Classes Of Pathogenesis-related Proteins Research Articles

Pathogenesis-Related Proteins Limit the Retention of Condensed Tannin Additions to Red Wines

Exogenous additions of condensed tannin (CT) to must or wine are a common winemaking practice, but many studies have reported inexplicably low and variable retention of added CT. We observed that additions of purified CT to red wines can result in the formation of an insoluble precipitate with high nitrogen content. Proteomic analysis of the precipitant identified several classes of pathogenesis-related proteins. Proteins in juices and red wines were quantitated by SDS-PAGE and were highest in native Vitis spp., followed by interspecific hybrids and Vitis vinifera. Wine protein was positively correlated with the ratio of juice protein to the quantity of tannin derived from fruit. The binding of added CT by wine protein could be well modeled by the Freundlich equation. These observations may explain the poor CT retention in previous studies, particularly for interspecific hybrids, and also indicate that protein removal during winemaking may improve exogenous CT retention.

Overexpression of Camellia sinensis Thaumatin-Like Protein, CsTLP in Potato Confers Enhanced Resistance to Macrophomina phaseolina and Phytophthora infestans Infection

Thaumatin-like proteins (TLPs), a class of pathogenesis related proteins are induced in response to pathogens and exhibit antifungal property when overexpressed in transgenic plants. In the present study, we have raised transgenic potato plants overexpressing TLP gene of Camellia sinensis (CsTLP). Fungal resistance assays of transgenic potato elucidated the potential role of CsTLP in imparting tolerance to fungal pathogens, Macrophomina phaseolina (necrotrophic) and Phytophthora infestans (hemi-biotrophic). Transgenic tubers with higher resistance to M. phaseolina, showed a concomitant and significant increase in transcripts of StPAL, StLOX, and StTLP genes involved in phenylpropanoid, lipoxygenase, and general defense response pathway, respectively after infection. Importantly, leaves of CsTLP transgenic lines inoculated with P. infestans spores under in vitro conditions also showed a resistant phenotype. The resistant phenotype recorded for the two important fungal pathogens by CsTLP transgenic potato plants is remarkable, since no effective control methods and no resistant cv. against M. phaseolina has been identified so far in potato.

Accumulated Evidence Substantiates a Role for Three Classes of Wheat Xylanase Inhibitors in Plant Defense

Since their discovery in 1997, work on proteinaceous wheat xylanase inhibitors (XIs) led to the identification and thorough biochemical and structural characterization of three classes of XIs, namely TAXI-type (Triticum aestivum xylanase inhibitor), XIP-type (xylanase inhibiting protein), and TLXI-type (thaumatin-like xylanase inhibitor) XIs. Already early on, a role in plant defense has been put forward for these proteins, mainly based on the observation that these XIs are only active against xylanases of microbial origin and can not inhibit the plant endogenous xylanases known so far. Considerable effort has been devoted to substantiate this plant defense hypothesis. Data at the genomic, transcriptomic and proteomic levels increasingly provide evidence that XIs, occurring as large polymorphic families, do indeed participate in plant defense. This review summarizes the current knowledge on XIs and the accumulated evidence on their role in plant defense. TLXI and XIP can be classified as pathogenesis-related (PR) proteins of classes PR-5 and PR-8, respectively, based on their homology with thaumatin-like proteins and chitinases. For TAXI, more evidence on the induction of TAXI proteins by a pathogen in tissues that normally do not express TAXI is required before it can be categorized as a new class of PR proteins. At the moment, TAXI proteins should be referred to as inducible defense-related proteins.

Identification and monitoring of Ulmus americana transcripts during in vitro interactions with the Dutch elm disease pathogen Ophiostoma novo-ulmi

Molecular mechanisms underlying the interaction leading to Dutch elm disease were studied in vitro using Ulmus americana L. callus culture inoculated with budding cells of the fungal pathogen Ophiostoma novo-ulmi (Brasier). An interaction cDNA library employing suppression subtractive hybridization was constructed from infected elm callus tissue 72 h post-inoculation. Five hundred and thirty-five expressed sequence tags, mostly from the host, were grouped into 314 unisequences and distributed into functional categories. After differential screening, 53 U. americana unisequences were considered upregulated during the interaction. The expression profiles at six time points of a subset of 18 elm transcripts were analyzed in more detail by quantitative reverse-transcriptase polymerase chain reaction. Upregulated sequences included transcripts with sequence similarity to genes coding for different classes of pathogenesis-related proteins and enzymes belonging to different branches of the phenylpropanoid pathway. The possible association with compartmentalization-related compounds and phytoalexin production is discussed. This study provides, for the first time, snapshots of molecular mechanisms involved in the compatible interaction between U. americana and O. novo-ulmi. The interaction library dataset also represents a valuable genomic resource for the highly appreciated urban tree U. americana.

