Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma

Toxoplasma gondii is a zoonotic protist pathogen that infects up to 1/3 of the human population. This apicomplexan parasite contains three genome sequences: nuclear (63 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear DNA of mitochondrial origin) and NUPTs (nuclear DNA of plastid origin) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome; the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma [~]28 MY ago, revealed that the movement and fixation of 5 NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb) and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen. Significance StatementThis study reveals how DNA located in cellular compartments called organelles can be transferred to the nucleus of the cell and inserted into the nuclear genome of apicomplexan parasite Toxoplasma. Insertions alter the DNA sequence and may lead to significant changes in how genes function. Unexpectedly, we found that the human protist pathogen, Toxoplasma gondii and closely-related species have the largest observed organellar genome fragment content (>11,000 insertion comprising over 1 Mb of DNA) inserted into their nuclear genome sequence despite their compact 65 Mb nuclear genome. Insertions are occurring at a rate that makes them a significant mutational force that deserves further investigation when examining causes of adaptation and virulence of these parasites.