Hypersensitive Defense Response Research Articles

Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection.

Lipid droplets (LDs) have classically been viewed as seed storage particles, yet they are now emerging as dynamic organelles associated with developmental and stress responses. Nevertheless, their involvement in plant immunity has still been little studied. Here, we found LD accumulation in Arabidopsis thaliana leaves that induced a hypersensitive response (HR) after Pseudomonas infection. We established a protocol to reproducibly isolate LDs and to analyze their protein content. The expression of GFP fusion proteins in Nicotiana benthamiana and in transgenic Arabidopsis lines validated the LD localization of glycerol-3-phosphate acyltransferase 4 (GPAT4) and 8 (GPAT8), required for cutin biosynthesis. Similarly, we showed LD localization of α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3), involved in the synthesis of fatty acid derivatives, and that of phytoalexin-deficient 3 (PAD3), which is involved in camalexin synthesis. We found evidence suggesting the existence of different populations of LDs, with varying protein contents and distributions. GPAT4 and GPAT8 were associated with LDs inside stomata and surrounding cells of untreated leaves, yet they were mainly confined to LDs in guard cells after bacterial inoculation. By contrast, α-DOX1 and PAD3 were associated with LDs in the epidermal cells of HR-responding leaves, with PAD3 mostly restricted to cells near dead tissue, while CLO3 had a more ubiquitous distribution. As such, the nature of the proteins identified, together with the phenotypic examination of selected mutants, suggests that LDs participate in lipid changes and in the production and transport of defense components affecting the interaction of plants with invading pathogens.

Research advances in plant-insect molecular interaction.

Acute and precise signal perception and transduction are essential for plant defense against insects. Insect elicitors—that is, the biologically active molecules from insects’ oral secretion (which contains regurgitant and saliva), frass, ovipositional fluids, and the endosymbionts—are recognized by plants and subsequently induce a local or systematic defense response. On the other hand, insects secrete various types of effectors to interfere with plant defense at multiple levels for better adaptation. Jasmonate is a main regulator involved in plant defense against insects and integrates with multiple pathways to make up the intricate defense network. Jasmonate signaling is strictly regulated in plants to avoid the hypersensitive defense response and seems to be vulnerable to assault by insect effectors at the same time. Here, we summarize recently identified elicitors, effectors, and their target proteins in plants and discuss their underlying molecular mechanisms.

A maize polygalacturonase functions as a suppressor of programmed cell death in plants

BackgroundThe hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7.ResultsExpression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field.ConclusionsZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.

Identification of a Spotted Leaf Sheath Gene Involved in Early Senescence and Defense Response in Rice.

Lesion mimic mutants (LMMs) commonly exhibit spontaneous cell death similar to the hypersensitive defense response that occurs in plants in response to pathogen infection. Several lesion mimic mutants have been isolated and characterized, but their molecular mechanisms remain largely unknown. Here, a spotted leaf sheath (sles) mutant derived from japonica cultivar Koshihikari is described. The sles phenotype differed from that of other LMMs in that lesion mimic spots were observed on the leaf sheath rather than on leaves. The sles mutant displayed early senescence, as shown, by color loss in the mesophyll cells, a decrease in chlorophyll content, and upregulation of chlorophyll degradation-related and senescence-associated genes. ROS content was also elevated, corresponding to increased expression of genes encoding ROS-generating enzymes. Pathogenesis-related genes were also activated and showed improved resistance to pathogen infection on the leaf sheath. Genetic analysis revealed that the mutant phenotype was controlled by a single recessive nuclear gene. Genetic mapping and sequence analysis showed that a single nucleotide substitution in the sixth exon of LOC_Os07g25680 was responsible for the sles mutant phenotype and this was confirmed by T-DNA insertion line. Taken together, our results revealed that SLES was associated with the formation of lesion mimic spots on the leaf sheath resulting early senescence and defense responses. Further examination of SLES will facilitate a better understanding of the molecular mechanisms involved in ROS homeostasis and may also provide opportunities to improve pathogen resistance in rice.