Characterization of genes associated with induced resistance against Penicillium expansum in apple fruit treated with quercetin

Penicillium expansum causes blue mould, a serious postharvest disease of apples, and is the main producer of the mycotoxin patulin. Since control by synthetic fungicides is less accepted by consumers, the demand for alternative means is pressing. In a recent study, the flavonoid quercetin, although scarcely effective in in vitro assays against P. expansum growth, significantly reduced blue mould rots on ‘Golden Delicious’ apples, suggesting an enhancement of host disease resistance. To confirm or reject this hypothesis, genes differentially expressed in quercetin-treated ‘Golden Delicious’ apples were identified by suppression subtractive hybridization (SSH). A pool of 88 unique gene transcripts was obtained. Several sequences revealed high similarities with different classes of pathogenesis-related proteins (RNase-like PR10 and PR8), or with proteins expressed under stress conditions. Other transcripts had high similarity to genes of unknown function or genes coding for proteins having a role in pathogen recognition and in signalling pathways. SSH data were validated by analysing the expression of 14 genes by quantitative real-time PCR (qPCR). Eleven genes proved to be up-regulated at a medium-high level in freshly harvested apples. Among these, 5 genes selected for temporal expression profiling revealed the existence of a combined effect, particularly at 24 or 48 h, between wounding and phenolic treatment. These results provide evidence that quercetin induces resistance to P. expansum in apples, by acting on the transcription level of genes involved in several distinct metabolic processes.

Isolation of a thermostable legume chitinase and study on the antifungal activity

Chitinases are listed as one class of pathogenesis-related proteins, and they have become a popular research topic because of their resistance to plant-pathogenic diseases. A chitinase with antifungal activity was isolated from the Canadian cranberry beans (Phaseolus vulgaris). The procedure included extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, CM-Sephadex C-50, and Sephadex G-75. There was an almost 108-fold increase in specific activity of the purified chitinase compared with that of the crude extract. The enzyme exhibited a molecular mass of 30.6 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and non-reducing conditions, indicating that it was a monomeric protein. The pI was determined to be 7.6 by isoelectric-focusing electrophoresis. The optimum pH and the optimum temperature for activity towards N-acetyl-glucosamine was 5.4 and 40-55 degrees C, respectively. It exerted a potent inhibitory action toward fungal species including Botrytis cinerea, Physalospora piricola, Fusarium oxysporum, and Pythium aphanidermatum. The chitinase was thermostable up to 58 degrees C in both enzymatic reaction and antifungal activity. The present findings demonstrated a thermostable chitinase from cranberry beans with potentially exploitable significance.

PURIFICATION AND CHARACTERIZATION OF A CHITINASE FROM PEANUT (ARACHIS HYPOGAEA L.)

ABSTRACT A chitinase was isolated from peanut (Arachis hypogaea L.) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, and high-performance liquid chromatography on POROS 20 HQ. There was a 133-fold increase in specific activity of purified chitinase compared with that of the crude extract. The protein exhibited a molecular mass of 34.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and nonreducing conditions, indicating that it is a monomeric protein. The isoelectric point was 5.1 by isoelectric focusing electrophoresis. Optimal pH activity was 5.4 and optimal temperature was 40–50C. The enzyme was stable below 55C, but was rapidly inactivated when incubated at temperatures above 60C. These results demonstrated that the purified protein was a kind of relatively thermostable chitinase from the peanut seeds. PRACTICAL APPLICATIONS This paper describes the isolation and characterization of a chitinase from Arachis hypogaea L. seeds. Chitinases are listed as the class of pathogenesis-related proteins, and they have become a popular research field because of their resistance to plant disease. The research we are presenting should help in understanding chitinase functions in plant defense mechanisms. A relatively thermostable enzyme newly obtained, which was stable below 55C, would certainly be significant for its utilization both in enzymology and in plant defense. In particular, a relatively heat-resistant enzyme from A. hypogaea L. seeds would certainly be helpful for elaborating strategies for constitutive expression of chitinase genes and their manipulation and general utilization in plant defense mechanisms through more recent techniques of genetic manipulation.

