related sequences found at other locations in
the genome, regardless of their pairing state.
Nine proteins are involved in MSUD, three of
which correspond to the core components of
the canonical RNAi pathway and the rest perform specific functions of this mechanism
(Hammond 2017). In the latter group are
included SAD-5 and SAD-6, which are the
only proteins known to participate in the identification of unpaired sequences (Samarajeewa
et al. 2014). Despite the poor characterization of
the mechanism of unpaired DNA detection, this
process is thought to trigger the production of
an aRNA, which is recognized in the perinuclear region by the proteins involved in the
silencing stage. Thus, a MSUD-specific RdRP
SAD-1 and the helicase SAD-3 (Hammond
et al. 2011, 2013a; Shiu and Metzenberg 2002;
Shiu et al. 2001) generate a dsRNA molecule
that is processed by Dicer DCL-1 into MSUDassociated small-interfering RNAs (masiRNAs)
(Alexander et al. 2008). These are then bound
by AGO SMS-2, and the passenger is removed
by the exonuclease QIP (Lee et al. 2003; Xiao
et al. 2010). The identification of masiRNAs
derived from an unpaired transposon in a sexual cross suggests that MSUD protects the
genome from transposons (Wang et al. 2015),
although another hypothesis points to the protection of the genome from meiotic drive elements, called spore killers, which rearrange
large genomic segments as part of the driving
mechanism (Hammond 2017).
Spore killers, described in Ascomycota phylum, are
single genes or complexes of genes that favor their
own propagation through meiosis and/or gametogenesis by killing the meiotic products not containing
them. The best characterized spore killer is sk-2 of N.
crassa (Turner and Perkins 1979). The sk-2 drive
mechanism requires that at least two distantly
located genes, a resistance gene called rsk and a killer
gene called rfk, inherit together during meiosis
(Campbell and Turner 1987). Tight linkage between
these two genes appears to be derived from chromosome rearrangements within the sk-2 element (Harvey et al. 2014). The sk-2, and also sk-3, suppresses
MSUD (Raju et al. 2007), suggesting that this blockage allows the evolution of chromosome rearrangements, which would be MSUD targets (Hammond
2017).
3. Antiviral Small-Interfering RNAs
The use of the RNAi pathway as an innate
immune system against viruses was one of the
first roles associated with this mechanism. Triggering of siRNA production by dsRNA or
ssRNA viruses has been found in plants, flies,
worms, mammals, and fungi (Harvey et al.
2011; Jeang 2012; Segers et al. 2007; Wilkins
et al. 2005; Zambon et al. 2006). Among fungi,
this response has been intensively studied in
the ascomycete Cryphonectria parasitica, a filamentous fungus that is the causal agent of
chestnut blight (Segers et al. 2007), and also
described in Colletotrichum higginsianum
(Campo et al. 2016), Aspergillus nidulans
(Hammond et al. 2008), Fusarium graminearum (Yu et al. 2018), and Sclerotinia sclerotiorum (Mochama et al. 2018). C. parasitica
produces an RNAi-mediated antiviral response
by the production of virus-derived siRNAs
(vsRNAs) (Table 5.1) that target and destroy
viral sequences. Although this fungus has two
dicer-like genes, four ago-like genes, and four
rdrp-like genes, only genes dcl2 and agl2 are
involved in the RNAi-mediated antiviral
response. Thus, single deletion mutants in any
of these two genes were defective in the production of vsRNAs and highly susceptible to
mycovirus infections, resulting in a severely
debilitated growth (Segers et al. 2007; Sun
et al. 2009). Interestingly, the C. parasitica
RNAi mechanism also promotes recombination of viral genomic RNA, a central component of virus evolution that contributes to the
emergence of new viruses (Sun et al. 2009;
Zhang and Nuss 2008).
The importance of the RNAi-mediated antiviral defense is reinforced by the fact that some
viruses present mechanisms of RNAi suppression. Thus, the hypovirus Cryphonectria hypovirus 1 (CHV1-EP173), a mycovirus that infects
C. parasitica, expresses the protein p29, a
papain-like protease, that inhibits the RNAi
pathway by repressing the transcriptional
induction of agl2 in response to virus infections
(Sun et al. 2009). Likewise, the existence of a
viral suppressor has also been demonstrated in
A. nidulans (Hammond et al. 2008).
5 Small RNAs in Fungi
111
the genome, regardless of their pairing state.
