1.3.4
HOST PROTEIN PHOSPHORYLATION IS ASSOCIATED WITH HAUSTORIUM FORMATION OF PERONOSPORA PARASITICA IN JAPANESE RADISH

K YOSHIDA1 and T OHGUCHI2

1National Research Institute of Vegetables, Ornamental Plants and Tea, Kanaya, Haibara, Shizuoka, 428-8501, Japan; 2College of Agriculture, Ehime University, Matsuyama, 790, Japan

Background and objectives
Peronospora parasitica Pers. ex Fr. is the causal agent of Japanese radish downy mildew. This fungus is one of the obligate parasites which form haustoria in living host cells to absorb nutrients. Haustorium formation is the most important phase for successful colonization of this fungus. We reported previously that haustorium formation of the fungus in root tissues of Japanese radish was suppressed by pretreatment with inhibitors of calcium ion-dependent metabolism [1]. In the present study, we tried to elucidate the correlation of host protein phosphorylation with haustoria formation in the tissues.

Materials and methods
P. parasitica and roots of Japanese radish (Raphanus sativus L. var. horteinsis Backer, cv. Shirokubimiyashige) cultivated for 3 months in the field, were used. Root discs 20 mm in diameter and 0.5 mm thick were prepared aseptically and inoculated with the fungus. Fractions of membrane and cytoskeleton [2] were isolated from root discs inoculated after pretreatment with chemicals or heat. Changes in the pattern of in vitro protein phosphorylation in these fractions were estimated by SDS-PAGE and autoradiography. Activities of protein kinase and protein phosphatase in these fractions were also investigated.

Results and conclusions
Metabolic disturbances in the host cell, induced by pretreatment with heat (50C, 60 s), cytochalasin B and protein kinase inhibitors, suppressed haustorium formation and, instead of the haustorium, a hypha-like structure similar to the germ tube was formed. Suppression was not related to known host-resistance reactions against the fungus. The suppression by heat treatment partially disappeared by treatment with calcium ionophore or a protein kinase activator. In vitro phosphorylation experiments revealed that specific polypeptides were phosphorylated when the fungus began to form a haustorium. Pretreatment with heat and cytochalasin B inhibited the specific phosphorylation of these peptides. Activities of membrane- and cytoskeleton-binding protein kinase depended on calcium ions and calmodulin. The activity of protein phosphatase was not changed dramatically in infected tissues. These results suggest that host calcium- and calmodulin-dependent protein phosphorylation are associated with the establishment of infection of P. parasitica.

References
1. Yoshida K, Ohguchi T, 1994. Annals of the Phytopathological Society of Japan 60, 253-256. 2. Tan Z, Boss WF, 1992. Plant Physiology 100, 2116-2120.