Structure and expression of two polygalacturonase genes of Claviceps purpurea oriented in tandem and cytological evidence for pectinolytic enzyme activity during infection of rye

Two putative polygalacturonase (PG) genes were isolated from strain T5 of Claviceps purpurea, using the pgaII gene of Aspergillus niger as a probe. The two genes (pg1 and pg2) are closely linked and arranged head-to-tail. They are highly homologous even in the upstream noncoding sequences, possess one intron each in the same position, and have cleavage sites for processing enzymes. They probably code for mature proteins of 343 and 344 amino acids, respectively, and share significant homology with endo-PGs of other filamentous fungi. Expression of pg1 and pg2 in axenic culture and during various stages of infection of rye was demonstrated using reverse transcription-polymerase chain reaction. The potential substrate of the putative products of pg1 and pg2 (poly- galacturonic acid), for the first time, was shown to be a component of the host cell walls in rye ovaries. This was achieved by immunogold trans-mission electron microscopy with the monoclonal antibody (MAb) JIM 5, specific for nonmethyl-esterified epitopes of pectin. This homogalacturonan was localized along the usual infection path in healthy carpels together with its methyl-esterified galacturonan type in the same cell walls with another MAb, JIM 7. At the interface of the penetrating hyphae and the host ovary epidermis. JIM 5 label density was locally enhanced and very high above hyphal sheaths. In the vicinity of intercellularly growing hyphae, label density was highly increased, and gold label occurred not only above the middle lamella area but also throughout the entire host cell wall. Chemical demethylation and immunogold labeling indicated a high total content of galacturonan and a conversion of pectic compounds at the host-parasite interface. During late infection phases, the lack of any JIM label, which previously occurred at the interface of intracellular hyphae, emphasized the complete utilization of homogalacturonan together with other plant polysaccharides. The observed host wall alterations provide evidence for secretion and activity of extracellular pectinolytic enzymes in planta. Both the expression of the two genes during infection of rye and the modification and degradation of homogalacturonan detected only at fungal sites indicate the fungal origin of pectinolytic enzymes, the activities of which have been documented previously in infected ovaries by B.I. Shaw and P.G. Mantle