Identification And Characteristics Of Inducible Extracellular Proteases Expressed By Zoophthora Radicans (Entomophthorales: Entomophthoraceae) Under Different Culture Conditions

Extracellular protease is an important virulence factor, playing a critical role in the degradation of cuticle structural protein during infection of entomopathogenic fungal into insect. The protease exhibits highly substrate-specific and can be easily induced by substrate. In this study, the activities and electrophoresis patterns of extracellular proteases which are expressed by strains of original and the various passages of Zoophthora radicans against Plutella xylostella under different culture conditions have been analyzed. Different types of extracellular protease have been identified by using protease inhibitors. The result indicated that Z. radicans produces abundant of extracellular proteases which can be selectively expressed based on different substrate, while the difference expression of extracellular protease between original and adapted strains suggested that the specific extracellular proteases may associated with the virulence against Pieris brassicae and P. xylostella.Analysis of protease activity under different culture conditions a high level of protease activities were expressed both by Zoophthora radicans original strain (Ro) and host-adapted strains (R₁ and R5) when they grow in MS medium containing insect cuticle or gelatin. It appeared that extracellular protease can be easily induced by proteins in the medium. On the contrary, very low level of protease activities were detected in extremely poor nutrient medium (MS). Interestingly, the level of protease activities in rich nutrient medium (Sabouraud's dexteose broth, SDB) are also very low in 5-15 day and it increased after 30 days, especially for R₅ isolates which exhibited a peak in 30 days. It implied that fungi did not synthesize protease when extra nutrient could be used. The result also indicated that the inducible expression of protease may be influenced by the lack of nutrient in the medium. Analysis of the electrophoresis patterns of the extracellular proteases under different culture conditions A 37KD band which belongs to serine protease was found to be inducedin any cultural condition, suggesting it is a conservative sequence coding protease for three strains. On the other hand, a 46KD band belonging to metalloprotease was expressed only in MS medium containing gelatin, however its expression could be inhibited by MS medium containing glucose. Interestingly, a protease band with molecular weight of 67KD was disappeared, whereas the bands of 46KD and 117KD become more obvious for isolates after several passages of subculture through P. xylostella when isolates grow in SDB medium The results indicated that inducible high substrate-specific extracellular protease was greatly expressed to adapt to new host during passages of subculture through P. xylostella. It suggested that the band of 67KD was likely associated to the virulence against P. brassicae, whereas the bands of 46KD and 117KD were probably related to the fungal virulence against P. xylostella.The identification of extracellular proteases different types of extracellular proteases could be identified by using protease inhibitors. The result showed that the band of 37KD was disappeared after treatment of serine protease inhibitor (PMSF), thus it belongs to a serine protease since the band. Similarly the band of 46KD belongs to metaloprotease since the band was disappeared after treatment of metalloprotease inhibitor (EDTA).