Nine proteins are involved in MSUD, three of
which correspond to the core components of
the canonical RNAi pathway and the rest perform specific functions of this mechanism
(Hammond 2017). In the latter group are
included SAD-5 and SAD-6, which are the
only proteins known to participate in the identification of unpaired sequences (Samarajeewa
et al. 2014). Despite the poor characterization of
the mechanism of unpaired DNA detection, this
process is thought to trigger the production of
an aRNA, which is recognized in the perinuclear region by the proteins involved in the
silencing stage. Thus, a MSUD-specific RdRP
SAD-1 and the helicase SAD-3 (Hammond
et al. 2011, 2013a; Shiu and Metzenberg 2002;
Shiu et al. 2001) generate a dsRNA molecule
that is processed by Dicer DCL-1 into MSUDassociated small-interfering RNAs (masiRNAs)
(Alexander et al. 2008). These are then bound
by AGO SMS-2, and the passenger is removed
by the exonuclease QIP (Lee et al. 2003; Xiao
et al. 2010). The identification of masiRNAs
derived from an unpaired transposon in a sexual cross suggests that MSUD protects the
genome from transposons (Wang et al. 2015),
although another hypothesis points to the protection of the genome from meiotic drive elements, called spore killers, which rearrange
large genomic segments as part of the driving
mechanism (Hammond 2017).
Spore killers, described in Ascomycota phylum, are
single genes or complexes of genes that favor their
own propagation through meiosis and/or gametogenesis by killing the meiotic products not containing
them. The best characterized spore killer is sk-2 of N.
crassa (Turner and Perkins 1979). The sk-2 drive
mechanism requires that at least two distantly
located genes, a resistance gene called rsk and a killer
gene called rfk, inherit together during meiosis
(Campbell and Turner 1987). Tight linkage between
these two genes appears to be derived from chromosome rearrangements within the sk-2 element (Harvey et al. 2014). The sk-2, and also sk-3, suppresses
MSUD (Raju et al. 2007), suggesting that this blockage allows the evolution of chromosome rearrangements, which would be MSUD targets (Hammond
2017).
3. Antiviral Small-Interfering RNAs
The use of the RNAi pathway as an innate
immune system against viruses was one of the
first roles associated with this mechanism. Triggering of siRNA production by dsRNA or
ssRNA viruses has been found in plants, flies,
worms, mammals, and fungi (Harvey et al.
2011; Jeang 2012; Segers et al. 2007; Wilkins
et al. 2005; Zambon et al. 2006). Among fungi,
this response has been intensively studied in
the ascomycete Cryphonectria parasitica, a filamentous fungus that is the causal agent of
chestnut blight (Segers et al. 2007), and also
described in Colletotrichum higginsianum
(Campo et al. 2016), Aspergillus nidulans
(Hammond et al. 2008), Fusarium graminearum (Yu et al. 2018), and Sclerotinia sclerotiorum (Mochama et al. 2018). C. parasitica
produces an RNAi-mediated antiviral response
by the production of virus-derived siRNAs
(vsRNAs) (Table 5.1) that target and destroy
viral sequences. Although this fungus has two
dicer-like genes, four ago-like genes, and four
rdrp-like genes, only genes dcl2 and agl2 are
involved in the RNAi-mediated antiviral
response. Thus, single deletion mutants in any
of these two genes were defective in the production of vsRNAs and highly susceptible to
mycovirus infections, resulting in a severely
debilitated growth (Segers et al. 2007; Sun
et al. 2009). Interestingly, the C. parasitica
RNAi mechanism also promotes recombination of viral genomic RNA, a central component of virus evolution that contributes to the
emergence of new viruses (Sun et al. 2009;
Zhang and Nuss 2008).
The importance of the RNAi-mediated antiviral defense is reinforced by the fact that some
viruses present mechanisms of RNAi suppression. Thus, the hypovirus Cryphonectria hypovirus 1 (CHV1-EP173), a mycovirus that infects
C. parasitica, expresses the protein p29, a
papain-like protease, that inhibits the RNAi
pathway by repressing the transcriptional
induction of agl2 in response to virus infections
(Sun et al. 2009). Likewise, the existence of a
viral suppressor has also been demonstrated in
A. nidulans (Hammond et al. 2008).
5 Small RNAs in Fungi
111