Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of PBANKA_0413400 and expresses RFP and GFP in female and male gametocytes, respectively.
Protein (function)
The gene was selected in a screen for identifying genes essential for for the formation of either male or female gametocytes
Phenotype
Strongly reduced numbers of male gametocytes; however, few (fertile) males are present; females fertile. Oocyst production not different from wild type.
From the paper:
'.....a transmission experiment and a cross with a cloned line of md3-KO showed that fertile male gametocytes were still present. However, consistent with the screen result, male fertility of mdr-KO was reduced to <1% when measured in competition with fertile mutants. We hypothesise that the md3 mutant produces fertile microgametes at a level that is much reduced, but still sufficient to give rise to a relative abundance of oocysts, probably because optimised laboratory infections of P. berghei produce an excess of zygotes, so that oocyst numbers begin to saturate at low input levels. ....'.
'... transcriptomes of FACS sorted mdr-ko parasites were essentially indistinguishable from wild type, suggesting deletion of md3 does not affect male differentiation, but sex ratio ....'.
Additional information
Ten genes, selected in a screen for identifying genes essential for for the formation of either male or female gametocytes, were targeted for gene deletion in the parent line that produces green-fluorescent males and red-fluorescent females.
From the paper:
'Flow cytometry analysis of parasites of individual knockout lines confirmed the biased expression of fluorescent sex reporters for all screen hits and further showed sex-specific losses of marker expression. Depending on the affected sex, we refer to the validated genes as “male development” (md1 to md5) or “female development” (fd1 to fd4. As expected, mutants showed a complete or nearly complete loss in their ability to form oocysts in mosquitoes, with the exception of md3, in which oocysts were merely reduced. Genetic crosses using either individual mutants or barcoded single-sex pools showed that for most genes, fertility was sex-specifically affected precisely as predicted by reporter expression. Two notable deviations from the screen results were observed. A cloned mutant in PBANKA_0828000, which by FACS only lacked parasites expressing the female marker, additionally suffered from male infertility; due to its broader gametocyte development (gd) phenotype we refer to this gene as gd1. The second mutant requiring further consideration is md3, for which both a transmission experiment and a cross with a cloned line showed that fertile male gametocytes were still present. However, consistent with the screen result, male fertility of all male mutants, including md3, was reduced to <1% when measured in competition with fertile mutants. We hypothesise that the md3 mutant produces fertile microgametes at a level that is much reduced, but still sufficient to give rise to a relative abundance of oocysts, probably because optimised laboratory infections of P. berghei produce an excess of zygotes, so that oocyst numbers begin to saturate at low input levels.'
For genetic crosses (to analyse male and female fertility) md1, md3, md4, md5, gd1, fd1, fd2, fd3 and fd4 knockout lines were mixed at a 1:1 ratio of iRBC with a nek4 knockout or a hap2 knockout prior to infection with the mixed parasite infected blood intraperitoneally.
Phenotypes of 10 gene-deletion mutants:
gd1 PBANKA_0828000 - no females, infertile males (PbGEM-091835)
fd1 PBANKA_1454800 - infertile females (N/A)
fd2 PBANKA_0902300 - infertile females (PbGEM-287226)
fd3 PBANKA_1418100 - infertile females (PbGEM-064794)
fd4 PBANKA_1435200 - infertile females (PbGEM-067210)
md1 PBANKA_1302700 - no males, females fertile (N/A)
md2 PBANKA_1447900 - no males, females fertile (PbGEM-340675)
md3 PBANKA_0413400 - few (fertile) males, females fertile (PbGEM-329075)
md4 PBANKA_0102400 - infertile males (PbGEM-327555)
md5 PBANKA_0716500 - infertile males (PbGEM-282066)
Other mutants |