Generation of Plasmodium yoelii malaria parasite for conditional degradation of proteins

Highlights

• Generation of two transgenic P. yoelii parasite lines stably expressing Tir1 for AID application.

• Validation of auxin-induced conditional protein degradation in multiple parasite developmental stages.

Abstract

The auxin-inducible degron (AID) system is a robust chemical-genetic method for manipulating endogenous protein level by conditional proteasomal degradation via a small molecule. So far, this system has not been adapted in the P. yoelii, an important and widely used Plasmodium rodent parasite model for malaria biology. Here, using the CRISPR/Cas9 genome editing method, we generated two marker-free transgenic P. yoelii parasite lines (eef1a-Tir1 and soap-Tir1) stably expressing the Oryza sativa gene tir1 under the promoters of eef1a and soap respectively. These two lines develop normally during the parasite life cycle. In these backgrounds, we used the CRISPR/Cas9 method to tag two genes (cdc50c and fbxo1) with the AID motif and interrogate the expression of these two proteins with auxin. The eef1a-Tir1 line allows efficient degradation of the AID-tagged endogenous protein in the asexual schizont and sexual gametocyte stages, while the soap-Tir1 line allows protein degradation in the ookinetes. These two lines will be a useful resource for studying the Plasmodium parasite biology based on the P. yoelii.

Introduction

Malaria remains one of the most serious infectious diseases worldwide. Rodent malaria parasites Plasmodium berghei and Plasmodium yoelii have been widely used as model systems to study malaria pathology and parasite biology. These species possess many biological features similar to those of the human malaria parasites in vertebrate hosts and mosquito vectors during life cycle. Recent years has witnessed great progress in developing research tools, in particular the CRISPR/Cas9 genome editing technology [[1], [2], [3]], which enables more efficient interrogation of gene function in malaria parasites. Because asexual blood stage of the parasites is the only time window suitable for DNA transfection, the lack of robust method hampers the functional investigation of genes essential for asexual blood stage development of Plasmodium parasites [4]. In the past decade or so, the conditional gene knockout approach was demonstrated in both the P. falciparum and P. berghei, wherein the genetic alteration at the genomic DNA level is irreversible and thereby the functional analysis is allowed only at its first point of protein action [[5], [6], [7]]. Methods to inducible control transcription have also been reported in the P. falciparum and P. berghei, but suffer from slow onset kinetics and were tested only for asexual blood stage [[8], [9], [10], [11]]. Different from the strategies targeting in the DNA or mRNA, manipulation of protein levels by chemical-genetic strategies is often more advantageous in inducibility, reversibility, and specificity. Two such regulatory systems composing of specific pair of ligands and associated protein binding domains have been developed in the P. falciparum to regulate protein stability using either FKBP protein destabilization domain or dihydrofolate reductase destabilization domain [[12], [13], [14]].

Plants evolved a unique system in which the plant hormone auxin induces rapid proteasomal degradation of certain proteins by a specific E3 ubiquitin ligase [15]. Kohei

Nishimura et al. creatively utilized the auxin-inducible degron (AID) in the non-plant cells and achieved conditional degradation of proteins of interest (POI) via a small molecule [16]. The system relies on the evolutionarily conserved eukaryotic SCF ubiquitin ligase complex composing of the Skp1, Cullin1, Rbx1, and F box protein, the last of which recruits protein substrates for degradation. Recently, the AID system has also been successfully adapted in the apicomplexan parasites, including P. berghei [17,18] and Toxoplasma gondii [19]. To implement this system, only two transgenic components are needed: a plant auxin receptor called transport inhibitor response 1 (TIR1) and a POI tagged with an AID. In these engineered parasite lines stably expressing the plant TIR1 [18,19], the auxin functions as a molecular glue promoting specific interaction between the ubiquitin ligase complex and AID-tagged POI, which triggers proteasomal degradation of the latter.

So far, the application of AID-mediated protein degradation has not been reported in the P. yoelii. Inability to manipulate the protein essential for parasite asexual blood stage development prompts us to develop an AID system in the P. yoelii. Here, using the CRISPR/Cas9 method, we generated two marker-free transgenic parasite lines eef1a-Tir1 and soap-Tir1, which stably express the Oryza sativa gene tir1 under the promoters of eef1a and soap genes respectively. These two lines develop normally during the life cycle. While the eef1a-Tir1 line allows efficient degradation of the engineered endogenous protein containing an AID motif in the schizonts and gametocytes, the soap-Tir1 line enables protein degradation in the ookinetes.