Early defence responses of cowpea (Vigna sinensis L.) induced by non-pathogenic rust fungi

In cowpea (Vigna sinensis L.) leaves the pathogenic rust fungus Uromyces vignae and the non-pathogens U. appendiculatus and U. viciae-fabae developed similarly to give rise to more than 80% haustorial mother cells. Whereas U. vignae was able to sporulate, the non-pathogens were stopped either after formation of some haustoria (U. appendiculatus) or immediately after formation of haustorial mother cells (U. viciae-fabae). Approximately 30% of the cells in contact with haustorial mother cells of the two non-pathogens showed autofluorescence and deposition of phloroglucinol/HCl-positive material. The early defence reactions of V. sinensis include induction of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and extracellular peroxidase (POD, EC 1.11.1.7) activity as early as 10 h and 24 h after inoculation, respectively. Probing Western blots with heterologous monospecific anti-PAL serum showed that pathogenesis-induced increases in enzyme activity are the result of de novo synthesis. Native polyacrylamide gel electrophoresis revealed the specific induction of two extracellular acidic POD forms in cowpea inoculated with the non-pathogens. Both PAL and apoplasmic POD activities were highest in U. viciae-fabae-, intermediate in U. appendiculatus- and low in U. vignae-inoculated or talcum-treated control plants. The timing of increasing PAL and extracellular POD activities in relation to deposition of lignin or lignin-like material in mesophyll cell walls indicates the involvement of lignification in the early defence of V. sinensis against biotrophic fungal parasites. Analysis of the substrate specificity of the inducible POD forms, separated and partially purified by chromatofocusing, showed that apoplasmic PODs preferentially oxidize the naturally occurring substrate coniferyl alcohol. These results support the assumption that POD-mediated lignification is involved in the expression of non-host resistance of cowpea to rust fungi.