Extracellular and intracellular infection of Botryosphaeria dothidea and resistance mechanism in apple cells

Apple ring rot, which is caused by Botryosphaeria dothidea, severely affects apple production. The mechanisms employed in apple cells against B. dothidea remain unknown. In this research, the pathogen infection mode and the relationship between cell death and disease resistance in ‘Fuji’/B. dothidea interaction pathosystem were investigated. By using transmission electron microscopy (TEM), our research showed that the pathogen infects apple cells both intracellularly and extracellularly. However, compared with that in immature fruit, the incidence of hyphae in the interior of mature apple fruit cells increased dramatically, suggesting that cell wall-mediated penetration resistance could be important in apple resistance against B. dothidea. TEM ultrastructural characterization identified the nuclear morphology of programmed cell death induction in both apple fruit and callus cells under B. dothidea infection. Overexpression of MdVDAC2 (MDP0000271281), which encodes an outer-membrane localized anion channel protein in mitochondria, significantly promoted cell death under B. dothidea infection and simultaneously inhibited pathogen infection, suggesting that cell death represents a disease resistance mechanism in apple against B. dothidea infection. Furthermore, BdCatalase (KAF4307763), a cytochrome P450 family protein BdCYP52A4 (KAF4300696), and subtilisin-domain containing proteins were identified from B. dothidea-secreted proteins, which suggested the potential involvement of active oxygen species and phytoalexins in combating B. dothidea infection and triggering or dampening apple resistance. Collectively, our research suggested that cell wall-mediated penetration resistance, programmed cell death machinery and microbial effector-interrelated signaling were among strategies recruited in apple to combat B. dothidea. The current research laid the foundation for further investigations into resistance mechanisms in apple.