Integrated Sensing Chip for Ultrasensitive Label-Free Detection of the Products of Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that has been widely used for the detection of pathogens in many organisms. Current LAMP-based sensors usually require the LAMP products to be labeled in order for them to be detected. Here, we present a novel label-free LAMP chip, which consists of a nanopore thin-film sensor embedded inside a LAMP reaction chamber. A fraction of LAMP primers is immobilized on the sensor surface, allowing the LAMP products to be synthesized and bound to the sensor surface via immobilized primers. After the LAMP reaction components are removed from the reaction chamber, the amplified LAMP products bound to the sensor surface give rise to significantly increased transducing signals, which can be measured by a portable optical spectrometer through an optical fiber probe. As a demonstration, we used the LAMP chip to detect the causal agent of late blight, Phytophthora infestans, which is one of the most devastating plant pathogens and poses a major threat to sustainable crop production worldwide. We show that this chip can detect as low as 1 fg/µL of P. infestans DNA in 30 min, which corresponds to an attomolar level of 1.6 × 10-6 attomole/µL and is at least 10 times more sensitive than the currently available methods. This label-free sensing technology holds great promise to open up a new avenue for ultrasensitive, highly specific, rapid, and cost-effective point-of-care diagnostics of plant, animal, human, and foodborne pathogens.

A Clickable NAD+ Analog-Based Assay of Poly(ADP-Ribosyl)ated Proteins.

Poly(ADP-ribosyl)lation (PARylation) is a posttranslational modification that plays an important role in a variety of biological processes in both animals and plants. Identification of PARylated substrates is the key to elucidating the regulatory mechanism of PARylation. Several approaches have been developed to identify PARylated substrates over the past decade; however, a reliable and efficient method is needed to demonstrate PARylated proteins. Here, we report a simple and sensitive assay of PARylated proteins using a clickable 6-alkyne-NAD+ analog. The 6-alkyne-NAD+ is incorporated into substrate proteins in the in vitro PARylation assay. The labeled proteins are covalently captured by disulfide azide agarose beads through copper-catalyzed azide-alkyne cycloaddition (CuAAC), cleaved under reducing conditions, and analyzed by immunoblotting. The covalent bonds between the PARylated proteins and azide beads allow high stringent washing to eliminate nonspecific binding. Furthermore, the disulfide linker permits efficient cleavage and recovery of highly enriched PARylated proteins. Therefore, this approach can detect proteins that undergo PARylation at very low levels.

Development of a Ligation-Independent Cloning-Based Dual Vector System for RNA Interference in Plants.

RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing mechanism that responds to double-stranded RNA (dsRNA) by sequence-specific downregulation of target genes. The dsRNA-mediated RNAi technology has become one of the most widely used and powerful tools for functional genomic studies in diverse organisms. However, its application has been limited due to the technical difficulty of making RNAi constructs caused by the inverted repeat structure that is required for the formation of hairpin RNA. Here, we present a ligation-independent cloning-based dual vector-mediated RNAi system for silencing specific genes in plants. This approach is simple, efficient, and cost-effective and can be readily adapted to other binary vectors for functional analysis of target genes and the development of sustainable disease and pest control strategies in a broad range of plant species.

NLR immune receptor RB is differentially targeted by two homologous but functionally distinct effector proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) receptors mediate immune responses by directly or indirectly sensing pathogen-derived effectors. Despite significant advances in the understanding of NLR-mediated immunity, the mechanisms by which pathogens evolve to suppress NLR activation triggered by cognate effectors and gain virulence remain largely unknown. The agronomically important immune receptor RB recognizes the ubiquitous and highly conserved IPI-O RXLR family members (e.g., IPI-O1) from Phytophthora infestans, and this process is suppressed by the rarely present and homologous effector IPI-O4. Here, we report that self-association of RB via the coiled-coil (CC) domain is required for RB activation and is differentially affected by avirulence and virulence effectors. IPI-O1 moderately reduces the self-association of RB CC, potentially leading to changes in the conformation and equilibrium of RB, whereas IPI-O4 dramatically impairs CC self-association to prevent RB activation. We also found that IPI-O1 associates with itself, whereas IPI-O4 does not. Notably, IPI-O4 interacts with IPI-O1 and disrupts its self-association, therefore probably blocking its avirulence function. Furthermore, IPI-O4 enhances the interaction between RB CC and IPI-O1, possibly sequestering RB and IPI-O1 and subsequently blocking their interactions with signaling components. Taken together, these findings considerably extend our understanding of the underlying mechanisms by which emerging virulent pathogens suppress the NLR-mediated recognition of cognate effectors.

