Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality Johnathan J. Dalzell a , Neil D. Warnock a , Michael A. Stevenson a , Angela Mousley a , Colin C. Fleming b , Aaron G. Maule a, * a Molecular Biosciences – Parasitology, School of Biological Sciences, Queen’s University Belfast, Belfast, UK b Agri-Food Biosciences Institute, Belfast, UK article info Article history: Received 19 February 2010 Received in revised form 23 March 2010 Accepted 24 March 2010 Keywords: Small interfering RNA (siRNA) RNAi Meloidogyne incognita Drosha Dicer Pasha Plant parasitic nematode abstract Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control. On silenc- ing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (si)RNA-soaked M. incog- nita eggs. Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH 2 )-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal. Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhi- bition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually lead- ing to embryonic lethality. To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cyto- solic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abun- dance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberra- tion of the J2s, which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M. incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-eri-1 transcript which encodes an RNAi-inhibiting siRNA exonuclease. We observe a marked up-regulation of Mi-eri-1 transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RNAi-based reverse genetic screens in plant parasitic nematodes. Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction A number of Caenorhabditis elegans mutants which display aber- rant revisions of normal developmental patterns have been identi- fied; the resultant defects may present in any tissue and may impact on cell division patterns, cell cycle phase lengths or termi- nal differentiation. In this way, heterochronic mutants suffer from temporal disruption of cellular fate, which may impact on other maturation events and tissue patterning (Moss, 2007). These heterochronic genes were first discovered in screens of C. elegans stem cell-like seam cells (Nimmo and Slack, 2009). Lin-4, the first such gene was found to encode an RNA hairpin-loop struc- ture which was processed to form a mature anti-sense RNA with complementarity to the 3 0 untranslated region (UTR) of lin-14 (Lee et al., 1993), a transcription factor which positively regulates the development of certain neuronal cells, in part through regula- tion of the cytoplasmic zinc-finger protein lin-28. Lin-4 mutants embryonate and pass L1 as wild type, however the inception of L2 developmental circuits results in the reiteration of L1-specific cell lineage differentiation patterns. It transpired that lin-4 was 0020-7519/$36.00 Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2010.03.010 * Corresponding author. Address: Molecular Biosciences – Parasitology, School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL Northern Ireland, UK. Tel.: +44 (0)28 9097 2059; fax: +44 (0)28 9097 5877. E-mail address: a.maule@qub.ac.uk (A.G. Maule). International Journal for Parasitology 40 (2010) 1303–1310 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara