| Summary: | Verticillium chlamydosporium is a fungal pathogen of eggs and females of plantparasitic nematodes. The fungus produced an alkaline serine protease in submerged culture. This enzyme, VCPI, was characterized as a class II subtilisin, based on amino acid sequenceh omology. Several of its characteristics, e.g. molecular mass (33 kDa), pI (ca 10) and broad substrate utilisation, are typical of fungal subtilisins. Although some immunological cross-reactivity existed with other enzymes of this class, an antigenic fingerprint was obtained that was distinct, even from the subtilisin that was its closest homologue based on amino acid sequence, PrI from the entomogenous fungus Metarhizium anisopliae. There was circumstantial evidence, suggesting that this fungal protease was involved in the infection of nematode eggs, which have a largely proteinaceous eggshell. First of all, the enzyme was able to remove the outer protein layer from eggs of the susceptible root-knot nematode, Meloidogyne incognita, exposing the underlying chitin layer. Scanning electron microscopy revealed that fungal hyphae on the egg surface left an imprint, presumably through enzymatic action. There was also evidence of the protease weakening the eggshell, as enzyme-treated nematode eggs were more easily lysed and infected by the fungus than those not pre-incubated in the enzyme. A polyclonal antibody against VCPI demonstrated protease production by the fungus, prior to, or concurrent with, penetration. The enzyme was associated with appressoria, i.e. fungal infection structures. In contrast to the susceptible root-knot nematode, VCPI had little impact on the egg shell of the potato cyst nematode Globodera rostochiensis. It is suggested that the limited in situ hydrolysis of G. rostochiensis egg shell proteins is a factor contributing to its relative resistance to the fungus. Regulation studies in batch culture showed that production of the protease VCPI was repressed by high carbon and nitrogen levels. Its basic regulatory mechanism was that of repression/derepression. However, the highest protease titre was obtained when M incognita eggs were present in the medium, suggesting induction by the host. Collagen and chitin were possibly responsible for this inductive effect. In conclusion, it is believed that VCPI is a protease with a dual role for V chlamydosporium. During saprotrophic growth, VCP1 would allow the fungus to scavenge nutrients from a wide range of protein sources. However, the enzyme also has a designated function in penetration of the host, which makes it a versatile tool for a fungus that can switch trophic modes during its life-cycle. The achievements of this research include the first demonstration in a nematode-attacking fungus of: -a well-characterized protease, including data on stability, kinetics and isoforms; -a subtilisin-like protease in an egg-parasitic nematophagous fungus; -a pathogenicity-related enzyme in V chlamydosporium; -a determinant of host specificity; - enzyme regulation in general, and induction by the host, in particular.
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