A Genome-Wide Association Study for Partial Resistance to Maize Common Rust.

Quantitative resistance to maize common rust (causal agent Puccinia sorghi) was assessed in an association mapping population of 274 diverse inbred lines. Resistance to common rust was found to be moderately correlated with resistance to three other diseases and with the severity of the hypersensitive defense response previously assessed in the same population. Using a mixed linear model accounting for the confounding effects of population structure and flowering time, genome-wide association tests were performed based at 246,497 single-nucleotide polymorphism loci. Three loci associated with maize common rust resistance were identified. Candidate genes at each locus had predicted roles, mainly in cell wall modification. Other candidate genes included a resistance gene and a gene with a predicted role in regulating accumulation of reactive oxygen species.

Similarities and Differences in Global Gene Expression Profiles between Herbicide and Pathogen-Induced PSII Inhibition

Plant pathogens, and photosynthesis inhibiting herbicides, can both damage photosystem II (PSII), causing it to be highly sensitive to damage by light energy, and to release high levels of reactive oxygen species (ROS). This photoinhibition of PSII could possibly be the source of the strong oxidative burst associated with the pathogeninduced, hypersensitive defense response (HR). To examine a possible mechanism of how the HR-associated ROS burst could originate from PSII inhibition, we compared the transcriptome responses in soybean undergoing photoinhibition induced by HR, to soybean undergoing photoinhibition induced by the herbicide bentazon, which specially stops PSII electron flow by preventing QB from binding to D1. Most genes shared similar expression patterns between HR and bentazon treatments; however, interesting differences were also observed. The most striking differences were seen with genes related to photosynthesis, where these genes were uniformly down regulated in HR, but were mostly up in response to bentazon. Another interesting difference was seen in genes of the phenylpropanoid pathway. These defense-related genes were mostly down or non-responsive to bentazon, but were generally induced in response to pathogen-induced HR, showing that soybeans activate the phenylpropanoidbased phytoalexins independent of PSII inhibition. We conclude that the PSII inhibition occurring during the HR is not being triggered simply by the inhibition of electron flow through the photosystem centers. Instead, it is more likely that the initial triggers of the HR halt the repair of damaged PSII which leads to enhancing photoinhibition and contributing the rapid production of ROS, sealing the fate of cells undergoing HR; and the triggers independently induce specific aspects of defenses such as the phenylpropanoid pathway.

A genome-wide association study of the maize hypersensitive defense response identifies genes that cluster in related pathways.

Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.

Capsicum annuum WRKY transcription factor d (CaWRKYd) regulates hypersensitive response and defense response upon Tobacco mosaic virus infection

WRKY transcription factors regulate biotic, abiotic, and developmental processes. In terms of plant defense, WRKY factors have important roles as positive and negative regulators via transcriptional regulation or protein–protein interaction. Here, we report the characterization of the gene encoding Capsicum annuum WRKY transcription factor d (CaWRKYd) isolated from microarray analysis in the Tobacco mosaic virus (TMV)-P0–inoculated hot pepper plants. CaWRKYd belongs to the WRKY IIa group, a very small clade in the WRKY subfamily, and WRKY IIa group has positive/negative regulatory roles in Arabidopsis and rice. CaWRKYd transcripts were induced by various plant defense-related hormone treatments and TMV-P0 inoculation. Silencing of CaWRKYd affected TMV-P0-mediated hypersensitive response (HR) cell death and accumulation of TMV-P0 coat protein in local and systemic leaves. Furthermore, expression of some pathogenesis-related (PR) genes and HR-related genes was reduced in the CaWRKYd-silenced plants compared with TRV2 vector control plants upon TMV-P0 inoculation. CaWRKYd was confirmed to bind to the W-box. Thus CaWRKYd is a newly identified Capsicum annuum WRKY transcription factor that appears to be involved in TMV-P0-mediated HR cell death by regulating downstream gene expression.

