QDE-1 with RNA/DNA-dependent RNA polymerase activities, which produces an aberrant
single-stranded RNA (aRNA) and subsequently
the dsRNA that activates the RNAi pathway
(Fig. 5.1) (Liu et al. 2010).
In addition to transgenes, fungal genomes
naturally contain two main types of tandem
repetitive sequences, TE arrays, and ribosomal
genes (rDNA), which are known to be a major
source of genome instability (Butler 1992; Bzymek and Lovett 2001; Vader et al. 2011). In fact,
several studies have revealed large amounts of
sRNAs derived from both types of loci in different fungi (Dumesic et al. 2013; Janbon et al.
2010; Nicolas et al. 2010), including qiRNA
(QDE-2-interacting small RNAs) and rasiRNAs
(repeat-associated small-interfering RNA) originated from rDNA in N. crassa (Lee et al. 2009)
and transposons in the basidiomycete Pleurotus ostreatus (Borgognone et al. 2018), respectively. Even though qiRNA and the transgenesinduced siRNAs have different origins, they
require the same RNAi components and are
the result of DNA damage (Chang et al. 2012;
Zhang et al. 2013). In addition, the RNAi pathway suppresses transposon proliferation in several fungi (Borgognone et al. 2018; Janbon et al.
2010; Nolan et al. 2005; Wang et al. 2010), and it
is required to maintain transgene tandem
repeats (Yang et al. 2015) and retrotransposon
arrays found in centromeres (Yadav et al.
2018).
An alternative mechanism to control TEs
during vegetative growth has been described
in C. neoformans. This mechanism identifies
transposon transcripts due to the presence of
suboptimal introns, which provokes that they
stall on spliceosomes. A spliceosome-coupled
and nuclear RNAi (SCANR) complex is able to
recognize these transposons transcripts stuck
on spliceosomes promoting siRNA production
that leads to control of transposons (Dumesic
et al. 2013).
Together, these observations suggest that
the principal function of the siRNA produced
from repeat regions is to maintain genome
stability, which is supported by the identification of DNA damage-induced sRNAs and the
involvement of Dicer enzymes in the maintenance of genome stability in plants and animals
(Bonath et al. 2018; Francia et al. 2013; Lu et al.
2018; Michalik et al. 2012; Wei et al. 2012).
2. Defense Responses in the Sexual Cycle
In addition to the RNAi mechanisms that operate in the vegetative growth to protect the
genome integrity, other RNAi pathways carry
out this function during sexual reproduction.
One of such RNAi mechanisms, called sexinduced silencing (SIS), was described in C.
neoformans. This is a PTGS mechanism that is
triggered by tandem integration of a transgene
array both in opposite-sex mating and unisexual reproduction. It shares the basic RNAi
machinery required for MIS, including Ago,
Dicer, and RdRP proteins (Wang et al. 2010,
2013). Beyond silencing exogenous transgenes,
SIS plays a critical role in transposon control
because RNAi mutants show an increase in
transposition/mutation rate and elevated levels
of siRNAs derived from repetitive TEs (Wang
et al. 2010, 2013). Interestingly, the higher
robustness of SIS compared to MIS might be
related to the fact that transposons in C. neoformans are more active during mating than during vegetative growth (Wang et al. 2010, 2013).
An additional RNAi mechanism involved in
defense of the genome during the sexual cycle
has been described in species of the Ascomycota phylum, such as N. crassa, Neurospora
tetrasperma, and Gibberella zeae (anamorph
Fusarium graminearum) (Ramakrishnan et al.
2011; Shiu et al. 2001; Son et al. 2011), suggesting that it has evolved recently (Hammond
2017). This RNAi mechanism protects genome
integrity through silencing of all those DNA
sequences that remain unpaired during the
meiotic prophase I. Consequently, it was
named Meiotic Silencing by Unpaired DNA
(MSUD) (Shiu et al. 2001). This mechanism
seems to be mechanistically distinct from the
SIS of C. neoformans because SIS is not triggered by unpaired DNA structures (Wang et al.
