Mutant/mutation
The mutant expresses a mutated version of DHC3. In this mutated version the microtubule-binding domain (MTBD), a globular fragment of 80-130 amino acids (3559-3648 amino acid) in the stalk tip is deleted. in addition, it and  expresses a C-terminal 6xHA-tagged version of DHC3

Protein (function)
There are 7 genes encoding putative dynein heavy chain (DHC) proteins of Plasmodium parasites. In the rodent malaria parasite P. yoelii, they are PY17X_0418900 (dhc1), PY17X_0618400 (dhc2), PY17X_0215400 (dhc3), PY17X_0508400 (dhc4), PY17X_0927400 (dhc5), PY17X_0603800 (dhc6), and PY17X_1333900 (dhc7).
Each dynein contains at least one dynein heavy chain (DHC) and other subunits including the intermediate chain (IC), light intermediate chain (LIC), and light chain (LC).

The invasive “zoite” stages of Plasmodium, including the ookinete, sporozoite, and merozoite, are morphologically polarized and possess a unique cortical pellicle underneath the parasite plasma membrane. From outside to inside, the pellicle consists of a double membrane organelle inner membrane complex (IMC) and a cytoskeleton layer of apically radiating subpellicular microtubules (SPMTs), both of which associate with each other and span along the periphery of the zoite parasites. Besides SPMTs, the invasive zoites of apicomplexan parasites possess a highly specialized structure called the apical polar ring (APR) at the cell apical cortex. APR is recognized as an electron-lucent region beneath the apical IMC. it is believed that APR functions as a microtubule-organizing center (MTOC) for nucleating SPMTs at the Plasmodium zoites. In Plasmodium, the SPMT cytoskeleton functions as a scaffold supporting parasite morphogenesis, maintaining the polarized cell shapes and providing parasite rigidity during gliding and invasion. In the process of ookinete growth, the parasite undergoes massive expansion of the plasma and cortex membrane. In addition, the ookinete acquires a complete set of apical organelles and structures via de novo assembly. IMC is assembled at the apical site of the initial protrusion and extends along the expanding plasma membrane to the basal end. After biogenesis,APR nucleates the assembly of apical SPMTs underling the IMC. Meanwhile, the apical tubulin ring (ATR), another compacted structure of MTs, emerges at the apex of ookinetes.While the IMC, SPMT, APR, ATR, and microneme are essential for either development, gliding, or midgut invasion of ookinetes, the mechanisms for de novo assembly of these organelles and structures at the apical distal area are largely unknown. We hypothesize that besides playing a cytoskeleton role, the apically radiating SPMTs may function as the tracks for the apical transport of cargoes containing the contents required for the assembly of apical organelles and structures in the ookinetes. In this scenario, the cytoplasmic dynein could be the primary motor moving towards the minus end of SPMTs for cargo transport to the apical distal area of ookinetes. However, the SPMT-based dynein transport machinery has not been identified in Plasmodium. Cytoplasmic dynein is a large multi-subunit protein complex, and the core is a homodimer of two heavy chain subunits (DHCs) interacting with intermediate, light intermediate, and light chain subunits

Phenotype
The microtubule-binding domain (MTBD), a globular fragment of 80-130 amino acids in the stalk tip, is responsible for the MT binding of DHC. To validate the association of DHC3 with SPMTs, we deleted the MTBD and investigated the effect on the SPMT. localization of DHC3. In the P. yoelii, the MTBD (3559-3648 amino acid) is located between the AAA4 and AAA5 of the pseudo-hexameric ring composed of 6 ATPase modules.
We used CRISPR-Cas9 to delete the genomic sequences encoding MTBD of DHC3 in the dhc3::6HA parasite and obtained a mutant line designated as ΔMTBD. Removal of MTBD had little effect on the protein level of DHC3 in the gametocytes, however, ΔMTBD showed reduced  formation of mature ookinetes compared to the parental line. The truncated DHC3 lost the peripheral localization in the ΔMTBD ookinetes.

