Pathogenesis-related Responses Research Articles

Epsilon-poly-l-lysine (ε-PL) exhibits multifaceted antifungal mechanisms of action that control postharvest Alternaria rot

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial poly-cationic peptide widely applied as a natural preservative in the food industry, whereas its application in preventing postharvest loss of fruit was largely absent. This study investigated the antifungal activity of ε-PL and determined the possible mechanisms involved. The in vivo results indicated that 500 mg L−1 exogenous ε-PL treatment significantly inhibited black spot rot in apple, jujube, and tomato. The lesion diameter inhibition rate was range from 20.11% to 29.09% by 500 mg L−1 ε-PL treatment. ε-PL exerts antifungal activity against A. alternata in vitro, the half-inhibition concentration is 160.1 mg L−1. ε-PL induced morphology and ultrastructure change on the pathogen, which resulted in the inhibition of A. alternata. This was accomplished by disturbing pathogen membrane integrity and functionality. The fluorometric assay confirmed that ε-PL induced endogenous reactive oxygen species formation and accumulation in A. alternata and the elicited severe lipid peroxidation that caused membrane lesions. Further, ε-PL treatment enhanced the expression of genes involved in antioxidant metabolism and pathogenesis-related responses in apple fruit. These findings illustrated that ε-PL exhibits multifaceted antifungal activity by the direct effect on the pathogen as well as induce host defense responses. ε-PL may be conducive as a promising alternative for Alternaria rot management.

The protein phosphatase subunit PP2A‐B′γ is required to suppress day length‐dependent pathogenesis responses triggered by intracellular oxidative stress

Oxidative stress responses are influenced by growth day length, but little is known about how this occurs. A combined reverse genetics, metabolomics and proteomics approach was used to address this question in Arabidopsis thaliana. A catalase-deficient mutant (cat2), in which intracellular oxidative stress drives pathogenesis-related responses in a day length-dependent manner, was crossed with a knockdown mutant for a specific type 2A protein phosphatase subunit (pp2a-b'γ). In long days (LD), the pp2a-b'γ mutation reinforced cat2-triggered pathogenesis responses. In short days (SD), conditions in which pathogenesis-related responses were not activated in cat2, the additional presence of the pp2a-b'γ mutation allowed lesion formation, PATHOGENESIS-RELATED GENE1 (PR1) induction, salicylic acid (SA) and phytoalexin accumulation and the establishment of metabolite profiles that were otherwise observed in cat2 only in LD. Lesion formation in cat2 pp2a-b'γ in SD was genetically dependent on SA synthesis, and was associated with decreased PHYTOCHROME A transcripts. Phosphoproteomic analyses revealed that several potential protein targets accumulated in the double mutant, including recognized players in pathogenesis and key enzymes of primary metabolism. We conclude that the cat2 and pp2a-b'γ mutations interact synergistically, and that PP2A-B'γ is an important player in controlling day length-dependent responses to intracellular oxidative stress, possibly through phytochrome-linked pathways.

Proteomic analysis of Fusarium oxysporum f. sp. cubense tropical race 4-inoculated response to Fusarium wilts in the banana root cells.

