Genomic landscape reveals the dominance of self-catalytic, high-copy group II introns in PMU-deficient complete genomes of PWB phytoplasmas.

Phytoplasmas are wall-less obligate parasites of plants and insects. Several phytoplasma strains within the Peanut Witches' Broom (PWB; 16SrII) group are associated with significant disease losses across diverse crops and weeds. We present complete, single contig genome assemblies for two Indian parthenium phyllody strains, 'Candidatus Phytoplasma asiaticum' PR34 and 'Ca. P. australasiaticum' PR08, generated through host DNA depletion and hybrid Illumina-Nanopore sequencing. Both genomes display characteristic features of reductive evolution (∼614 kb and 589 kb, respectively) but show notable differences from previously sequenced PWB phytoplasmas. In contrast to most of PMU-rich phytoplasma genomes, neither PR34 nor PR08 retains intact Potential Mobile Units. Instead, both harbor numerous open reading frames encoding group II intron reverse transcriptase/ maturase proteins, predominantly of the mitochondrial-like type, with PR34 containing 52 and PR08 28 such loci that together constitute > 4% of each genome. These observations support the hypothesis that intron-associated processes may contribute to genome variability in the absence of canonical PMUs. Comparative analyses support the classification of PR34 as a distinct species within the PWB complex and reveal both conserved Sec-dependent effectors (SAP05, SAP11, and SAP54/PHYL1) and lineage-specific secreted proteins with predicted nuclear localization. Additional retained features include functional sodA genes and multiple truncated HlyB-like transporters. Collectively, these high-quality genomes illustrate a genomic configuration in which extensive genome reduction and loss of PMUs coexist with the retention of core virulence factors and an expanded repertoire of group II introns, providing a framework for future investigation of genome plasticity in phytoplasmas.