When transformation of Botrytis cinerea occurred in mononucleated protoplasts the hygromycin resistance phenotype was stable and integrated plasmid DNA although rearranged was transmitted through meiosis. We observed that transformants were often heterokaryotic and using serial conidial transfer, we showed failure of expression of the entire copies of integrated plasmids in some conidial isolates. A non-Mendelian segregation of the hygromycin resistance phenotype was observed in most crosses between these transformants and sensitive strains. However, a 1:1 segregation ratio of plasmid DNA hybridisation was observed. Mechanisms of gene silencing in B. cinerea, in both the asexual and the sexual cycle, are discussed.

We describe a strategy for systematic amplification of chitin synthase genes (chs) in the filamentous ascomycetes plant-pathogen Botrytis cinerea using PCR with multiple degenerate primers designed on specific and conserved sequence motifs. Eight distinct chs genes were isolated, named Bcchs I, II, IIIa, IIIb, IV, V, VI and VII. They probably constitute the entire chs multigenic family of this fungus, as revealed by careful analysis of six euascomycetes genomes. Bcchs I, IIIa, IIIb, IV and VI genes were subjected to DNA walking and their deduced amino acid sequences were compared by hydrophobic cluster analysis (HCA) to localize putative residues critical for CHS activity. HCA also enabled us to highlight three different transmembrane topologies of the CHS membranous isoenzymes. We found that the N-terminal region of the BcCHSI isoenzyme, and its orthologues in other euascomycetes, probably contain folded peptide motifs with conserved tyrosine residues. Their putative role is discussed. The BcCHSVII isoenzyme appeared to belong to a new class of CHS orthologues that was demonstrated by phylogenetic study to branch apart from division 1 and 2 of CHS.

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.

Rishitin accumulation as well as its biosynthesis from added [2-14C]MVA was observed in tomato cell suspension cultures elicited by yeast extract.

The qualitative and quantitative influence of 3mM phosphonate on the lipid classes and component fatty acids was studied in Phytophthora capsici. The treatment increased the phospholipid content of the mycelium while the concentration of neutral lipids, and particularly TAG, was lowered. The distribution of fatty acids in neutral and polar lipids was affected: the proportion of saturated C16, C18 and polyunsaturated 18:3n-6 increased; on the contrary, the polyunsaturated C20 fatty acids concentration decreased suggesting alterations of the fatty acid desaturation processes. The minor alternate pathway leading to a biosynthesis of 20:5n-3, observed after addition of labelled 18:3n-3, was totally inhibited after phosphonate treatment.

The effects of infection by Phytophthora capsici on the repartition and fatty acid composition of polar lipids in three different foliar tissues of Lycopersicon esculentum (May seedlings) were investigated. The tissues studied were: (i) highly infected tissues, under explant, 6 mm diameter; (ii) infected tissues, margin of the lesions, 9 mm width; and (iii) adjacent peripheral tissues, about 52% total area, apparently sound, compared with uninfected control leaflets. The fatty acid composition of each lipid class was drastically altered in the highly infected area. The desaturation pathway of polyunsaturated fatty acids, specific to plants, was either partly inhibited for glycoglycerolipids, or replaced by the desaturation pathway for the formation of parasite phospholipids. Consequently, the long chain fatty acids characterizing P. capsici increased progressively from peripheral to highly infected tissues and the phosphatidylcholine (PC) of the plant was replaced by the PC of the parasite. The increase in the percentage of parasite PC could be used as an infection index. The amounts of all polar lipid classes were increased in the peripheral zone, except phosphatidic acid. A possible distance effect of infection is enhancement of membrane biosynthesis. Phosphonate treatment was found to favour the synthesis of all polar lipid classes in the control leaflet but to be inactive in preserving the integrity of the fatty acid composition of the lipid classes in infected tissues.

Le dosage autoradiographique de l'acide phosphorique montre l'accumulation de ce produit au voisinage des hyphes du pathogene. Cette substance pourrait etre liee a des mecanismes de defense des plants