Background
Fungal pathogens are the main factors responsible for the most severe diseases affecting
plants, leading to significant reduction in yield and crop quality and causing enormous
economic losses worldwide. It is estimated that around 30% of the emerging diseases
are caused by fungi (Giraud et al., 2010) thus requiring new strategies to improve
their management. Biological control approach, frequently referred to the use of non-pathogenic
microbial antagonists or products derived from their metabolism, represents a valid
and promising alternative under a more ecological perspective to reduce the activities
and to control populations of target pathogens (Singh, 2016). However, although the
use of antagonists belonging to species different from that of the pathogen has been
successfully reported, the use of competitors belonging to the same species of the
pathogen is not widespread. A biocontrol strategy based on competition for space and
nutrients and/or the induction of plant defenses against virulent pathogens performed
by attenuated or avirulent pathogens (Ghorbanpour et al., 2018) could, therefore,
be considered a valid alternative.
The Results So Far
Veloso et al. (2015) reported the use of an avirulent isolate of Fusarium oxysporum
to reduce Verticillium wilt severity in pepper, through competition and induction
of the plant defense responses. A similar approach was described by Salazar et al.
(2012) for the management of anthracnose in strawberries. The avirulent isolate F7
of Colletotrichum fragariae conferred full protection from the infection caused by
C. acutatum and also enhanced plant resistance against Botrytis cinerea through the
induction of plant defense responses. Similarly, the use of an attenuated Verticillium
nigrescens isolate reduced cotton wilt caused by a virulent isolate of V. dahliae
(Vagelas and Leontopoulos, 2015).
(Aimé et al., 2013) used an avirulent isolate of F. oxysporum to combat F. oxysporum
f. sp. lycopersici to reduce Fusarium wilt by priming a Salicylic-dependant signaling
defense on tomato plants. The use of an avirulent strain of Valsa mali var. mali reduced
the infection rate of apple tree canker caused by the virulent strain LXS080601 from
97 to 41% (Zhang et al., 2014) on apple callus. In 1993 as regards the mycotoxigenic
fungi, Cotty and Bayman (1993) suggested the use of a non-aflatoxigenic isolate of
Aspergillus flavus to control the development of aflatoxigenic strains in maize kernels
by competitive exclusion and this strategy today is commercially applied in several
countries (Ojiambo et al., 2018).
However, the selection of suitable isolates to be used as potential antagonists from
the local fungal community often takes (long) time for identification and screening.
Selection within a great number of isolates based on morphological, physiological
and genetic features is usually required, followed by an in vivo screening against
the pathogen on a real disease scenario. An interesting alternative to easily and
quickly obtain new genotypes able to act as biocontrol agents, could be the induction
of genetic mutations in the virulent genotypes, providing new avirulent strains that
can compete directly with the virulent ones or induce plant defense responses (Ghorbanpour
et al., 2018). The application of genetic transformation techniques to silencing genes
putatively involved in pathogenicity has been widely used to uncoil the role of these
genes in the establishment and development of the infection processes (Johnson et
al., 2018). However, the disruption of a gene function usually involves the integration
in the genome of foreign DNA sequences used as reporter genes in order to select transformants,
leading to the generation of antibiotic-resistant or fluorescent strains. These genetic
modifications represent a major constraint for their use in field.
The Genome-Editing Era: State of the Art and Perspectives for the Management of Plant
Diseases
The arrival of the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic
Repeats – CRISPR Associated protein 9) genome-editing technique enabled researchers
to modify genomic sequences in a more precise way (Knott and Doudna, 2018). CRISPR-Cas9
Type II system uses two principal components for gene targeting and cleavage: the
RNA guide (sgRNA) and the Cas9 endonuclease. The sgRNA consists of a simple chimeric
strand of RNA, which leads Cas9 up to the localization in the genome of the target
gene, whose expression has to be blocked. Cas9 is able to bind the DNA and to produce
a double strand break (DSB) in the target gene. The DSB then induces the activation
of one of the DNA cellular reparation systems, the Non-Homologous End-Joining (NHEJ)
system, that can re-join the ends of the DSB without introducing errors or, unlikely,
giving rise to insertions or deletions of nucleotides during the repair. These InDels
led to changes in the gene reading frame producing non-sense sequences or causing
the appearance of premature stop codons, thus blocking the transcription of the target
gene (Bono et al., 2015). In most cases the application of the technique in filamentous
fungi consisted of a proof of concept of its feasibility (see Table 1). The system
has the advantage that once implemented in the organism, it is possible to change
the target gene by changing the sgRNA spacer sequence, as well as silencing several
genes simultaneously by transforming the cell with different sgRNAs along with Cas9
(Hsu et al., 2015). Nevertheless, the most interesting advantage is that it allows
to perform marker-free deletions by using transient expression plasmids that can self-replicate
only under antibiotic pressure (Katayama et al., 2015, Nødvig et al., 2015; Schuster
et al., 2016; Zhang et al., 2016; Liu et al., 2017; Wenderoth et al., 2017; Weyda
et al., 2017; Wang et al., 2018). The use of CRISPR-Cas not only provides a time-saving
path to perform genomic functional analyses, but also could provide new fungal genotypes,
that can be used as potential competitors of plant pathogens and/or in the priming
of plant defense responses.
Table 1
Application of CRISPR-Cas9 for gene-silencing in filamentous fungi.
Fungal species
Edited gene
Aim
References
Alternaria alternata
Polyketide synthase A (pksA) 1,3,8-THN reductase (bmr2)
Proof of concept
Wenderoth et al., 2017
Aspergillus aculeatus
Aspergillus brasilensis
Aspergillus carbonarius
Aspergillus luchuensis
Aspergillus nidulans
Aspergillus niger
Polyketide synthase (albA) Laccase (yA)
Proof of concept
Nødvig et al., 2015
Aspergillus carbonarius
Pigment biosynthetic gene (ayg1)
Proof of concept
Weyda et al., 2017
Aspergillus fumigatus
Polyketide synthase P (pksP)
Proof of concept
Zhang et al., 2016
Aspergillus nidulans
Aspergillus niger
Aspergillus oryzae
Laccase (yA) Polyketide synthase (albA) Polyketide synthase (wA)
Proof of concept
Nødvig et al., 2018
Aspergillus niger
AMP-dependent synthetase and ligase α-aminoadipate-semialdehyde dehydrogenase Aldo/keto
reductase Alcohol dehydrogenase, zinc-binding Short-chain dehydrogenase/reductase
FAD-dependent oxidoreductase Mandelate racemase/muconate lactonizing enzyme d-isomer
specific 2-hydroxyacid dehydrogenase
Production of galactaric acid
Kuivanen et al., 2016
Aspergillus niger
Polyketide synthase (albA)
Proof of concept
Zheng et al., 2018
Aspergillus oryzae
Polyketide synthase (wA) Conidial laccase (yA) Orotidine 5-phosphate decarboxylase
(pyrG)
Proof of concept
Katayama et al., 2015
Fusarium graminearum
Histidine kinase 1 (os1) Trichodiene synthase (tri5)
Proof of concept
Gardiner and Kazan, 2018
Fusarium oxysporum
Orotate phosphoribosiltransferase (ura3, ura5) Polyketide synthase 4 (pks4)
Proof of concept
Wang et al., 2018
Ganoderma lucidum
Ganoderma lingzhi
Orotate phosphoribosyltransferase (ura3)
Proof of concept
Qin et al., 2017
Myceliopthora thermophila
Myceliopthora heterotalica
Carbon catabolite repression transcription factor (cre-1) Endoplasmic reticulum stress
regulator (res-1) β-glucosidase (gh1-1) Alkaline protease (alp-1)
Enhancement of lignocellulase production
Liu et al., 2017
Neurospora crassa
Carbon catabolism repressor (cre-1)
Enhancement of cellulase production
Matsu-ura et al., 2018
Penicillium chrysogenum
Polyketide synthase (pks17)
Proof of concept
Pohl et al., 2016, 2018
Pyricularia oryzae
Scytalone dehydratase (sdh) suppressor of RAD six (sdr2)
Proof of concept
Arazoe et al., 2015
Trichoderma reesei
Transcription factor in cellulase biosynthesis (clr2) Orotate phosphoribosyltransferase
(ura5) Gene putatively involved in glucose signaling and carbon catabolism repression
(vib1) Methyltransferase (lae1)
Proof of concept
Liu et al., 2015
Sclerotinia sclerotiorum
Oxaloacetate acetylhydrolase (Ssoah1) Polyketide synthase (Sspks13)
Proof of concept Pathogenicity test
Li et al., 2018
Ustilago maydis
Central regulator of pathogenic development (bW2) (bE1)
Proof of concept
Schuster et al., 2016
One possible scenario for the application of CRISPR-Cas9 silenced mutants could be
Fusarium Head Blight (FHB), one of the most destructive diseases of grain cereal crops
worldwide caused by different Fusarium spp., with F. graminearum and F. culmorum as
the most common and aggressive agents. In FHB, while yield loss derives from sterility
of infected florets, grain quality reduction is mainly due to the accumulation of
trichothecenes—coded by the fungal tri genes cluster—highly toxic for humans and animals.
Previous studies reported that iRNA (interference RNA) Δtri6 mutants of F. culmorum
showed reduced disease indices ranging from 40 to 80% on durum wheat (Scherm et al.,
2011). In addition, classic knocked-out Δtri5 and Δtri6 mutants of F. graminearum
were unable to spread the disease to the adjacent spikelets and grains on wheat and
corn, respectively, and also induced plant defense responses (Ravensdale et al., 2014).
Likewise, Δmap1 mutants of F. graminearum showed two-fold reduction of mycotoxin production
and were unable to produce perithecia as well as to penetrate in wheat tissues, while
the ability to colonize the straw was not affected (Urban et al., 2003). Considering
that competition for space and nutrients between virulent and non-virulent strains
could reduce the disease, the field release of non-virulent CRISPR-mutant strains
of F. graminearum and F. culmorum might help to control the incidence of FHB
Another contribution of CRISPR-Cas9 is the production of well-known antagonists with
enhanced biocontrol aptitudes achieved through genome-editing (Vicente Muñoz et al.,
2017). For example, species belonging to genus Trichoderma have been considered outstanding
biocontrol agents able to reduce the disease severity (Sarrocco et al., 2013), not
only by constraining the growth of the phytopathogens (Sarrocco et al., 2009), even
killing them, but also by eliciting the plant defense responses (Fiorini et al., 2016;
Sarrocco et al., 2017). One of the mechanisms through which these fungi can antagonize
phytopathogenic fungi is the release of a wide arsenal of cell-wall degrading enzymes
and secondary metabolites such as antibiotics, among others (Khalid, 2017).
Genetic engineering of the metabolic pathways that trigger the biosynthesis of secreted
proteins and secondary compounds could provide new fungal strains with enhanced biocontrol
activity. Previous studies reported that it is possible to achieve the same effect
through the silencing of negative regulatory elements, signal-transduction components
or genes belonging to contiguous metabolic networks, thus, redirecting metabolite
flow and biosynthesis or supressing the feedback inhibition by which its production
could be regulated (Bailey, 1991). For example, Δtvk1 mutants of T. virens displayed
enhanced biocontrol activity against R. solani, in addition to an increased expression
of mycoparasitism-related genes and overproduction of lytic enzymes (Mendoza-Mendoza
et al., 2003). Likewise, four knockout mutants in SSCPs (small secreted cysteine rich
proteins)-encoding genes of T. virens showed greater ability to induce ISR (Induced
Systemic Resistance) on corn against Cochliobolus heterostrophus than the wild type
(Lamdan et al., 2015). Another example of biocontrol enhanced ability was described
by Reithner et al. (2005) in T. atroviride, in which the Δtga1 mutants exhibited an
overproduction of antifungal secondary metabolites. Similarly, the Δtmk1 mutants of
T. atroviride showed overproduction of 6-pentyl-pyrone and peptaibols, resulting in
an enhanced antifungal activity and increased protection of bean plants against Rhizoctonia
solani (Reithner et al., 2007). On the other hand, biosynthesis of secondary metabolites
is often carried out by clustered genes whose expression could be induced by environmental
conditions. However, in many cases these clusters are silent and their activation
cannot be achieved (Osbourn, 2010). Bok et al. (2009) demonstrated that the silencing
of a transcription factor involved in the methylation of lysine 4 of the histone H3
in Aspergillus nidulans activated the expression of cryptic clusters and yielded novel
secondary metabolites. The silencing of ace1 gene induces the up-regulation of four
polyketide biosynthetic gene clusters in T. atroviride, leading to an increase in
the production of antibiotics and other secondary metabolites that clearly enhanced
its potential as biocontrol agent against F. oxysporum and R. solani (Fang and Chen,
2018). Following this approach, it is possible to induce the activation of unknown
clusters in beneficial fungi by using CRISPR-Cas9, allowing the discovery of new secondary
metabolites that could interact with plants or phytopathogens. This could result in
new interesting biocontrol strains to be released in field avoiding the introduction
of transgenes in the environment.
Conclusions
The availability of novel or the improvement of known techniques that are safer for
people and the environment is of outmost importance to guarantee food safety and security
especially in those countries where famine is still an important issue (Vurro et al.,
2010). A novel technique that allows the production of precise knock-out mutants without
the insertion of foreign DNA in a saprotrophic/pathogenic fungus opens new possibilities
of controlling plant pathogens. The use of such edited fungal strains needs a correct
strategy to minimize possible risks. The major risk related to the release of a mutant
strain is the rise, in the field, of novel combinations of pathogenesis/fitness related
genes following the sexual or parasexual cycle. The genetic background of an edited
isolate and its wild type is exactly the same, except for the edited gene, thus novel
combinations of genes are not conceivable. Anyway, to further reduce such possibility,
we can imagine a strategy of deployment that includes 1) the gene edit of the most
prevalent genotype of the pathogen/saprotroph in the release area; 2) the editing
of more than one gene in the same metabolic pathway and 3) the editing also of the
idiomorphs and /or the HET genes, to make sexual or parasexual recombination (including
the re-gain of virulence) even less likely.
Anyway, the application of novel techniques and the release of new products need,
as usual, to be evaluated for their safety and to be accepted by populations. A recent
sentence of the Court of Justice of the EU stated that edited organisms, even if they
do not contain alien DNA, have to be subjected to the rules set up for Genetically
Modified Organisms. This is not the place to discuss this issue, but it is high time,
in EU at least, to reconsider the whole GMO legislation.
Author Contributions
All authors listed have made a substantial, direct and intellectual contribution to
the work, and approved it for publication.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.