See also mutant RMgm-5620 that lacks expression of DHC3 (Δdhc3). Analysis of this mutant showed the following:
Normal development/growth of asexual blood stages of Δdhc3 and and gametocyte formation in mice. The Δdhc3 showed normal gamete formation and fertilization in vitro. and developed from diploid to tetraploid during ookinete development. The in vitro assay for zygote to ookinete differentiation revealed that Δdhc3 had a dramatic decrease in ookinete formation (59% in 17XNL, 14% in Δdhc3). Time-course analysis revealed that DHC3 deficiency caused developmental arrestment mainly at stages I and II, and a small proportion of parasites developed into mature looking ookinetes. The defective morphology of the Δdhc3 ookinetes was als oobserved under scanning electron microscopy (SEM). Compared to the 17XNL ookinetes with characteristic crescent shapes, the mature-looking ookinetes of Δdhc3 lost cell bending.We further assessed the gliding activity of ookinetes in vitro and found that the mature-looking ookinetes of Δdhc3 displayed a significantly reduced gliding speed. No oocyst formation. No salivary gland sporozoites.....

Additional information
The seven dhc-genes were tagged with a sextuple HA epitope (6HA) to analyse the
expression and localization of these proteins. See the mutants RMgm-5613 (PY17X_0418900-6HA; dhc1), RMgm-5614 (PY17X_0618400-6HA; dhc2), RMgm-5615 (PY17X_0215400-6HA; dhc3, RMgm-5616 (PY17X_0508400-6HA; dhc4), RMgm-5617 (PY17X_0927400-6HA; dhc5), RMgm-5618 (PY17X_0603800-6HA; dhc6), RMgm-5619 PY17X_1333900-6HA; dhc7). These 6HA-tagged parasite lines showed normal asexual blood stage proliferation and gametocyte differentiation in mice, suggesting that the addition of 6HA did not affect parasite viability. Immunofluorescence assay (IFA) showed that all 7 DHCs displayed no detectable expression in the asexual blood stages. Only DHC3 was expressed in female gametocytes, ookinetes, and sporozoites. The other 6 DHCs were specifically expressed in male gametocytes.

DHC3 was evenly distributed along the periphery of ookinetes, suggesting the existence of SPMT-based dynein in the ookinetes. The localization of DHC3 was analysed relative to proteins known to be expressed within specific localizations in ookinetes. Parasite clones were generated with additional proteins tagged with quadruple Myc epitope (4Myc) or triple V5 epitope (3V5) from the dhc3::6HA parasite. These proteins included P28 (plasma membrane), GAP45 (IMC), MyosinB and SAS6L (apical tubulin ring, ATR), APR2 (apical polar ring, APR), GCβ (ookinete extrados site, OES), and CTRP and chitinase (microneme). Among these proteins, P28 and GAP45 showed overlapping signals with DHC3, further supporting the peripheral localization of DHC3 in the ookinetes.

Two parasite lines dhc3::3V5 and 4Myc::dhc3 were generated with endogenous DHC3 tagged with 3V5 at the C-terminus and with 4Myc at the N-terminus, respectively. Both DHC3::3V5 and 4Myc::DHC3 proteins displayed similar localization at the ookinetes.
To visualize DHC3 localization in living ookinetes, a parasite line was generated, dhc3::mScarlet, with DHC3 C-terminally tagged with a red fluorescence protein mScarlet (see mutant RMgm-5621). The mScarlet-tagged DHC3 was also distributed along the periphery of ookinetes.These results suggested the existence of the SPMT-based cytoplasmic dynein in the ookinetes and DHC3 is likely a subunit of the dynein complex.

 From the paper:
The cortical cytoskeleton of subpellicular microtubules (SPMTs) supports the Plasmodium ookinete morphogenesis during mosquito transmission of malaria. SPMTs are hypothesized to function as the cytoskeletal tracks in motor-driven cargo transport for apical organelle and structure assembly in ookinetes. We identify the core subunit DHC3 and other subunits of dynein showing co-localizing with SPMTs in the ookinete. In-depth phenotypical and functional analyses demonstrate that SPMT-based dynein plays an essential role in ookinete morphogenesis, shape, and gliding motility. DHC3 disruption impairs ookinete development, shape, and gliding, leading to failure in mosquito infection of Plasmodium. The DHC3-deficient ookinetes display defective formation or localization of apical organelles and structures. Rab11A and Rab11B interact with DHC3 at SPMTs in a DHC3-dependent manner, likely functioning as the receptors for the cargoes driven by SPMT-dynein. Disturbing Rab11A or Rab11B phenocopies DHC3 deficiency in ookinete morphogenesis. This study confirms the existence of the SPMT-based cytoplasmic dynein motor in the ookinetes and reveals its importance in intracellular cargo transport in ookinete morphogenesis.

Other mutants