BackgroundFusarium wilt of banana is one of the most destructive diseases in the world. This disease has caused heavy losses in major banana production areas. Except for molecular breeding methods based on plant defense mechanisms, effective methods to control the disease are still lacking. Dynamic changes in defense mechanisms between susceptible, moderately resistant, and highly resistant banana and Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4) at the protein level remain unknown. This research reports the proteomic profile of three banana cultivars in response to Foc4 and transcriptional levels correlated with their sequences for the design of disease control strategies by molecular breeding.ResultsThirty-eight differentially expressed proteins were identified to function in cell metabolism. Most of these proteins were positively regulated after Foc4 inoculation. These differentially regulated proteins were found to have important functions in banana defense response. Functional categories implicated that these proteins were associated with pathogenesis-related (PR) response; isoflavonoid, flavonoid, and anthocyanin syntheses; cell wall strengthening; cell polarization; reactive oxygen species production and scavenging; jasmonic acid-, abscisic acid-, and auxin-mediated signaling conduction; molecular chaperones; energy; and primary metabolism. By comparing the protein profiles of resistant and susceptible banana cultivars, many proteins showed obvious distinction in their defense mechanism functions. PR proteins in susceptible ‘Brazil’ were mainly involved in defense. The proteins related to PR response, cell wall strengthening and antifungal compound synthesis in moderately resistant ‘Nongke No.1’ were mainly involved in defense. The proteins related to PR response, cell wall strengthening, and antifungal compound synthesis in highly resistant ‘Yueyoukang I’ were mainly involved in defense. 12 differentially regulated genes were selected to validate through quantitative real time PCR method. Quantitative RT-PCR analyses of these selected genes corroborate with their respective protein abundance after pathogen infection.ConclusionsThis report is the first to use proteomic profiling to study the molecular mechanism of banana roots infected with Foc4. The differentially regulated proteins involved in different defense pathways are likely associated with different resistant levels of the three banana cultivars.

Combined effects of the plant growth-promoting bacterium Pseudomonas putida UW4 and salinity stress on the Brassica napus proteome

The influences of two strains of Pseudomonas putida UW4, a plant growth-promoting bacterium (PGPB) on the shoot and root proteomic profiles of Brassica napus (canola) were examined under salinity stress using two-dimensional difference gel electrophoresis. The two strains (wild-type UW4 and an AcdS minus mutant strain) differ in the availability of a functional 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, a bacterial protein known to play a role in host plant's stress responses. In total, ninety protein spots (representing 76 different proteins) with significantly altered expression levels in the presence of salt and/or bacteria were identified by mass spectrometry. Many of the identified proteins are involved in photosynthesis, anti-oxidative processes, transportation across membranes, and pathogenesis-related responses. The effects of salt and bacteria on the canola proteome were shown to be quite diverse, with salinity stress causing more dramatic plant protein expression changes than bacteria. In addition, bacteria were demonstrated to moderate some of the salt effects on plant protein differential expression. This work contributes to the understanding of how plant protein expression is affected by various environmental signals.

Regulation of NB-LRR-type UNI and its related signaling pathway

Activation of NB-LRR-related UNI proteins by uni-1D mutation, a gain-of-function mutation of the UNI gene, induces some pathogenesis-related responses and also affects morphology through modulation of meristem activities. In a recent study we reported that the uni-1D phenotypes require cooperative action of ERECTA (ER) receptor kinase family members in UNI-expressing cells, suggesting that an intracellular signaling crosstalk between ER-family-dependent and UNI-triggered signaling pathways plays a significant role in the phenotypes. Further we recently succeeded in the establishment of a methodology for rapid identification of factors involved in the UNI function. EMS-induced causal mutations that suppress the uni-1D phenotypes could be identified using whole-genome-sequencing technologies with much less labor compared with the conventional map-based cloning method that is generally time-consuming and labor-intensive. Thus it would be now possible to intensively identify factors that play significant roles in regulation of UNI proteins and/or UNI-related signaling pathways.

Differential disease resistance response in the barley necrotic mutant nec1

BackgroundAlthough ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.Resultsnec1 mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating nec1 disease resistance demonstrated positive effect of nec1 mutation on non-host resistance against Pseudomonas syringae pv. tomato (Pst) at high inoculum density, whereas at normal Pst inoculum concentration nec1 resistance did not differ from wt. In contrast to augmented P. syringae resistance, penetration resistance against biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew, was not altered in nec1. The nec1 mutant significantly over-expressed race non-specific Bgh resistance-related genes BI-1 and MLO. Induction of BI-1 and MLO suggested putative involvement of nec1 in race non-specific Bgh resistance, therefore the effect of nec1on mlo-5-mediated Bgh resistance was assessed. The nec1/mlo-5 double mutant was as resistant to Bgh as Nec1/mlo-5 plants, suggesting that nec1 did not impair mlo-5 race non-specific Bgh resistance.ConclusionsTogether, the results suggest that nec1 mutation alters activation of systemic acquired resistance-related physiological markers and non-host resistance in barley, while not changing rapid localized response during compatible interaction with host pathogen. Increased resistance of nec1 against non-host pathogen Pst suggests that nec1 mutation may affect certain aspects of barley disease resistance, while it remains to be determined, if the effect on disease resistance is a direct response to changes in SA signaling.

Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models

Hydrogen peroxide (H(2)O(2)) is an important signal molecule involved in plant development and environmental responses. Changes in H(2)O(2) availability can result from increased production or decreased metabolism. While plants contain several types of H(2)O(2)-metabolizing proteins, catalases are highly active enzymes that do not require cellular reductants as they primarily catalyse a dismutase reaction. This review provides an update on plant catalase genes, function, and subcellular localization, with a focus on recent information generated from studies on Arabidopsis. Original data are presented on Arabidopsis catalase single and double mutants, and the use of some of these lines as model systems to investigate the outcome of increases in intracellular H(2)O(2) are discussed. Particular attention is paid to interactions with cell thiol-disulphide status; the use of catalase-deficient plants to probe the apparent redundancy of reductive H(2)O(2)-metabolizing pathways; the importance of irradiance and growth daylength in determining the outcomes of catalase deficiency; and the induction of pathogenesis-related responses in catalase-deficient lines. Within the context of strategies aimed at understanding and engineering plant stress responses, the review also considers whether changes in catalase activities in wild-type plants are likely to be a significant part of plant responses to changes in environmental conditions or biotic challenge.

Genes associated with resistance to wheat yellow rust disease identified by differential display analysis

This study is focused on the identification of early response genes involved in the resistance mechanism of one of the most important diseases of wheat, yellow rust. The strategy undertaken was to use differential display reverse transcriptase-PCR method (DDRT-PCR) on two of the yellow rust differential lines of wheat, were infected with the virulent and the avirulent Puccinia striiformis f. sp. tritici races together with appropriate control inoculations. Upon infection of the plant materials (resistant and/or susceptible), different time points were evaluated on DDRT-PCR banding profiles. The expression level differences of 33 genes in DDRT-PCR were further analyzed using quantitative real-time PCR (qRT-PCR). Among which only those (14 gene fragments) resulting reproducible induction or repression levels were reported with measured fold changes. Based on the sequence similarity analysis, all but one were absent in NCBI databank, thus they are newly found wheat sequences. Among those, two genes were identified as full ORF, including 5′ and 3′ end untranslated regions (UTR); namely cyclophilin like protein (putative antifungal activity) and ubiquitin-conjugating enzyme (E2) or Rad6. The sequence homology analysis of the cloned gene fragments revealed that the genes detected may have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance responses, pathogenesis related responses, and including a few with no known function.

Temporal Analysis of Sucrose-induced Phosphorylation Changes in Plasma Membrane Proteins of Arabidopsis

Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants. In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied. Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply. Among the identified phosphorylation sites, the well described phosphorylation site at the C terminus of plasma membrane H(+)-ATPases showed a relative increase in phosphorylation level in response to sucrose. This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. A new phosphorylation site was identified in the plasma membrane H(+)-ATPase AHA1 and/or AHA2. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment. Seven response classes were identified including non-responsive, rapid increase (within 3 min), slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.

Tissue-specific mRNA expression profiling in grape berry tissues

BackgroundBerries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions.ResultsOverall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented.ConclusionThese results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality.

MICROARRAY EXPRESSION PROFILING OF NAGAMI KUMQUAT IN RESPONSE TO CANKER

Citrus canker disease, caused by a bacterial pathogen Xanthomonas axonopodis pv. citri, affects citrus species and citrus relatives in many areas of the world. Kumquats have apparent resistance to canker, the mechanism of which has not been determined. Forward and reverse subtractive cDNA libraries were constructed using Nagami kumquat mRNA to identify genes that are differentially expressed during the interaction with the canker pathogen. Some cDNA clones were selected for sequencing, and the sequences were analyzed using NCBI BLAST. Homologues to transcription factors, receptor and resistance genes known to be involved in plant-pathogen interaction were identified. cDNA microarrays containing 2304 of these genes were analyzed for expression profiles during the interaction with the canker pathogen. The results demonstrate how the use of microarray-based expression profiling methods can help elucidate plant pathogenesis-related response mechanisms and assign roles for previously uncharacterized genes. This can be considered to be a case study in citrus that has used these high throughput technologies to understand the defense mechanisms in Fortunella and citrus at the molecular level.

