Genome-wide mapping and biophysical characterization of G-quadruplex structures in phytoplasma and their prospective role in host pathogenesis.

UNLABELLED: G-quadruplexes are non-canonical DNA structures formed by guanine-rich sequences that regulate gene expression and genome stability. Despite AT-rich phytoplasma genomes, their G-quadruplex potential remains unexplored. We performed genome-wide mapping, comparative analyses and biophysical validation of putative G-quadruplex forming sequences (PGQSs) in four phytoplasma strains: Onion yellows phytoplasma, Aster yellows witches'-broom phytoplasma, 'Candidatus Phytoplasma australiense' and 'Candidatus Phytoplasma mali'. We identified 376 (OY-M, previously reported by Singh and Lakhanpaul 2019), 286, 340 and 129 PGQSs in OY-M, AY-WB, AUSGY and AT, respectively, with densities of 0.2143-0.4407 PGQSs/Kbp. Strong positive correlations between GC content and PGQS density occurred in whole genomes and genic regions, while regulatory and repeat-rich regions showed weak correlations, suggesting functional enrichment beyond GC bias. PGQSs were enriched in putative regulatory regions and essential Sec translocation pathway genes (secA and secY). Circular dichroism spectroscopy and native PAGE confirmed parallel and mixed G4 topologies in vitro, while mutated controls failed to form stable structures. These findings highlight non-random distribution and potential regulatory significance of G-quadruplexes in phytoplasma genomes, providing insights into molecular mechanisms underlying phytoplasma pathogenicity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s12298-026-01801-8.