2010). The molecular mechanism of MSUD has
been studied in detail in N. crassa, where it can
be divided into a detection stage of unpaired
DNA between homologous chromosomes and a
silencing stage of unpaired DNA and any
110
F. E. Nicola ´s et al.
single-stranded RNA (aRNA) and subsequently
the dsRNA that activates the RNAi pathway
(Fig. 5.1) (Liu et al. 2010).
In addition to transgenes, fungal genomes
naturally contain two main types of tandem
repetitive sequences, TE arrays, and ribosomal
genes (rDNA), which are known to be a major
source of genome instability (Butler 1992; Bzymek and Lovett 2001; Vader et al. 2011). In fact,
several studies have revealed large amounts of
sRNAs derived from both types of loci in different fungi (Dumesic et al. 2013; Janbon et al.
2010; Nicolas et al. 2010), including qiRNA
(QDE-2-interacting small RNAs) and rasiRNAs
(repeat-associated small-interfering RNA) originated from rDNA in N. crassa (Lee et al. 2009)
and transposons in the basidiomycete Pleurotus ostreatus (Borgognone et al. 2018), respectively. Even though qiRNA and the transgenesinduced siRNAs have different origins, they
require the same RNAi components and are
the result of DNA damage (Chang et al. 2012;
Zhang et al. 2013). In addition, the RNAi pathway suppresses transposon proliferation in several fungi (Borgognone et al. 2018; Janbon et al.
2010; Nolan et al. 2005; Wang et al. 2010), and it
is required to maintain transgene tandem
repeats (Yang et al. 2015) and retrotransposon
arrays found in centromeres (Yadav et al.
2018).
An alternative mechanism to control TEs
during vegetative growth has been described
in C. neoformans. This mechanism identifies
transposon transcripts due to the presence of
suboptimal introns, which provokes that they
stall on spliceosomes. A spliceosome-coupled
and nuclear RNAi (SCANR) complex is able to
recognize these transposons transcripts stuck
on spliceosomes promoting siRNA production
that leads to control of transposons (Dumesic
et al. 2013).
Together, these observations suggest that
the principal function of the siRNA produced
from repeat regions is to maintain genome
stability, which is supported by the identification of DNA damage-induced sRNAs and the
involvement of Dicer enzymes in the maintenance of genome stability in plants and animals
(Bonath et al. 2018; Francia et al. 2013; Lu et al.
2018; Michalik et al. 2012; Wei et al. 2012).
2. Defense Responses in the Sexual Cycle
In addition to the RNAi mechanisms that operate in the vegetative growth to protect the
genome integrity, other RNAi pathways carry
out this function during sexual reproduction.
One of such RNAi mechanisms, called sexinduced silencing (SIS), was described in C.
neoformans. This is a PTGS mechanism that is
triggered by tandem integration of a transgene
array both in opposite-sex mating and unisexual reproduction. It shares the basic RNAi
machinery required for MIS, including Ago,
Dicer, and RdRP proteins (Wang et al. 2010,
2013). Beyond silencing exogenous transgenes,
SIS plays a critical role in transposon control
because RNAi mutants show an increase in
transposition/mutation rate and elevated levels
of siRNAs derived from repetitive TEs (Wang
et al. 2010, 2013). Interestingly, the higher
robustness of SIS compared to MIS might be
related to the fact that transposons in C. neoformans are more active during mating than during vegetative growth (Wang et al. 2010, 2013).
An additional RNAi mechanism involved in
defense of the genome during the sexual cycle
has been described in species of the Ascomycota phylum, such as N. crassa, Neurospora
tetrasperma, and Gibberella zeae (anamorph
Fusarium graminearum) (Ramakrishnan et al.
2011; Shiu et al. 2001; Son et al. 2011), suggesting that it has evolved recently (Hammond
2017). This RNAi mechanism protects genome
integrity through silencing of all those DNA
sequences that remain unpaired during the
meiotic prophase I. Consequently, it was
named Meiotic Silencing by Unpaired DNA
(MSUD) (Shiu et al. 2001). This mechanism
seems to be mechanistically distinct from the
SIS of C. neoformans because SIS is not triggered by unpaired DNA structures (Wang et al.
2010). The molecular mechanism of MSUD has
been studied in detail in N. crassa, where it can
be divided into a detection stage of unpaired
DNA between homologous chromosomes and a
silencing stage of unpaired DNA and any
110
F. E. Nicola ´s et al.