.

Peroxynitrite (ONOO-) exhibits a well-documented nitration activity in relation to proteins and lipids; however, the interaction of ONOO- with nucleic acids remains unknown in plants. The study uncovers RNA and mRNA nitration as an integral event in plant metabolism intensified during immune response. Using potato-avr/vr Phytophthora infestans systems and immunoassays we documented that potato immunity is accompanied by two waves of boosted ONOO- formation affecting guanine nucleotides embedded in RNA/mRNA and protein tyrosine residues. The early ONOO- generation was orchestrated with an elevated level of protein nitration and a huge accumulation of 8-nitroguanine (8-NO2-G) in RNA and mRNA pools confirmed as a biomarker of nucleic acid nitration. Importantly, potato cells lacking ONOO- due to scavenger treatment and attacked by the avr pathogen exhibited a low level of 8-NO2-G in the mRNA pool correlated with reduced symptoms of programmed cell death (PCD). The second burst of ONOO- coincided both with an enhanced level of tyrosine-nitrated proteins identified as subtilisine-like proteases and diminished protease activity in cells surrounding the PCD zone. Nitration of both RNA/mRNA and proteins via NO/ONOO- may constitute a new metabolic switch in redox regulation of PCD, potentially limiting its range in potato immunity to avr P. infestans.

The Dynamics of the Defense Strategy of Pea Induced by Exogenous Nitric Oxide in Response to Aphid Infestation.

The aim of this study was to investigate the effect of exogenous nitric oxide (NO), i.e., S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the metabolic status of Pisum sativum L. cv. Cysterski leaves infested by Acyrthosiphon pisum Harris, population demographic parameters and A. pisum feeding activity. A reduction in the level of semiquinone radicals in pea seedling leaves pretreated with exogenous NO occurred 24 h after A. pisum infestation, which was earlier than in non-pretreated leaves. A decrease in the level of O2•- was observed in leaves pretreated with GSNO and infested by aphids at 48 and 72 h post-infestation (hpi). Directly after the pretreatment with GSNO, an increase in the level of metal ions was recorded. NO considerably induced the relative mRNA levels for phenylalanine ammonia-lyase in 24-h leaves pretreated with NO donors, both non-infested and infested. NO stimulated the accumulation of pisatin in leaves until 24 h. The Electrical Penetration Graph revealed a reduction in the feeding activity of the pea aphid on leaves pretreated with NO. The present study showed that foliar application of NO donors induced sequentially defense reactions of pea against A. pisum and had a deterrent effect on aphid feeding and limited the population growth rate.

The multifunctional face of plant carbonic anhydrase.

Although most studies on the ubiquitous enzyme carbonic anhydrase (CA) have indicated its significant role in plants to facilitate the diffusion of CO2 to the site of inorganic carbon fixation, it is becoming increasingly likely that carbonic anhydrase isoforms also have diverse unexplored functions in plant cells. This review lays emphasis on additional roles of CA associated with many physiological, biochemical and structural changes in plant metabolism. The presented findings have revealed essential functions of CA isoforms in plant adjustment to both abiotic and biotic agents and developmental stimuli. However, sometimes it is difficult to separate the non-photosynthetic from the photosynthetic-related role of CAs during post-stress impaired metabolism, and the preventive CA outcome might be due to the effect of these enzymes on improvement of photosynthetic capacity. Finally, taking into account the experimental evidence, the direct and indirect functional roles of CAs in mitigating negative effects of environmental conditions are presented.

Contrasting Regulation of NO and ROS in Potato Defense-Associated Metabolism in Response to Pathogens of Different Lifestyles.

Our research provides new insights into how the low and steady-state levels of nitric oxide (NO) and reactive oxygen species (ROS) in potato leaves are altered after the challenge with the hemibiotroph Phytophthora infestans or the necrotroph Botrytis cinerea, with the subsequent rapid and invader-dependent modification of defense responses with opposite effects. Mainly in the avirulent (avr) P. infestans-potato system, NO well balanced with the superoxide level was tuned with a battery of SA-dependent defense genes, leading to the establishment of the hypersensitive response (HR) successfully arresting the pathogen. Relatively high levels of S-nitrosoglutathione and S-nitrosothiols concentrated in the main vein of potato leaves indicated the mobile function of these compounds as a reservoir of NO bioactivity. In contrast, low-level production of NO and ROS during virulent (vr) P. infestans-potato interactions might be crucial in the delayed up-regulation of PR-1 and PR-3 genes and compromised resistance to the hemibiotrophic pathogen. In turn, B. cinerea triggered huge NO overproduction and governed inhibition of superoxide production by blunting NADPH oxidase. Nevertheless, a relatively high level of H2O2 was found owing to the germin-like activity in cooperation with NO-mediated HR-like cell death in potato genotypes favorable to the necrotrophic pathogen. Moreover, B. cinerea not only provoked cell death, but also modulated the host redox milieu by boosting protein nitration, which attenuated SA production but not SA-dependent defense gene expression. Finally, based on obtained data the organismal cost of having machinery for HR in plant resistance to biotrophs is also discussed, while emphasizing new efforts to identify other components of the NO/ROS cell death pathway and improve plant protection against pathogens of different lifestyles.

The combined nitrate reductase and nitrite-dependent route of NO synthesis in potato immunity to Phytophthora infestans.

In contrast to the in-depth knowledge concerning nitric oxide (NO) function, our understanding of NO synthesis in plants is still very limited. In view of the above, this paper provides a step by step presentation of the reductive pathway for endogenous NO generation involving nitrate reductase (NR) activity and nitrite implication in potato defense to Phytophthora infestans. A biphasic character of NO emission, peaking mainly at 3 and then at 24 hpi, was detected during the hypersensitive response (HR). In avr P. infestans potato leaves enhanced NR gene and protein expression was tuned with the depletion of nitrate contents and the increase in nitrite supply at 3 hpi. In the same time period a temporary down-regulation of nitrite reductase (NiR) and activity was found. The study for the link between NO signaling and HR revealed an up-regulation of used markers of effective defense, i.e. Nonexpressor of PR genes (NPR1), thioredoxins (Thx) and PR1, at early time-points (1-3 hpi) upon inoculation. In contrast to the resistant response, in the susceptible one a late overexpression (24-48 hpi) of NPR1 and PR1 mRNA levels was observed. Presented data confirmed the importance of nitrite processed by NR in NO generation in inoculated potato leaves. However, based on the pharmacological approach the potential formation of NO from nitrite bypassing the NR activity during HR response to P. infestans has also been discussed.