Coordinated regulation of plant immunity by poly(ADP-ribosyl)ation and K63-linked ubiquitination.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification reversibly catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) and plays a key role in multiple cellular processes. The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes. Here we show that Arabidopsis UBC13A and UBC13B, the major drivers of lysine 63 (K63)-linked polyubiquitination, directly interact with PARPs/PARGs. Activation of pathogen-associated molecular pattern (PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13. Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related (PR) proteins but decreased accumulation of secreted PR proteins. Protein disulfide-isomerases (PDIs), essential components of endoplasmic reticulum quality control (ERQC) that ensure proper folding and maturation of proteins destined for secretion, complex with PARPs/PARGs and are PARylated upon PAMP perception. Significantly, PARylation of UBC13 regulates K63-linked ubiquitination of PDIs, which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion. Taken together, these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.

Virus-Induced Gene Silencing in Diploid and Tetraploid Potato Species.

Potato is the world's fourth largest food crop and a vegetatively propagated model polyploid plant. To facilitate genomic studies in potato, here we describe detailed protocols to silence genes in both diploid potato Solanum bulbocastanum and tetraploid potato cultivars such as Maris Bard, Arran Pilot, Ancilla, and Serrana using tobacco rattle virus (TRV)- or potato virus X (PVX)-induced gene silencing (VIGS) system, respectively. The established VIGS system represents an efficient and powerful approach for functional analysis of genes involved in growth, development, metabolism, and responses to biotic and abiotic stresses in potato.

Virus-Based microRNA Silencing in Plants.

Virus-based microRNA silencing (VbMS) is an efficient, powerful, and high-throughput approach to screen and investigate the function of microRNAs (miRNAs) in plants. The VbMS system was originally developed in Nicotiana benthamiana and tomato (Solanum lycopersicum) and has been extended to various other plant species such as Arabidopsis, cotton, and wheat with different virus vectors. VbMS is generally designed to use virus vectors to direct the expression of miRNA target mimic (TM) molecules which can complementarily pair to target miRNAs and block their function. Here, we describe the TRV- and PVX-based VbMS approaches to silence endogenous miRNAs in N. benthamiana and tomato plants by Agrobacterium infiltration. This method can be further applied to other plant species using suitable virus vectors in combination with diverse TM strategies, which will facilitate functional studies of miRNAs in plants.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

PBS3 Protects EDS1 from Proteasome-Mediated Degradation in Plant Immunity.

Plant immunity is controlled by both positive regulators such as PBS3 and EDS1 and negative regulators such as NPR3 and NPR4. However, the relationships among these important immune regulators remain elusive. In this study, we found that PBS3 interacts with EDS1 in both the cytoplasm and the nucleus, and is required for EDS1 protein accumulation. NPR3 and NPR4, which function as salicylic acid receptors and adaptors of Cullin3-based E3 ligase, interact with and mediate the degradation of EDS1 via the 26S proteasome. We further discovered that PBS3 inhibits the polyubiquitination and subsequent degradation of EDS1 by reducing the association of EDS1 with the Cullin3 adaptors NPR3 and NPR4. Furthermore, we showed that PBS3 and EDS1 also contribute to PAMP-triggered immunity in addition to effector-triggered immunity. Collectively, our study reveals a novel mechanism by which plants fine-tune defense responses by inhibiting the degradation of a positive player in plant immunity.

3-Aminobenzamide Blocks MAMP-Induced Callose Deposition Independently of Its Poly(ADPribosyl)ation Inhibiting Activity.

Cell wall reinforcement with callose is a frequent plant response to infection. Poly(ADP-ribosyl)ation is a protein post-translational modification mediated by poly(ADP-ribose) polymerases (PARPs). Poly(ADP-ribosyl)ation has well-known roles in DNA damage repair and has more recently been shown to contribute to plant immune responses. 3-aminobenzamide (3AB) is an established PARP inhibitor and it blocks the callose deposition elicited by flg22 or elf18, two microbe-associated molecular patterns (MAMPs). However, we report that an Arabidopsis parp1parp2parp3 triple mutant does not exhibit loss of flg22-induced callose deposition. Additionally, the more specific PARP inhibitors PJ-34 and INH2BP inhibit PARP activity in Arabidopsis but do not block MAMP-induced callose deposition. These data demonstrate off-target activity of 3AB and indicate that 3AB inhibits callose deposition through a mechanism other than poly(ADP-ribosyl)ation. POWDERY MILDEW RESISTANT 4 (PMR4) is the callose synthase responsible for the majority of MAMP- and wound-induced callose deposition in Arabidopsis. 3AB does not block wound-induced callose deposition, and 3AB does not reduce the PMR4 mRNA abundance increase in response to flg22. Levels of PMR4-HA protein increase in response to flg22, and increase even more in flg22 + 3AB despite no callose being produced. The callose synthase inhibitor 2-deoxy-D-glucose does not cause similar impacts on PMR4-HA protein levels. Beyond MAMPs, we find that 3AB also reduces callose deposition induced by powdery mildew (Golovinomyces cichoracearum) and impairs the penetration resistance of a PMR4 overexpression line. 3AB thus reveals pathogenesis-associated pathways that activate callose synthase enzymatic activity distinct from those that elevate PMR4 mRNA and protein abundance.

