Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues.

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.

Analysis of the beta-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum.

The biocontrol agent Trichoderma harzianum IMI206040 secretes beta-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of beta-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular beta-1,3-glucanases upon induction with laminarin, a soluble beta-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible beta-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-beta-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.

Subcellular localization, abundance and stability of chitin synthetases 1 and 2 from Saccharomyces cerevisiae.

The existence of more than one chitin synthetase in fungal cells poses the question of whether these enzymes have similar or different localization. The subcellular distribution of chitin synthetases 1 and 2 (Chs1 and Chs2) was determined in cell-free extracts of Saccharomyces cerevisiae fractionated by sucrose density gradient sedimentation. Chs1 was examined in two strains: ATCC 26109, a wild-type strain, and D3C (MAT alpha ura3-52). Chs2 was investigated in a strain (D3B) freed of Chs1 by gene disruption (MATa his4 ura3-52 chs1::URA3). A prolonged, strong centrifugation (20 h at 265000 g) was necessary to cleanly resolve two major populations of chitin synthetase particles: chitosomes (a population of microvesicles of low buoyant density, d = 1.15 g ml-1) and plasma membrane (a population of vesicles of high buoyant density, d = 1.21 g ml-1). Chs1 and Chs2 were both present in chitosomes and plasma membrane, but the relative distribution of each chitin synthetase in these two membranous populations varied. Chs2 was much less abundant than Chs1 and required Co2+ rather than Mg2+ as a cofactor. A salient finding was the high sensitivity of chitosomal Chs2 to high centrifugal forces. The subcellular distribution of 1,3-beta-glucan synthetase was the same in the three strains studied, i.e. unaffected by the presence or absence of Chs1. Culture conditions affected the profiles of chitin and glucan synthetases: the relative abundance of Chs1 in chitosomes or plasma membrane was quite different in cells grown on two different media but the buoyant density was not affected; in contrast, there was shift in the buoyant density of the two peaks of 1,3-beta-glucan synthetase. We concluded that the subcellular localization of Chs1 and Chs2 remains the same despite genetic and other differences in the properties of these enzymes. We confirmed that 1,3-beta-glucan synthetase and chitin synthetase exhibit a partially different subcellular distribution-an indication that these two enzymes are mobilized through different secretory pathways.

Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane.

We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.