Detection of peroxynitrite accumulation in Arabidopsis thaliana during the hypersensitive defense response

Nitric oxide (NO) is synthesized in plants in response to stress, and its role in signaling is well-documented. In contrast, very little is known about the physiological role of its derivate peroxynitrite (ONOO −), which forms when NO reacts with O 2 - and induces protein modification by tyrosine nitration. Infection with an avirulent pathogen triggers the simultaneous production of NO and reactive oxygen species, as well as an increase in tyrosine nitration, so peroxynitrite could be physiologically relevant during this process. To gain insight into the role of peroxynitrite in plants, we measured its accumulation during the hypersensitive response in Arabidopsis thaliana using the specific peroxynitrite-sensitive fluorescent dye HKGreen-2 in a leaf disc assay. The avirulent pathogen Pseudomonas syringae pv. tomato, carrying the AvrB gene (Pst AvrB), induced a strong increase in fluorescence 3–4 h post-infiltration (hpi) which peaked 7–8 hpi. The increase in HKGreen-2 fluorescence was inhibited by co-injecting the peroxynitrite-scavenger urate together with the pathogen, and was almost completely eliminated by co-infiltrating urate with HKGreen-2, confirming that HKGreen-2 fluorescence in planta is induced specifically by peroxynitrite. This establishes a link between peroxynitrite synthesis and tyrosine nitration, and we therefore propose that peroxynitrite transduces the NO signal by modifying protein functions.

Identification of a Maize Locus That Modulates the Hypersensitive Defense Response, Using Mutant-Assisted Gene Identification and Characterization

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called Mutant-Assisted Gene Identification and Characterization is used to identify naturally occurring loci modulating the hypersensitive defense response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a "reporter." In this study the reporter phenotype was caused by a partially dominant autoactive disease resistance gene, Rp1-D21, which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally occurring useful genetic variation.

Improving Antioxidant Capacity of Fresh‐Cut Mangoes Treated with UV‐C

The effect of UV-C irradiation time on total phenol, flavonoids, beta-carotene, ascorbic acid contents, and antioxidant capacity (ORAC, DPPH(*)) of fresh-cut "Tommy Atkins" mango stored for 15 d at 5 degrees C was investigated. Fresh-cut mango was irradiated for 0, 1, 3, 5, and 10 min prior to storage at 5 degrees C. UV-C irradiation for 10 min induced a hypersensitive defense response resulting in the phenols and flavonoids accumulation which was positively correlated with ORAC and DPPH(*) values. However, beta-carotene and ascorbic acid content of fresh-cut mangos decreased with irradiation time during storage. Antioxidant capacity (ORAC, DPPH(*)) was increased in fresh-cut mangoes treated with UV-C irradiation. In conclusion, UV-C irradiation appears to be a good technique to improve the total antioxidant capacity of fresh-cut mango.

Effect of Processing Under Ultraviolet Light on the Shelf Life of Fresh-Cut Cantaloupe Melon

ABSTRACT: The effect of processing cantaloupe melon under ultraviolet-C (UV-C) radiation on storage properties of the cut fruit at 10 °C was compared with post-cut UV-C fruit treatment and the untreated control. Cutting fruit under UV-C light induced a hypersensitive defense response that resulted in increased accumulation of ascorbate peroxidase relative to the other 2 treatments. Fruit processed under UV-C radiation had the lowest esterase activity throughout the storage period. Lipase activity was higher in post-cut treated fruit than fruit processed under UV-C light and the control fruit. Lipase activity, however, decreased rapidly in fruit processed under UV-C and was undetectable after 7 d of storage. Human sensory aroma evaluation indicates reduced rancidity, and instrumental texture measurements suggested improved firmness retention in fruit cut under UV-C radiation. The treatment also reduced respiration during cut fruit storage. UV-C was effective in reducing yeast, mold, and Pseudomonas spp populations in both treatments. Fresh-cut pieces from whole melon cut under UV light had lower populations of aerobic mesophilic and lactic acid bacteria relative to the control and post-cut treated pieces. Results indicate that while post-cut application of UV improved shelf life of cut cantaloupe melon, cutting fruit under UV-C radiation further improves product quality.