Isolation and characterization of centromeric repetitive DNA sequences in Saccharum spontaneum.

Sugarcane (Saccharum hybrids spp.) is the most important sugar crop that accounts for ~75% of the world's sugar production. Recently, a whole-genome sequencing project was launched on the wild species S. spontaneum. To obtain information on the DNA composition of the repeat-enriched region of the centromere, we conducted a genome-wide analysis of the DNA sequences associated with CenH3 (a mutant of histone H3 located in eukaryote centromeres) using chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. We demonstrate that the centromeres contain mainly SCEN-like single satellite repeat (Ss1) and several Ty3/gypsy retrotransposon-related repeats (Ss166, Ss51, and Ss68). Ss1 dominates in the centromeric regions and spans up to 500 kb. In contrast, the Ty3/gypsy retrotransposon-related repeats are either clustered spanning over a short range, or dispersed in the centromere regions. Interestingly, Ss1 exhibits a chromosome-specific enrichment in the wild species S. spontaneum and S. robustum, but not in the domesticated species S. officinarum and modern sugarcane cultivars. This finding suggests an autopolyploid genome identity of S. spontaneum with a high level of homology among its eight sub-genomes. We also conducted a genome-wide survey of the repetitive DNAs in S. spontaneum following a similarity-based sequence clustering strategy. These results provide insight into the composition of sugarcane genome as well as the genome assembly of S. spontaneum.

Disease resistance through impairment of α-SNAP-NSF interaction and vesicular trafficking by soybean Rhg1.

α-SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] and NSF proteins are conserved across eukaryotes and sustain cellular vesicle trafficking by mediating disassembly and reuse of SNARE protein complexes, which facilitate fusion of vesicles to target membranes. However, certain haplotypes of the Rhg1 (resistance to Heterodera glycines 1) locus of soybean possess multiple repeat copies of an α-SNAP gene (Glyma.18G022500) that encodes atypical amino acids at a highly conserved functional site. These Rhg1 loci mediate resistance to soybean cyst nematode (SCN; H. glycines), the most economically damaging pathogen of soybeans worldwide. Rhg1 is widely used in agriculture, but the mechanisms of Rhg1 disease resistance have remained unclear. In the present study, we found that the resistance-type Rhg1 α-SNAP is defective in interaction with NSF. Elevated in planta expression of resistance-type Rhg1 α-SNAPs depleted the abundance of SNARE-recycling 20S complexes, disrupted vesicle trafficking, induced elevated abundance of NSF, and caused cytotoxicity. Soybean, due to ancient genome duplication events, carries other loci that encode canonical (wild-type) α-SNAPs. Expression of these α-SNAPs counteracted the cytotoxicity of resistance-type Rhg1 α-SNAPs. For successful growth and reproduction, SCN dramatically reprograms a set of plant root cells and must sustain this sedentary feeding site for 2-4 weeks. Immunoblots and electron microscopy immunolocalization revealed that resistance-type α-SNAPs specifically hyperaccumulate relative to wild-type α-SNAPs at the nematode feeding site, promoting the demise of this biotrophic interface. The paradigm of disease resistance through a dysfunctional variant of an essential gene may be applicable to other plant-pathogen interactions.

PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses.

Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.

Microbial pathogens trigger host DNA double-strand breaks whose abundance is reduced by plant defense responses.

Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs) in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS) is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.

Salicylic acid activates DNA damage responses to potentiate plant immunity.

DNA damage is normally detrimental to living organisms. Here we show that it can also serve as a signal to promote immune responses in plants. We found that the plant immune hormone salicylic acid (SA) can trigger DNA damage in the absence of a genotoxic agent. The DNA damage sensor proteins RAD17 and ATR are required for effective immune responses. These sensor proteins are negatively regulated by a key immune regulator, SNI1 (suppressor of npr1-1, inducible 1), which we found is a subunit of the structural maintenance of chromosome (SMC) 5/6 complex required for controlling DNA damage. Elevated DNA damage caused by the sni1 mutation or treatment with a DNA-damaging agent markedly enhances SA-mediated defense gene expression. Our study suggests that activation of DNA damage responses is an intrinsic component of the plant immune responses.

Mutations in FLS2 Ser-938 dissect signaling activation in FLS2-mediated Arabidopsis immunity.

