Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this fermtin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.Cuticle constitutes the first barrier between a higher plant and its environment. The mode of penetration of this barrier by phytopathogenic fungi has been a controversial subject for a long time (4). Penetration is either by the mere physical force of fungal growth or by enzymic dissolution of the cuticle. The nature of the opening created by the penetrating fungus has been used as a supporting evidence for the enzymic penetration theory (5). However, in the absence of convincing evidence to support either side, the arguments remained unsettled. The structural component of plant cuticle is a biopolyester, cutin, derived from hydroxy and epoxy fatty acids (3) and therefore this polymer constitutes the major physical barrier between the invading fungus and the plant. Recently we have isolated in homogeneous form cutinases from the extracellular fluid of several fungal pathogens grown on cutin as the sole source of carbon (6, 7). We were able to prepare antibodies against these pure enzymes and using ferritin-labeled antibody a specific test was performed to determine whether a fungal pathogen excretes cutinase during the cuticular penetration phase of the infection process. The results presented in this communication provide convincing evidence that Fusarium solani f. pisi excretes cutinase during infection of the host tissue. MATERIALS AND METHODSCutinase-I was isolated in homogeneous form from the extracellular fluid of F. solani f. pisi grown on apple cutin as the sole source of carbon (6). Rabbit antiserum was prepared against the pure cutinase (7) and the immunoglobulin fraction was isolated from this antiserum by repeated ammonium sulfate fractionation (2). Ferritin was conjugated to the immunoglobulin using xylylene metadiisocyanate as the coupling agent (1 Epicotyls from 5-day-old etiolated Pisum sativum seedlings were excised and rinsed in distilled H20 several times. The cuticular surface of these epicotyl segments, 0.5 cm long, was inoculated with small (about 40-,ul) droplets of a thick suspension of micro-and macroconidia of F. solani f. pisi from 15-to 20-day-old cultures grown on 2% potato dextrose agar. The inoculated segments of epicotyl were placed in a Petri dish in humid atmosphere. After varying periods of incubation at room temperature the infected segments were immersed in the ferritin-conjugated anticutinase-I preparation for 30 min. Controls were similarly treated with nonconjugated ferritin or ferritinconjugated immunoglobulin fraction prepared from a rabbit which was not immunized with cutinase-I. The incubated specimens were rinsed in 0.1 M Na-phosphate buffer (pH 7.2) for 30 min and fix...

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The growth-related parameters of Thiobacillus intermedius, cultured in glutamate-CO.2-S20O2medium, have been determined. After centrifugation at 48,000 x g for 1 h, 24% of the n-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity ofthe disrupted-cell suspensions obtained from C02-S2032-and glutamate-CO.,-S.,203-grown cells could be sedimented, and the specific activities of this enzyme in the supernatant fractions were almost equivalent. The enzyme was stable in T. intermedius starved of thiosulfate in the presence and absence of glutamate, but a progressive decrease was evident in several growth cycles, each cycle supported by resupplementation of cells with thiosulfate. Polyhedral inclusion bodies were present in CO.._S.,O:2-and glutamate-C02-S 0:1-grown cells. The number of polyhedral bodies per cell increased during mixotrophic growth approximately in proportion to the observed increase in the specific activity of RuBPCase. RuBPCase could not be detected in T. intermedius grown heterotrophically on yeast extract, nor could polyhedral bodies be found.