Genome-wide identification of the P-loop NTPase gene family in Pyrus betulifolia and functional characterization of PbePloopNTP21 in resistance to black spot disease.

Pear black spot disease caused by Alternaria alternata severely impacts the pear industry, and the disease resistance of Pyrus betulifolia rootstocks is critical for grafted tree health. We characterized the P-loop NTPase superfamily in P. betulifolia and identified 30 members, with fragment replication as the main expansion driver. Multispecies collinearity and stress expression analyses identified the key gene PbePloopNTP21 (encoding an EF-1α homolog). Comparative analysis of EF-1α proteins from P. betulifolia, Malus domestica and fungal pathogens (Colletotrichum fructicola, A. alternata, Cytospora mali) revealed strictly conserved core P-loop nucleotide-binding/hydrolysis domains and lineage-specific polymorphisms in surface regions. Pear and apple EF-1α proteins formed a distinct host clade that was phylogenetically separate from fungal clades, providing a molecular framework for host-pathogen interactions. PbePloopNTP21 localizes exclusively to the nucleus. Functional assays showed its overexpression enhanced P. betulifolia resistance to black spot disease while VIGS-mediated silencing increased susceptibility, confirming its positive regulatory role in disease resistance. Physiological tests demonstrated PbePloopNTP21 promotes ROS clearance and immune responses by inducing antioxidant (SOD, POD, CAT) and defense-related enzymes (PAL, CHI). We observed its downregulation post-infection and hypothesize that it may function as a host receptor/target for pathogenic effectors; this hypothesis remains to be verified in subsequent studies. This study provides novel genetic resources and a theoretical basis for elucidating the molecular mechanisms of pear black spot disease resistance, laying a practical foundation for disease-resistant pear molecular breeding.