Identification of differentially expressed genes in Malus domestica after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere

Biological control of plant diseases by the application of antagonistic micro-organisms to the plant phyllosphere is only marginally understood. Suppression subtractive hybridization (SSH) was used for the identification of genes expressed after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere of the apple scab-susceptible cultivar Malus domestica cv. Holsteiner Cox. In total, 157 expressed sequence tag (EST) clones were obtained. The sequencing of 113 ESTs which have a significantly elevated transcript level and the comparison of the obtained sequences with databases revealed similarities to different classes of pathogenesis-related proteins, for example, RNase-like PR10 protein and endochitinase, or similarities to proteins expressed under stress conditions that could have a protective function, for example, a germin-like protein, glutathione S-transferase, thioredoxin-like proteins, and heat shock proteins. In addition, several transcripts were identified that code for proteins which have a crucial role at different stages of pathogen recognition and in signalling pathways or an as yet unknown function in plant defence. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated after pathogen infection are also up-regulated after the application of a non-pathogenic bacterium to a M. domestica cultivar. The expression of these proteins might increase the plant resistance towards pathogen infection and damage.

A simplified procedure for the subtractive cDNA cloning of photoassimilate-responding genes: isolation of cDNAs encoding a new class of pathogenesis-related proteins.

Transgenic tobacco plants (ppa-1) constitutively expressing Escherichia coli pyrophosphatase behind the 35S CaMV promoter accumulate high levels of soluble sugars in their leaves [27]. These plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level. By differential hybridization of a subtractive library enriched for transcripts present in the transgenic plants 12 different cDNAs were isolated. By sequence analysis four cDNAs could be identified as 1-aminocyclopropane-1-carboxylate-oxidase and as three different pathogenesis-related proteins (PR-1b, PR-Q and SAR 8.2). Two cDNAs were homologous to a calmodulin-like protein from Arabidopsis and a human ribosomal protein L19 while six cDNA clones remained unknown. One of these clones (termed PAR-1 for photoassimilate-responsive) displayed features similar to pathogenesis-related proteins: Hybridizing transcripts, 1.2 and 1.0 kb in length, were strongly inducible by salicylate and accumulated in tobacco plants after infection with potato virus Y (PVY) both in infected and uninfected systemic leaves. PAR-1 transcripts also accumulated in wildtype leaves upon floating on glucose and sucrose whereas sorbitol and polyethylene glycol had no effect. Rescreening of the ppa-1 cDNA library with the PAR-1 cDNA as probe resulted in 25 hybridizing cDNAs which by homology were found to fall into three classes (PAR-1a, b, c). The cDNAs coding for PAR-1a and b were 90.6% homologous on the DNA level while both were less related to the PAR-1c cDNA (70.5% and 75.2% homologous, respectively). One open reading frame was identified in all three PAR-1 cDNA classes. Translation would result in proteins with a theoretical molecular mass of about 20 kDa. The N-terminal amino acid sequences resemble a signal peptide which would direct the proteins to the secretory pathway. Using selective 3′ hybridization probes of the three PAR-1 cDNAs it was possible to discriminate the different transcripts. Both PAR-1a and PAR-1c mRNAs are induced in plants treated with PVY.

Antifreeze Proteins in Winter Rye Are Similar to Pathogenesis-Related Proteins

The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.

CDNA cloning, structure and expression of a novel pathogenesis-related protein in bean.

We have isolated cDNA clones spanning the full length of a transcript (PvPR3) encoding a novel pathogenesis-related (PR) protein in bean (Phaseolus vulgaris). The PvPR3 transcript accumulates gradually over 24 hr in elicitor-treated cell suspensions. This pattern of expression is distinct from those of previously reported elicitor-induced transcripts in bean. Specifically, transcripts encoding two recently described acidic bean PR proteins, phenylpropanoid pathway enzymes, accumulate to maximal levels by 4-8 hr, while hydroxyproline-rich glycoprotein mRNA accumulation is delayed by several hours. The PvPR3 mRNA also accumulates after wounding of hypocotyls with kinetics comparable to those of mRNA encoding phenylpropanoid pathway mRNAs. PvPR3 appears to exist as a single gene within a family of approximately 15 related genes in the bean genome. The PvPR3 protein deduced from the cDNA sequences (14,950 Da pI = 10.0) lacks a putative signal peptide suggesting a cytosolic localization. Amino acid sequence comparisons with databases revealed that PvPR3 represents a new class of PR proteins without significant sequence homology to previously characterized PR proteins or other proteins.