Immunoelectron microscopic analysis of callose secretion and nuclear condensation associated with defense responses of plant pathogen-stressed plant cells

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Spores of Alternaria alternata Japanese pear pathotype germinated and formed appressoria at tips of germ tubes on surfaces of the susceptible and resistant pear leaves. Penetration pegs emerged from appressorial bottom made an attempt to invade cell walls of susceptible epidermal cells. The pegs penetrated into the cuticular layers and then were grown as infection hyphae along the pectin layers, but didn't invade directly the epidermal cytoplasm except for the completely disrupted cells. AK-toxin-induced plasma membrane modifications, associated with callose secretion, appeared at plasmodesmata of susceptible leaf cells before the fungal invasion in tissues, suggesting the hypothesis that the toxin was produced from germ tubes was also supported. On the contrary, the pathogen inoculated in resistant leaves was able to penetrate into cuticles, but fungal invasion was not limited after the cuticular penetration. These results indicated that resistant mechanism to black spot disease was e … More xpressed at the stage of cuticular invasion during pathogenesis. Papilla formation was found at the spaces between cell walls and plasma membranes irrespective of susceptible and resistant leaves against pathogen inoculation, but penetration pegs were observed, but not involved in the papillae themselves. This suggests that papillae are non-specific plant responses against cuticular wounding, but didn't contribute directly to disease resistance in black spot disease. Immunoelectron microscopic study showed that β-l,3-D-glucan accumulated both at papillae in the pathogen-infected susceptible and resistant leaves, and at callose in the toxin-treated susceptible ones. The glucan was shown to be be secreted from Golgi bodies of susceptible leaves via Golgi vesicles to damaged sites of plasma membranes, plasma membrane modifications. The generation of reactive oxygen species (ROS) wsa found at the membrane fragments of plasma modifications in the toxin-treated susceptible leaves, but not at the papillae. This results showed that ROS generation was associated with the toxin-induced plasma membrane modifications which were damaged plasmalemma which were spatially consistent with cellular ion-leakage sites. ROS generation also was recognized at the cell walls of penetration pegs during infection process in the pathogen-inoculated susceptible and resistant leaves. ROS might cause hardening cell walls by cross-reaction of cell wall proteins with ROS, suggesting increased aggressiveness of penetration pegs. Although nuclear condensation was observed in the toxin-treated and spore-inoculated susceptible leaves, the nuclear changes were not evaluated as one of apoptotic events. The changes were compatible with pknosis seen in necrotic tissues because of no laddering of DNA. Less