Fabienne Baillieul

Study of defense-related gene expression in grapevine leaves and berries infected with Botrytis cinerea ∗

Defense responses of grapevine towards Botrytis cinerea were investigated. The expression of genes coding for proteins involved in defense were studied: (a) phenylalanine ammonia-lyase (PAL) and stilbene synthase (StSy), (b) an acidic chitinase (VCH3) and a basic chitinase (VCHIT1b), and (c) a polygalacturonase inhibitor protein (PGIP). Since no PGIP was known in grapevine, a complete cDNA sequence was first characterized by PCR and RACE-PCR amplifications. RNAs isolated from infected leaves and infected berries were analysed by semi-quantitative and real-time RT-PCRs. In infected leaves, the expression of PAL, StSy, PGIP and VCH3 genes occurred 6 hours post inoculation (hpi). Increase of VCHIT1b gene expression was delayed (24 hpi). Maximum levels of induction of these genes were observed at 48 hpi, except for the VCH3 gene (24 hpi). Activation of these defense responses was not sufficient to stop B. cinerea spread. In berries, no VCH3 gene expression was detected. Maximum levels of induction were observed in stage 3 (loss of berry colour and abundant production of conidia) for the PAL and PGIP genes, and in stage 4 (shrivelled berry) for the StSy and VCHIT1b genes.

Spatial and Temporal Induction of Cell Death, Defense Genes, and Accumulation of Salicylic Acid in Tobacco Leaves Reacting Hypersensitively to a Fungal Glycoprotein Elicitor

We have analyzed the spatial and temporal expression of defense responses induced in attached tobacco leaves treated with a Phytophthora megasperma glycoprotein that was previously shown to be an efficient elicitor of the hypersensitive reaction. The infiltrated tissue (zone 1), the surrounding tissue (zone 2), which is 5 mm in width, and tissue at a distance >2 cm from zone 2 (zone 3) were analyzed separately. Cell death occurred only in zone 1 and was completed by 14 h. Defense gene expression was induced in zones 1 and 2 with striking differential patterns, but not in zone 3. There was a rapid (1 to 4 h) induction of genes of the phenylpropanoid, salicylic acid, and ses-quiterpenoid pathways in zones 1 and 2. However, it was strong and transient in the former and of lesser extent but sustained in the latter. High amounts of scopoletin, a phenylpropanoid metabolite, were found synthesized in zone 2. Pathogenesis-related (PR) transcripts and the corresponding PR proteins accumulated in high amounts in zo...

Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine.

Rhamnolipids produced by the bacteria Pseudomonas aeruginosa are known as very efficient biosurfactant molecules. They are used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants. In this paper, the role of rhamnolipids as novel molecules triggering defence responses and protection against the fungus Botrytis cinerea in grapevine is presented. The effect of rhamnolipids was assessed in grapevine using cell suspension cultures and vitro-plantlets. Ca(2+) influx, mitogen-activated protein kinase activation and reactive oxygen species production form part of early signalling events leading from perception of rhamnolipids to the induction of plant defences that include expression of a wide range of defence genes and a hypersensitive response (HR)-like response. In addition, rhamnolipids potentiated defence responses induced by the chitosan elicitor and by the culture filtrate of B. cinerea. We also demonstrated that rhamnolipids have direct antifungal properties by inhibiting spore germination and mycelium growth of B. cinerea. Ultimately, rhamnolipids efficiently protected grapevine against the fungus. We propose that rhamnolipids are acting as microbe-associated molecular patterns (MAMPs) in grapevine and that the combination of rhamnolipid effects could participate in grapevine protection against grey mould disease.

Tobacco Class I and II Catalases Are Differentially Expressed During Elicitor-Induced Hypersensitive Cell Death and Localized Acquired Resistance

Expression of tobacco class I (CAT1) and class II (CAT2) catalases was analyzed in leaves reacting hypersensitively to tobacco mosaic virus (TMV) or to a fungal glycoprotein elicitor. In TMV-infected plants, Cat1 transcript levels declined rapidly while Cat2 transcripts accumulated strongly. The spatial and temporal changes in catalase transcripts, proteins, and activity during the hypersensitive reaction (HR) were further investigated in tobacco leaves infiltrated with a glycoprotein elicitor. Two functionally different zones were discriminated: the infiltrated tissue in which cells undergo the HR, called the HR-zone 1; and the surrounding tissue showing strong induced defense responses, called the LAR (Localized Acquired Resistance)- zone 2. Levels of Cat1 and Cat2 mRNA and proteins and catalase activity decreased in the HR-zone 1. In the LAR-zone 2, Cat1 transcripts became rapidly undetectable, but levels of Cat2 mRNA and protein and catalase activity increased. Catalase expression in elicitorinfiltrat...

RHAMNOLIPID BIOSURFACTANTS AS NEW PLAYERS IN ANIMAL AND PLANT DEFENSE AGAINST MICROBES

Rhamnolipids are known as very efficient biosurfactant molecules. They are used in a wide range of industrial applications including food, cosmetics, pharmaceutical formulations and bioremediation of pollutants. The present review provides an overview of the effect of rhamnolipids in animal and plant defense responses. We describe the current knowledge on the stimulation of plant and animal immunity by these molecules, as well as on their direct antimicrobial properties. Given their ecological acceptance owing to their low toxicity and biodegradability, rhamnolipids have the potential to be useful molecules in medicine and to be part of alternative strategies in order to reduce or replace pesticides in agriculture.

