Transient silencing mediated by in vitro synthesized double-stranded RNA indicates that PsCdc14 is required for sporangial development in a soybean root rot pathogen

Abstract

In many eukaryotic organisms, Cdc14 phosphatase regulates multiple biological events during anaphase and is essential for mitosis. It has been shown that Cdc14 is required for sporulation in the potato blight pathogen Phytophthora infestans; however, the role that the Cdc14 homolog (PsCdc14) plays in the soil-borne soybean root rot pathogen P. sojae remains ambiguous. PsCdc14 is highly expressed in sporulation, zoospore, and cyst life stages, but not in vegetative mycelia and infection stages, suggesting that it contributes to asexual reproduction and thus the spread of the disease. Double-stranded RNA (dsRNA) mediates gene silencing, a post-transcriptional and highly conserved process in eukaryotes, involving specific gene silencing through degradation of target mRNA. We combined in vitro dsRNA synthesis and a polyethylene glycol-mediated transformation system to construct a dsRNA-mediated transient gene silencing system; and then performed a functional analysis of PsCdc14 in P. sojae. PsCdc14 mRNA was dramatically reduced in transformants after protoplasts were exposed in in vitro synthesized PsCdc14 dsRNA, resulting in low sporangial production and abnormal development in P. sojae silencing lines. Furthermore, dsRNA-mediated transient gene silencing could enable elucidation of P. sojae rapid gene function, facilitating our understanding of the development and pathogenicity mechanisms of this oomycete fungus. Phytophthora sojae, PsCdc14, sporangium, transient gene silencing Citation:Zhao W, Yang X Y, Dong S M, et al. Transient silencing mediated by in vitro synthesized double-stranded RNA indicates that PsCdc14 is required for sporangial development in a soybean root rot pathogen.

Cytokinesis-required Cdc14 is a signaling hub of asexual development and multi-stress tolerance in Beauveria bassiana

“…3A). The severe conidiation defect is consistent with those caused by knockdown cdc14 expression in two oomycetes116 and likely attributable to the repressed expression of hymA required for conidiophore development30, hyd1 / 2 essential for hydrophobin biosynthesis and spore coat rodlet layer assembly during conidiation31, and flbB−D as transcriptional factors required for morphogenesis and asexual development32333435. The exceptional up-regulation of flbA might contribute to brlA activation for the premature initiation of development36 but its effect on brlA could be shaded by the repressed flbB−D and fluG , another brlA activator37.…”

“…Deletion of cdc14p from Candida albicans affected late cell-cycle events and morphogenesis, such as large cell aggregates, decreased invasion into solid substrate and impaired hyphal growth15. Knockdown expression of Cdc14 orthologues may result in defective sporulation in Phytophthora 116. However, most of the previous studies have focused primarily on Cdc14 contribution to nuclear events but paid little attention to possible effects on multi-stress responses and other biological aspects, such as virulence, although Cdc14 may interact with other phosphatases and MAPKs in budding yeast8.…”

The MADS-box Transcription Factor PsMAD1 Is Involved in Zoosporogenesis and Pathogenesis of Phytophthora sojae

“…Among these genes, the protein phosphatase coding gene, PsCDC14 ( Ps108222 ), showed down-regulation. PsCDC14 and its homolog PiCDC14 have been revealed to be essential for zoosporogenesis in P. sojae and Phytophthora infestans , respectively ( Ah Fong and Judelson, 2003 ; Zhao et al, 2011 ). PiCDC14 acts as a transcription regulator to reprogram gene expression during zoospore formation ( Seidl et al, 2013 ).…”

The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst

“…Double-stranded RNA of PsLAC4 was synthesized according to the protocol of the Ambion MEGAscript RNAi kit. Transient transformation was carried out using the PEG-mediated method but with some modifications as described (Zhao et al, 2011). All phenotypic assays were carried out at day 8-15 after transformation.…”