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Eukaryotic Cell, October 2009, p. 1486-1497, Vol. 8, No. 10
1535-9778/09/$08.00+0     doi:10.1128/EC.00191-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Single dicer Gene Is Required for Efficient Gene Silencing Associated with Two Classes of Small Antisense RNAs in Mucor circinelloides{triangledown} ,{dagger}

Juan P. de Haro, Silvia Calo, María Cervantes, Francisco E. Nicolás,{ddagger} Santiago Torres-Martínez, and Rosa M. Ruiz-Vázquez*

Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, 30100 Murcia, Spain

Received 30 June 2009/ Accepted 29 July 2009

RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2– transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores.

* Corresponding author. Mailing address: Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, 30100 Murcia, Spain. Phone: 34 868 887136. Fax: 34 868 883963. E-mail: rmruiz{at}um.es

{triangledown} Published ahead of print on 7 August 2009.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} Present address: School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom.

Eukaryotic Cell, October 2009, p. 1486-1497, Vol. 8, No. 10
1535-9778/09/$08.00+0     doi:10.1128/EC.00191-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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