Reduced polysome levels and preferential recruitment of a defense gene transcript into polysomes in soybean cells treated with the syringolide elicitor

The bacterial syringolide elicitor induces a hypersensitive defense response (HR) in leaf tissue and cell suspensions of soybean cultivars carrying resistance gene Rpg4. To investigate the possible involvement of translational control of gene expression in the syringolide-induced HR, ribosome profiles and polysome-loading of two gene transcripts were investigated. Sucrose density gradient fractionation of soybean cell extracts was used to demonstrate a syringolide-induced, Rpg4-specific reduction in large polysomes and a concomitant increase in 80S ribosomes, presumably reflecting an overall inhibition of translation initiation. Syringolide treatment also led to an accumulation of chalcone synthase defense gene mRNA, which remained efficiently loaded into large polysomes in spite of a 22% reduction in cellular large polysome levels. In contrast, while mRNA levels of the housekeeping gene actin remained unchanged following syringolide treatment, the amount of actin mRNA loaded into large polysomes was decreased by 49%. Thus, the translational machinery appeared to selectively exclude a subpopulation of actin transcripts while simultaneously maintaining efficient loading of transcripts for the defense gene chalcone synthase. These data suggest that elicited soybean cells utilize translational control of gene expression during the shift from normal metabolism to defense response activation.

Arabidopsis SHMT1, a serine hydroxymethyltransferase that functions in the photorespiratory pathway influences resistance to biotic and abiotic stress

We found that a recessive mutation, shmt1-1, causes aberrant regulation of cell death resulting in chlorotic and necrotic lesion formation under a variety of environmental conditions. Salicylic acid-inducible genes and genes involved in H(2)O(2) detoxification were expressed constitutively in shmt1-1 plants in direct correlation with the severity of the lesions. The shmt1-1 mutants were more susceptible than control plants to infection with biotrophic and necrotrophic pathogens, developing severe infection symptoms in a high percentage of infected leaves. In addition, mutants carrying shmt1-1 or a loss-of-function shmt1-2 allele, were smaller and showed a greater loss of chlorophyll and greater accumulation of H(2)O(2) than wild-type plants when subjected to salt stress. SHMT1 was map-based cloned and found to encode a serine hydroxymetyltransferase (SHMT1) involved in the photorespiratory pathway. Our results indicate that this enzymatic activity plays a critical role in controlling the cell damage provoked by abiotic stresses such as high light and salt and in restricting pathogen-induced cell death, supporting the notion that photorespiration forms part of the dissipatory mechanisms of plants to minimize production of reactive oxygen species (ROS) at the chloroplast and to mitigate oxidative damage. Moreover, results shown here indicate that whereas production of ROS is an essential component of the hypersensitive defense response, the excessive accumulation of these toxic compounds impairs cell death containment and counteracts the effectiveness of the plant defenses to restrict pathogen infection.

Syringolide elicitor-induced oxidative burst and protein phosphorylation in soybean cells, and tentative identification of two affected phosphoproteins

The bacterial plant pathogen Pseudomonas syringae pv. glycinea carrying an active allele of avirulence gene D (avrD) elicits a hypersensitive defense response (HR) specifically in soybean cultivars carrying the disease resistance gene Rpg4. Expression of avrD leads to production of a low molecular weight elicitor known as syringolide. Infiltration of syringolide into soybean leaves elicits an Rpg4-specific HR and also induces Rpg4-specific Ca2+, H+, and K+ ion fluxes and cell death in soybean cell suspensions. We have demonstrated that syringolide treatment of soybean cell suspensions further induces an Rpg4-specific hydrogen peroxide burst and subsequent shifts in protein phosphorylation. Furthermore, proteins co-migrating with two affected phosphoproteins (45 and 20kDa) were collected from preparative 2D-PAGE gels and internal peptide amino acid sequences were obtained. Partial amino acid sequence from the 45kDa protein showed strong identity to a family of plant EF-hand calcium-binding proteins and sequences from the 20kDa protein showed strong identity to a family of plant glycine-rich RNA-binding proteins (GRPs). The soybean GRP cDNA was cloned and sequenced.

