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International audienceIt is well-known that standard predictive techniques are not very robust to parameter uncertainties and to external disturbances. Furthermore, they require the exact knowledge of the delay. In practice, these constraints are rarely satisfied. In this paper, solutions are presented to allow the use of predictive control in presence of external disturbances, parameter uncertainties and an unknown input delay. First, a recent predictive control method developed to attenuate the effect of external disturbances is shown to be also robust to parameter uncertainties. In addition, a delay estimator is presented to estimate unknown time-varying delays. Theoretical results are widely illustrated through experimental tests on a DC motor

BackgroundO-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae) in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk).ResultsBy comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length.ConclusionsAbout one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

The broad-range phytopathogenic fungus Botrytis cinerea secretes hundreds of proteins during infection of its plant hosts. One of these proteins, BcIEB1, is abundantly secreted and is able to elicit plant defenses, probably as a pathogen-associated molecular pattern, although its native function in B. cinerea biology remains unknown. Pull-down experiments designed to isolate the molecular target of BcIEB1 in tobacco resulted in the identification of osmotin, a pathogenesis-related protein of family 5 that shows antifungal activity. The expression of osmotin in Escherichia coli allowed the verification of the BcIEB1-osmotin interaction with pure proteins by pull-down and far Western blot experiments, as well as the confirmation of the activity of osmotin against B. cinerea. Interestingly, B. cinerea Δbcieb1 mutants are more susceptible than the wild-type to osmotin, and the external addition of pure BcIEB1 protects the Δbcieb1 mutants, as well as Saccharomyces cerevisiae, from the antifungal action of osmotin, thus pointing at PR5 inhibition as the primary native function of BcIEB1. The question of whether osmotin is also involved in the activation of plant defenses by BcIEB1 is also addressed, and the data suggest that osmotin does not participate in the elicitation process.

Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea.

Plants activate defense responses against a possible pathogen once pattern-recognition receptors (PRRs) perceive the presence of pathogen-associated molecular patterns (PAMPs). Glycosyl hydrolase family 11 (GH11) endoxylanases from Trichoderma, Fusarium and Botrytis species have been described as being able to induce the defense response in plants, in a way that is independent of its enzymatic activity. However, until now, it has not been possible to establish with certainty which regions of these enzymes are recognized by plants as PAMPs. We show here for the first time that a short 25-residue peptide (named Xyn25) from the Botrytis cinerea xylanase BcXyn11A can reproduce by itself all the effects observed for the treatment of plants with the whole BcXyn11A protein. These include necrosis on leaves, seedling growth inhibition, induction of a ROS burst, electrolyte leakage, cytoplasm shrinkage, autofluorescence, cell death, and induction of defense genes. Two highly conserved four-amino acid regions within Xyn25 were shown to be necessary for the elicitation activity by substituting them with tracts of four alanine residues.

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Cerato-platanin family proteins are secreted and have been found in both the fungal cell wall and the extracellular medium. They elicit defence responses in a variety of plants and have been proposed to be perceived as pathogen-associated molecular patterns (PAMPs) by the plant immune system, although, in the case of the necrotroph Botrytis cinerea, the cerato-platanin BcSpl1 contributes to fungal virulence instead of plant resistance. In this study, we report that BcSpl1, which was previously found in the secretome as an abundant protein, is even more abundant in the fungal cell wall. By fusion to green fluorescent protein (GFP), we also show that BcSpl1 associates with the plant plasma membrane causing rapid morphological changes at the cellular level, such as the disorganization of chloroplasts, prior to macroscopic necrosis in the treated tissue. By a combination of serial deletion studies, synthetic peptides and chimeric proteins, we mapped the eliciting activity to a two-peptide motif in the protein surface. The expression of a chimeric protein displaying this motif in B. cinerea mutants lacking BcSpl1 undoubtedly showed that the motif is responsible for the contribution of BcSpl1 to virulence.