A Novel Spo11 Homologue Functions as a Positive Regulator in Cyst Differentiation in Giardia lamblia

Abstract

Giardia lamblia persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during Giardia encystation, suggesting parasexual recombination processes of this protozoan. Giardia possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the cwp promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate cwp gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of cwp1-3 and myb2 genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Arabidopsis Spo11 reduced the levels of cwp1-3 and myb2 gene expression and cyst formation. Targeted disruption of spo11 gene with CRISPR/Cas9 system led to a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for Giardia differentiation into cyst.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

“…Recently, the catalytically inactive form of the CRISPR/Cas9 system, CRISPRi, has been introduced to G. intestinalis for the successful knockdown of several genes that encode cytoskeletal proteins [ 23 ] and recently, RNA-targeting Cas ribonuclease, CasRx, was experimentally used in G. intestinalis [ 24 ] . The ‘classical’ CRISPR/Cas9 approach was used to reduce the gene expression in G. intestinalis [ 25 , 26 ] but this strategy did not achieve full genomic knockout. Thus, the only reported complete removal of all four gene copies was achieved for the cwp1 gene using the Cre/loxP system [ 27 ].…”

A myeloid leukemia factor homolog is involved in tolerance to stresses and stress-induced protein metabolism in Giardia lamblia

“…CRISPR/Cas9 system has been shown as a useful method for target disruption in G. lamblia [ 19 , 42 , 102 , 103 ]. For our gene disruption strategy, a plasmid consists of a selection marker cassette flanking with two homologous sequences, is transfected into G. lamblia with a transient plasmid consisting of Cas9 expression cassette (Fig.…”