The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response.

In plants, salicylic acid (SA) plays an important role in signaling both local and systemic defense responses. Previous efforts to identify SA effector proteins in tobacco have led to the isolation of two soluble cytoplasmic SA-binding proteins (SABPs): catalase, SABP, and an approximately 25-kDa protein, SABP2. Here we describe the identification of an SA-binding protein, SABP3, in the stroma of tobacco chloroplasts. SABP3 bound SA with an apparent dissociation constant (K(d)) of 3.7 microM and exhibited much greater affinity for biologically active than inactive analogs. Purification and partial sequencing of SABP3 indicated that it is the chloroplast carbonic anhydrase (CA). Confirming this finding, recombinant tobacco chloroplast CA exhibited both CA enzymatic and SA-binding activities. Expression of this protein in yeast also demonstrated that CA/SABP3 has antioxidant activity. A second gene encoding CA was also cloned, and its encoded protein was shown to behave similarly to that purified as SABP3. Finally, silencing of CA gene expression in leaves suppressed the Pto:avrPto-mediated hypersensitive response in disease resistance. These results demonstrate that SA may act through multiple effector proteins in plants and shed further light on the function of CA in chloroplasts.

Chemoenzymatic synthesis of the microbial elicitor (-)-syringolide via a fructose 1,6-diphosphate aldolase-catalyzed condensation reaction.

Syringolide 2, an elicitor of the bacterial plant pathogen Pseudomonas syringae pv. tomato which triggers a hypersensitive defense response in resistant soybean plants, has been synthesized in five steps via a fructose 1,6-diphosphate aldolase reaction.

Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants.

Arabidopsis mutations at the RPS2 locus result in loss of resistance to Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

We isolated and characterized two Arabidopsis thaliana mutants that fail to mount a hypersensitive defense response (HR) when infiltrated with phytopathogenic Pseudomonas strains carrying the avirulence (avr) gene avrRpt2 but still mount an HR when infiltrated with strains carrying other avr genes. One of these mutants was isolated using a method we developed that enriches for Arabidopsis seedlings that survive vacuum-infiltration with a bacterial strain carrying an avr gene. Genetic analysis showed that the phenotypes of both mutants resulted from mutations at a single locus, RPS2. In contrast to the wild type, both rps2 mutants failed to limit the growth of Pseudomonas strains carrying avrRpt2. Heterozygous RPS2/rps2 plants displayed a phenotype intermediate between those of RPS2/RPS2 and rps2/rps2 homozygotes. These experiments show that the wild-type allele at the rps2 locus, RPS2, encodes a component of a signal transduction pathway that responds to a signal generated by avrRpt2 and that RPS2 is required for the elicitation of an HR. RPS2 was mapped near the restriction fragment length polymorphism marker PG11 on chromosome IV.

Molecular Characterization of Avirulence Gene D fromPseudomonas syringaepv.tomato

Avirulence gene D, cloned from Pseudomonas syringae pv. tomato, caused P. s. pv. glycinea to elicit a hypersensitive defense response on certain cultivars of soybean. Nucleotide sequence data for a 5.6-kb HindIII fragment containing avrD disclosed five long open-reading frames (ORFs) occurring in tandem. The phenotype conferred by avrD was expressed in P. s. pv. glycinea solely by the first of these ORFs (933 bases) that encoded a protein of 34,115 daltons. Neither a signal peptide sequence nor significant regions of hydrophobicity were present that would indicate secretion of the protein or its membrane association. Hybridization analyses revealed that some but not all P. syringae pathovars contained DNA homologous to avrD. This included weak hybridization to all tested races of P. s. pv. glycinea, although none of them express the phenotype conferred by avrD. The avrD gene occurred on an indigenous 75-kb plasmid in several P. s. pv. tomato isolates.