Bernard Fritig

Plant ‘pathogenesis-related’ proteins and their role in defense against pathogens

The hypersensitive reaction to a pathogen is one of the most efficient defense mechanisms in nature and leads to the induction of numerous plant genes encoding defense proteins. These proteins include: 1) structural proteins that are incorporated into the extracellular matrix and participate in the confinement of the pathogen; 2) enzymes of secondary metabolism, for instance those of the biosynthesis of plant antibiotics; 3) pathogenesis-related (PR) proteins which represent major quantitative changes in soluble protein during the defense response. The PRs have typical physicochemical properties that enable them to resist to acidic pH and proteolytic cleavage and thus survive in the harsh environments where they occur: vacuolar compartment or cell wall or intercellular spaces. Since the discovery of the first PRs in tobacco many other similar proteins have been isolated from tobacco but also from other plant species, including dicots and monocots, the widest range being characterized from hypersensitively reacting tobacco. Based first on serological properties and later on sequence data, the tobacco PRs have been classified in five major groups. Group PR-1 contains the first discovered PRs of 15-17 kDa molecular mass, whose biological activity is still unknown, but some members have been shown recently to have antifungal activity. Group PR-2 contains three structurally distinct classes of 1,3-beta-glucanases, with acidic and basic counterparts, with dramatically different specific activity towards linear 1,3-beta-glucans and with different substrate specificity. Group PR-3 consists of various chitinases-lysozymes that belong to three distinct classes, are vacuolar or extracellular, and exhibit differential chitinase and lysozyme activities. Some of them, either alone or in combination with 1,3-beta-glucanases, have been shown to be antifungal in vitro and in vivo (transgenic plants), probably by hydrolysing their substrates as structural components in the fungal cell wall. Group PR-4 is the less studied, and in tobacco contains four members of 13-14.5 kDa of unknown activity and function. Group PR-5 contains acidic-neutral and very basic members with extracellular and vacuolar localization, respectively, and all members show sequence similarity to the sweet-tasting protein thaumatin. Several members of the PR-5 group from tobacco and other plant species were shown to display significant in vitro activity of inhibiting hyphal growth or spore germination of various fungi probably by a membrane permeabilizing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological function of `pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-β-glucanase activity

Three of the ten acidic ‘pathogenesis‐related’ (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely −O, −N and −2, have been shown to have 1,3‐β‐glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross‐reacted with a basic protein which is also a 1,3‐β‐glucanase induced by virus infection and which can be considered as a new basic pathogenesis‐related protein of tobacco. Protein PR‐O and the basic 1,3‐β‐glucanase display about the same specific enzymatic activity, i.e. 50‐fold and 250‐fold higher than specific activities of proteins PR‐N and −2 respectively.

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoo-spore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.

Antimicrobial proteins in induced plant defense

During the past few years a wide spectrum of plant antimicrobial proteins has been detailed, and enhanced resistance has been obtained by introducing the corresponding genes into crop species to produce transgenic lines. With the aim of manipulating the plant signals that regulate an array of defense responses, the most intense research focuses on the avr-R-mediated recognition events and elucidation of the subsequent signaling pathways that govern the activation of genes encoding antimicrobial proteins.

Thaumatin-like pathogenesis-related proteins are antifungal

Tobacco pathogenesis-related (PR) proteins of group 5, namely PR-S and osmotin, were shown to be serologically related to zeamatin, an antifungal protein of maize seeds. These PR proteins, dubbed thaumatin-like because they show sequence homology with thaumatin, a sweet-tasting protein from fruits of Thaumatococcus daniellii, were demonstrated to have a direct antifungal activity, with specificity for different fungal species. Osmotin was particularly effective in inhibiting the growth of Candida albicans, Neurospora crassa and Trichoderma reesei. PR-S had no detectable activity against these fungi but was found the most potent antifungal protein against the plant pathogen Cercospora beticola. Osmotin caused rapid bursting of the hyphal tips of N. crassa, suggesting that tobacco thaumatin-like PR proteins are antifungal by a membrane permeabilization mechanism similar to that demonstrated previously for zeamatin. These results confirm and extend observations made by Woloshuk et al. [1] who showed that osmotin and a related protein from tomato had antifungal activity against the phytopathogen, Phytophthora infestans.

Downregulation of a Pathogen-Responsive Tobacco UDP-Glc:Phenylpropanoid Glucosyltransferase Reduces Scopoletin Glucoside Accumulation, Enhances Oxidative Stress, and Weakens Virus Resistance

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid– and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.

Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways

Summary:  In their environment, plants interact with a multitude of living organisms and have to cope with a large variety of aggressions of biotic or abiotic origin. To survive, plants have acquired, during evolution, complex mechanisms to detect their aggressors and defend themselves. Receptors and signaling pathways that are involved in such interactions with the environment are just beginning to be uncovered. What has been known for several decades is the extraordinary variety of chemical compounds the plants are capable to synthesize, and many of these products are implicated in defense responses. The number of natural products occurring in plants may be estimated in the range of hundreds of thousands, but only a fraction have been fully characterized. Despite the great importance of these metabolites for plant and also for human health, our knowledge about their biosynthetic pathways and functions is still fragmentary. Recent progress has been made particularly for phenylpropanoid and oxylipin metabolism, which are emphasized in this review. Both pathways are involved in plant resistance at several levels: by providing building units of physical barriers against pathogen invasion, by synthesizing an array of antibiotic compounds, and by producing signals implicated in the mounting of plant resistance.

A revised nomenclature for chitinase genes

The nomenclature for chitinase genes has been revised to correspond to the nomenclature of PR-proteins and to distinguish classes from families. Accordingly, there are now four families of chitinases, two of which are further divided in classes.

Mechanisms of Plant Defense Responses

Section 1: Genes for Incompatibility between Plants and Pathogens. Section 2: Pathogenicity Genes and Signals for Establishment of Infection. Section 3: Elicitors and Signal Transduction. Section 4: Plant Defence Response Genes and Signals for Their Regulation. Section 5: Results and Perspectives of Biotechnological Applications. Section 6: Other Aspects of Plant-Pathogen Interactions.

Phenylalanine Ammonia-Lyase in Tobacco (Molecular Cloning and Gene Expression during the Hypersensitive Reaction to Tobacco Mosaic Virus and the Response to a Fungal Elicitor)

A tobacco (Nicotiana tabacum L. cv Samsun NN) cDNA clone coding the enzyme phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library made from polyadenylated RNA purified from tobacco mosaic virus (TMV)-infected leaves. Southern analysis indicated that, in tobacco, PAL is encoded by a small family of two to four unclustered genes. Northern analysis showed that PAL genes are weakly expressed under normal physiological conditions, they are moderately and transiently expressed after wounding, but they are strongly induced during the hypersensitive reaction to TMV or to a fungal elicitor. Ribonuclease protection experiments confirmed this evidence and showed the occurrence of two highly homologous PAL messengers originating from a single gene or from two tightly co-regulated genes. By in situ RNA-RNA hybridization PAL transcripts were shown to accumulate in a narrow zone of leaf tissue surrounding necrotic lesions caused by TMV infection or treatment with the fungal elicitor. In this zone, no cell specificity was observed and there was a decreasing gradient of labeling from the edge of necrosis. Some labeling was also found in various cell types of young, healthy stems and was shown to accumulate in large amounts in the same cell types after the deposition of an elicitor solution at the top of the decapitated plant.