Identification of differentially regulated proteins in response to a compatible interaction between the pathogenFusarium graminearum and its host,Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.

Gene expression profiling of Arabidopsis thaliana in compatible plant-aphid interactions.

Phloem feeding involves unique biological interactions between the herbivore and its host plant. The economic importance of aphids, whiteflies, and other phloem-feeding insects as pests has prompted research to isolate sources of resistance to piercing-sucking insects in crops. However, little information exists about the molecular nature of plant sensitivity to phloem feeding. Recent discoveries involving elicitation by plant pathogens and chewing insects and limited studies on phloem feeders suggest that aphids are capable of inducing responses in plants broadly similar to those associated with pathogen infection and wounding. Our past work showed that compatible aphid feeding on leaves of Arabidopsis thaliana induces localized changes in levels of transcripts of genes that are also associated with infection, mechanical damage, chewing herbivory, or resource allocation shifts. We used microarray and macroarray gene expression analyses of infested plants to better define the response profile of A. thaliana to M. persicae feeding. The results suggest that genes involved in oxidative stress, calcium-dependent signaling, pathogenesis-related responses, and signaling are key components of this profile in plants infested for 72 or 96 h. The use of plant resistance to aphids in crops will benefit from a better understanding of induced responses. The establishment of links between insect elicitation, plant signaling associated with phloem feeding, and proximal resistance mechanisms is critical to further research progress in this area.

Probing plant-pathogen interactions and downstream defense signaling using DNA microarrays.

The interaction between a plant and a pathogen activates a wide variety of defense responses. The recent development of microarray-based expression profiling methods, together with the availability of genomic and/or EST (expressed sequence tag) sequence data for some plant species, has allowed significant progress in the characterization of plant pathogenesis-related responses. The small number of expression profiling studies completed to date have already identified an amazing number of genes that had not previously been implicated in plant defense. Some of these genes can be associated with defense signal transduction or antimicrobial action, but the functional contribution of many others remains uncertain. Initial expression profiling work has also revealed similarities and distinctions between different defense signaling pathways, and cross-talk (both overlap and interference) between pathogenesis-related responses and plant responses to other stresses. Potential transcriptional cis-regulatory elements upstream of co-regulated genes can also be identified. Whole-genome arrays are only now becoming available, and many interactions remain to be studied (e.g. different pathogen species, plant genotypes, mutants, time-points after infection). Expression profiling technologies, in combination with other genomic tools, will have a substantial impact on our understanding of plant-pathogen interactions and defense signaling pathways.

A novel and direct assay for measuring enzymatic depolymerisation of ?-1,3-glucans

Indirect colorimetric analyses of released reducing groups, as an indicator of induced β-1,3-glucanase activities, tend to be the assay methods of choice for the characterization of plant endo- and exo-β-1,3-glucanase. However, interfering low molecular weight reducing sugars found in unprocessed plant extracts often mask in vitro estimations of β-1,3-glucanase activity. The enzyme-catalysed hydrolysis of an optically active β-1,3-glucan polymer was monitored polarimetrically by measuring changes in the rotation of plane-polarized light of the reaction assay medium. A direct, non-radiochemical assay that detects changes in the optical rotation of released (13) β-D-oligoglucosides with low degrees of polymerisation (≪25) has been found to be highly reproducible, rapid (total analysis time 15 min), sensitive (detection limit 0.007 unit/mL glucanase), selective and non-destructive. This assay has been used to detect a salicylate-induced cell wall-bound tomato β-1,3-glucanase, which is a component of a pathogenesis-related response in plants. Copyright © 2000 John Wiley & Sons, Ltd.