Flagellin-sensing 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.

Light-dependent, dark-promoted interaction between Arabidopsis cryptochrome 1 and phytochrome B proteins.

Plant photoreceptors transduce environmental light cues to downstream signaling pathways, regulating a wide array of processes during growth and development. Two major plant photoreceptors with critical roles in photomorphogenesis are phytochrome B (phyB), a red/far-red absorbing photoreceptor, and cryptochrome 1 (CRY1), a UV-A/blue photoreceptor. Despite substantial genetic evidence for cross-talk between phyB and CRY1 pathways, a direct interaction between these proteins has not been observed. Here, we report that Arabidopsis phyB interacts directly with CRY1 in a light-dependent interaction. Surprisingly, the interaction is light-dissociated; CRY1 interacts specifically with the dark/far-red (Pr) state of phyB, but not with the red light-activated (Pfr) or the chromophore unconjugated form of the enzyme. The interaction is also regulated by light activation of CRY1; phyB Pr interacts only with the unstimulated form of CRY1 but not with the photostimulated protein. Further studies reveal that a small domain extending from the photolyase homology region (PHR) of CRY1 regulates the specificity of the interaction with different conformational states of phyB. We hypothesize that in plants, the phyB/CRY1 interaction may mediate cross-talk between the red/far-red- and blue/UV-sensing pathways, enabling fine-tuning of light responses to different spectral inputs.

DNA repair proteins are directly involved in regulation of gene expression during plant immune response.

Systemic acquired resistance (SAR), an inducible plant-defense response to local infection, requires the signaling molecule salicylic acid (SA) and the transcriptional coactivator NPR1, with concerted activation of pathogenesis-related (PR) genes. Arabidopsis sni1 is an npr1 suppressor and derepression of defense genes in sni1 causes reduced growth and fertility and increased homologous recombination. Characterizing suppressors of sni1, we identify the DNA damage repair proteins SSN2 and RAD51D as genetic and physical interactors with SNI1. During plant defense, SSN2 and possibly RAD51D replace the transcription repressor SNI1 at pathogenesis-related gene promoters. In the presence of SNI1, NPR1 is also required for SSN2 binding. Thus, coordinated action of SNI1, SSN2-RAD51D, and NPR1 ensures the tight control of plant immune gene expression. Given that the SSN2-RAD51D complex is conserved in eukaryotes, their dual function in homologous recombination and transcription regulation of plant-defense genes suggests a general link between these two stress responses.

Arabidopsis BRCA2 and RAD51 proteins are specifically involved in defense gene transcription during plant immune responses.

Systemic acquired resistance (SAR) is a plant immune response associated with both transcriptional reprogramming and increased homologous DNA recombination (HR). SNI1 is a negative regulator of SAR and HR, as indicated by the increased basal expression of defense genes and HR in sni1. We found that the sni1 phenotypes are rescued by mutations in BREAST CANCER 2 (BRCA2). In humans, BRCA2 is a mediator of RAD51 in pairing of homologous DNA. Mutations in BRCA2 cause predisposition to breast/ovarian cancers; however, the role of the BRCA2-RAD51 complex in transcriptional regulation remains unclear. In Arabidopsis, both brca2 and rad51 were found to be hypersusceptible not only to genotoxic substances, but also to pathogen infections. A whole-genome microarray analysis showed that downstream of NPR1, BRCA2A is a major regulator of defense-related gene transcription. ChIP demonstrated that RAD51 is specifically recruited to the promoters of defense genes during SAR. This recruitment is dependent on the SAR signal salicylic acid (SA) and on the function of BRCA2. This study provides the molecular evidence showing that the BRCA2-RAD51 complex, known for its function in HR, also plays a direct and specific role in transcription regulation during plant immune responses.

Plant immunity requires conformational changes [corrected] of NPR1 via S-nitrosylation and thioredoxins.

Changes in redox status have been observed during immune responses in different organisms, but the associated signaling mechanisms are poorly understood. In plants, these redox changes regulate the conformation of NPR1, a master regulator of salicylic acid (SA)-mediated defense genes. NPR1 is sequestered in the cytoplasm as an oligomer through intermolecular disulfide bonds. We report that S-nitrosylation of NPR1 by S-nitrosoglutathione (GSNO) at cysteine-156 facilitates its oligomerization, which maintains protein homeostasis upon SA induction. Conversely, the SA-induced NPR1 oligomer-to-monomer reaction is catalyzed by thioredoxins (TRXs). Mutations in both NPR1 cysteine-156 and TRX compromised NPR1-mediated disease resistance. Thus, the regulation of NPR1 is through the opposing action of GSNO and TRX. These findings suggest a link between pathogen-triggered redox changes and gene regulation in plant immunity.