Comparative analysis of defence responses induced by the endophytic plant growth-promoting rhizobacterium Burkholderia phytofirmans strain PsJN and the non-host bacterium Pseudomonas syringae pv. pisi in grapevine cell suspensions

Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that colonize the rhizosphere of many plant species and confer beneficial effects, such as an increase in plant growth. PGPR are also well known as inducers of systemic resistance to pathogens in plants. However, the molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain largely unknown. Burkholderia phytofirmans strain PsJN is an endophytic PGPR that colonizes grapevine and protects the plant against the grey mould disease caused by Botrytis cinerea. This report focuses on local defence events induced by B. phytofirmans PsJN after perception by the grapevine cells. It is demonstrated that, after addition to cell suspension cultures, the bacteria were tightly attaching to plant cells in a way similar to the grapevine non-host bacteria Pseudomonas syringae pv. pisi. B. phytofirmans PsJN perception led to a transient and monophasic extracellular alkalinization but no accumulation of reactive oxygen species or cell death were detected. By contrast, challenge with P. syringae pv. pisi induced a sustained and biphasic extracellular alkalinization, a two phases oxidative burst, and a HR-like response. Perception of the PGPR also led to the production of salicylic acid (SA) and the expression of a battery of defence genes that was, however, weaker in intensity compared with defence gene expression triggered by the non-host bacteria. Some defence genes up-regulated after B. phytofirmans PsJN challenge are specifically induced by exogenous treatment with SA or jasmonic acid, suggesting that both signalling pathways are activated by the PGPR in grapevine.

Differential Induction of Grapevine Defenses by Two Strains of Botrytis cinerea

ABSTRACT Even though Botrytis cinerea, the causal agent of gray mold, is a highly variable fungus with strains displaying very different degrees of virulence toward one given host plant species, no study has yet shown any correlation between the lack of aggressiveness of one given strain and its ability to stimulate a defense response from its host. Strains of B. cinerea collected from different host plant species were screened for their pathogenicity on grapevine to select two strains with similar morphological characteristics but different levels of virulence. In grapevine leaves, the less aggressive strain, T4, enhanced the accumulation of many defense products including secondary metabolites and the pathogenesis-related proteins, chitinase and beta-1,3-glucanase. Interestingly, secondary metabolites were formed in cells around a small group of dead cells. When compared with T4, the more aggressive strain, T8, had larger necrotic spots, no secondary metabolite biosynthesis, and accumulations of chitinases and beta-1,3-glucanases that were more delayed, yet only slightly weaker. The culture fluids of both strains mimicked the differential effect of each isolate in stimulating chitinase activity when infiltrated into grapevine leaves.

Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.

Plant pathogens have evolved by developing different strategies to infect their host, which in turn have elaborated immune responses to counter the pathogen invasion. The apoplast, including the cell wall and extracellular space outside the plasma membrane, is one of the first compartments where pathogen-host interaction occurs. The plant cell wall is composed of a complex network of polysaccharides polymers and glycoproteins and serves as a natural physical barrier against pathogen invasion. The apoplastic fluid, circulating through the cell wall and intercellular spaces, provides a means for delivering molecules and facilitating intercellular communications. Some plant-pathogen interactions lead to plant cell wall degradation allowing pathogens to penetrate into the cells. In turn, the plant immune system recognizes microbial- or damage-associated molecular patterns (MAMPs or DAMPs) and initiates a set of basal immune responses, including the strengthening of the plant cell wall. The establishment of defense requires the regulation of a wide variety of proteins that are involved at different levels, from receptor perception of the pathogen via signaling mechanisms to the strengthening of the cell wall or degradation of the pathogen itself. A fine regulation of apoplastic proteins is therefore essential for rapid and effective pathogen perception and for maintaining cell wall integrity. This review aims to provide insight into analyses using proteomic approaches of the apoplast to highlight the modulation of the apoplastic protein patterns during pathogen infection and to unravel the key players involved in plant-pathogen interaction.

Burkholderia phytofirmans PsJN Primes Vitis vinifera L. and Confers a Better Tolerance to Low Nonfreezing Temperatures

Several endophytic bacteria reportedly induce resistance to biotic stress and abiotic stress tolerance in several plant species. Burkholderia phytofirmans PsJN is a plant-growth-promoting rhizobacterium (PGPR) that is able to colonize grapevine tissues and induce resistance to gray mold. Further, PsJN induces physiological changes that increase grapevine tolerance to low nonfreezing temperatures. To better understand how bacteria induced the observed phenomena, stress-related gene expression and metabolite accumulation were monitored in 6-week-old Chardonnay grapevine plantlets after exposure to low nonfreezing temperatures. Under normal conditions (26°C), plantlet bacterization had no significant effect on the monitored parameters. By contrast, at 4°C, both stress-related gene transcripts and metabolite levels increased earlier and faster, and reached higher levels in PsJN-bacterized plantlets than in nonbacterized counterparts, in accordance with priming phenomena. The recorded changes may be correlated with the tolerance to cold stress conferred by the presence of PsJN. This is the first time that PGPR-induced priming has been shown to protect plants against low-temperature stress. Moreover, 1 week after cold exposure, levels of stress-related metabolites had declined more in PsJN-bacterized plants, suggesting that the endophyte is involved in the cold acclimation process via the scavenging system.

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.