Chapter 2 - Mycoviruses, RNA Silencing, and Viral RNA Recombination

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Abstract

In contrast to viruses of plants and animals, viruses of fungi, mycoviruses, uniformly lack an extracellular phase to their replication cycle. The persistent, intracellular nature of the mycovirus life cycle presents technical challenges to experimental design. However, these properties, coupled with the relative simplicity and evolutionary position of the fungal host, also provide opportunities for examining fundamental aspects of virus–host interactions from a perspective that is quite different from that pertaining for most plant and animal virus infections. This chapter presents support for this view by describing recent advances in the understanding of antiviral defense responses against one group of mycoviruses for which many of the technical experimental challenges have been overcome, the hypoviruses responsible for hypovirulence of the chestnut blight fungus Cryphonectria parasitica. The findings reveal new insights into the induction and suppression of RNA silencing as an antiviral defense response and an unexpected role for RNA silencing in viral RNA recombination.

Introduction

Although widely distributed throughout the diverse taxonomic groups of the filamentous fungi, mycoviruses (viruses of fungi) share surprisingly similar lifestyles (recently reviewed by Nuss, 2010). With two reported exceptions (Dawe and Kuhn, 1983, Yu et al., 2010), all characterized mycoviruses have genomes composed of double-stranded (ds) or single-stranded (ss) RNA. In contrast to viruses of plants and animals, mycoviruses uniformly lack an extracellular phase to their replication cycle. Consequently, they are not infectious in the classical sense. Infections cannot be initiated by exposure of uninfected hyphae to cell extracts prepared from an infected fungal strain. Rather, mycoviruses are transmitted by intracellular mechanisms such as anastomosis (fusion of hyphae) or through asexual spores. Since the absence of exposure to the extracellular environment reduces the need for the formation of particles that protect viral genetic information, a significant number of mycoviruses, for example, members of the taxonomic families Narnaviridae and Hypoviridae, do not even encode capsid proteins (reviewed in Nuss, 2005).

As a result of an exclusively intracellular lifestyle and dependence on the host for transmission, mycovirus infections are persistent and generally absent of severe symptoms and cell death. The constraints of this virus–host relationship have contributed to the development of novel phenotypic characteristics of fundamental interest and potential practical value. These include the recent report that the ability of the endophytic fungus Curvularia protuberance to confer heat tolerance to the panic grass Dichanthelium lanuginosum requires the presence of the mycovirus Curvularia thermal tolerance virus (Marquez et al., 2007). Moreover, the virus-infected endophyte was able to confer heat tolerance to a crop plant, tomato. A growing number of mycoviruses have been reported to alter the ability of plant pathogenic fungi to cause disease (recently reviewed by Ghabrial and Suzuki, 2009, Pearson et al., 2009). These mycovirus infections generally result in reduced virulence, termed hypovirulence, and offer the potential for development of biological control strategies for a range of fungal diseases.

The persistent, intracellular nature of the mycovirus life cycle presents technical challenges to experimental design but also provides opportunities for examining fundamental aspects of virus–host interactions from a perspective that is quite different from that pertaining for most plant and animal virus infections. Recent advances in the understanding of antiviral defense responses against one group of mycoviruses for which many of the technical experimental challenges have been overcome, the hypoviruses responsible for hypovirulence of the chestnut blight fungus Cryphonectria parasitica, will be developed in this chapter as support for this view. The findings have revealed new insights into the induction and suppression of RNA silencing as an antiviral defense response and an unexpected role for RNA silencing in viral RNA recombination.

Section snippets

Overcoming Technical Challenges to Mycovirus Research

Although initial interest in hypoviruses derived primarily from reports of virus-mediated control of chestnut blight in Europe and potential use in North America (reviewed by Anagnostakis, 1982, Dawe and Nuss, 2001, Heiniger and Rigling, 1994, Nuss, 1992), several key advancements in hypovirus and C. parasitica molecular biology and genomics subsequently led to the development of a robust experimental system that was able to overcome most of the technical challenges inherent in mycovirus

Vegetative incompatibility

Mycoviruses are able to readily spread through the hyphal network that comprises a fungal colony. The septa that compartmentalize the tube-shaped hyphae contain pores that allow free flow of virus particles or viral genetic information. However, transmission of mycoviruses between different strains of the same fungal species is often regulated by a genetic self/nonself recognition system termed heterokaryon or vegetative incompatibility (vic). Interactions between vegetative incompatible

Induction of the RNA silencing pathway in response to mycovirus infection

The requirement of a single Dicer gene, dcl2, and a single Argonaute gene, agl2, for RNA silencing antiviral defense in C. parasitica has allowed an examination of the activation of this response in the absence of potential contributions from multiple Dicers and Argonautes, as occurs in plants, and potentially in animal cells (Ding and Voinnet, 2007). Transcript levels for C. parasitica dcl1 and dcl2 were found to increase ∼ 1.5- and ∼ 15-fold, respectively, following either hypovirus or

RNA Silencing Contributes to Mycovirus RNA Recombination

One of the most unexpected observations to come from study of the RNA silencing antiviral defense response in C. parasitica was that RNA silencing contributes to hypovirus viral RNA recombination. Viral RNA recombination is one of the major components of viral evolution and a driving force behind the emergence of new viruses (reviewed by Nagy and Simon, 1997). Early molecular characterizations of hypovirus RNAs revealed the accumulation of significant levels of defective interfering (DI) RNAs (

Concluding Remarks

Filamentous fungi have featured prominently as experimental systems for advancing biochemistry, genetics, and molecular and cellular biology (reviewed in Borkovich et al., 2004, Davis and Perkins, 2002). Recent examples include the use of N. crassa in foundation studies on RNA silencing and the identification of the first RNAi pathway gene (reviewed in Li et al., 2010). The potential utility of fungi as experimental systems is further enhanced when one layers on the rich diversity of biological

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