Effect of phosducin silencing on the photokinetic motile response of Blepharisma japonicum

Abstract

The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of βγ-dimer of G-proteins (Gβγ) with β-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gβ (~36 kDa) was reduced, whereas the level of Gβ localized to the membrane (~32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.

The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

“…RNAi has been performed in two other ciliates, Paramecium tetraurelia and Blepharisma japonicum , using the method of feeding with bacteria expressing double-stranded RNA [20],[21]. However, in other ciliates, such as Tetrahymena thermophila , RNAi by feeding does not work.…”

Characterisation of the RNA-interference pathway as a Tool for Genetics in the Nascent Phototrophic Endosymbiosis,Paramecium bursaria

“…This means that a growing library of somatic transformants (featuring RNAi deficient mutations) would need to be maintained and propagated through mitosis to prevent these genetic changes from being lost upon regeneration of the somatic macronucleus. We therefore propose that delivery of exogenously derived dsRNA complementary to a target transcript, in the manner conducted in this study and others [48][49][50][51] , remains the optimal experimental approach for largescale gene knock-down surveys in this, and possibly other, ciliate systems. (Figure S1).…”

“…The ciliate-specific scan (scn)RNA (25-nt) and internal eliminated sequence (ies)RNA (~28-nt) pathways are endogenous, and function primarily to eliminate the bulk of noncoding DNA present in the germline micronuclear genome during development of the somatic macronucleus 35,[41][42][43]46,47 . Paramecium also encodes a shortinterfering (si)RNA (23-nt) pathway capable of processing both exogenously [48][49][50][51] and endogenously 32,44 derived RNA precursors. Although siRNA is believed to have evolved to protect against foreign genetic elements (such as viruses, transposons and transgenes) 52 , some siRNA-based RNAi factors have also been implicated in the regulation of endogenous transcriptome expression in the non-photo-endosymbiotic model system, P. tetraurelia 32 .…”

“…Paramecium also encodes a short-interfering (si)RNA (23-nt) pathway capable of processing both exogenously [48][49][50][51] and endogenously 32,44 derived RNA precursors. Although siRNA is believed to have evolved to protect against foreign genetic elements (such as viruses, transposons and transgenes) 52 , some siRNA-based RNAi factors have also been implicated in the regulation of endogenous transcriptome expression in the non-photo-endosymbiotic model system, P. tetraurelia 32 .…”

Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria

“…28-nt) pathways are endogenous, and function primarily to eliminate the bulk of non-coding DNA present in the germline micronuclear genome during development of the somatic macronucleus [35,[41][42][43]46,47]. Paramecium also encodes a short-interfering (si)RNA (23-nt) pathway capable of processing both exogenously [48][49][50][51] and endogenously [32,44] derived RNA precursors. Although siRNA is believed to have evolved to protect against foreign genetic elements (such as viruses, transposons and transgenes) [52], some siRNA-based RNAi factors have also been implicated in the regulation of endogenous transcriptome expression in the non-photo-endosymbiotic model system, P. tetraurelia [32].…”