6. Use of CRISPR to study phytohormone-regulated root
development
CRISPR technology offers great potential to study how individual genes
dictate plants’ developmental or adaptive processes (Table 1). Great
effort has been made to understand the role of individual phytohormones
in the developmental processes, but many questions remain to be answered
in the future. In recent years, CRISPR-Cas technology accelerated the
research on the developmental processes in rice, also focusing on many
targets involved in phytohormone-mediated regulation. Here, we give
examples of how CRISPR technology can help elucidate the
phytohormone-regulated root developmental processes.
Auxin signalling pathway
Auxin is believed to be the main regulator of root development in
plants. As such, many components of auxin biosynthesis, transport, or
signalling become targets of genome editing. A high number of members
represents the individual gene families involved in auxin signalling
pathways and auxin transport. Whole-genome identification studies
identified 31 AUX/IAA gene family members, 25 ARFs , 5AUX1/LIKE AUX1 (LAX ) and 12 PINs in rice. Members of
these gene families frequently possess redundant functions. To
understand the contributions of individual members to the resulting
phenotype, a comparison of single mutants and higher-order mutants is
often necessary. CRISPR-Cas tools brought new opportunities to study
these multigene families. With the CRISPR-Cas system, the production of
higher-order knockout mutants is faster and more precise. Li et
al . employed this strategy to analyse the PIN gene family in
rice. Single and double mutants of four paralogous OsPIN1 genes
were obtained by multiplex CRISPR-Cas9 system. Due to functional
redundancy, single pin1 mutants do not show profound differences
in the root phenotype compared to the control plants. Only the double
mutant pin1a pin1b shows reduced primary root length, crown root
and lateral root numbers, as well as reduced gravitropic responses,
suggesting overlapping roles of PIN1a and PIN1b genes in
auxin-regulated root development.
Similarly, five members of the rice OsTIR1/AFB auxin coreceptor family
were studied to understand whether they have similar or diversified
functions during plant development. Mutants in OsTIR1/AFB genes
were obtained by CRISPR-Cas9 technology. In this case, two gRNAs were
designed for each member of the OsTIR1/AFB family, one of which
was specifically targeting the F-box coding region. In agreement with
the findings from TIR1/AFB family in Arabidopsis, rice OsTIR1/AFBhomologous genes also possess partially overlapping functions in diverse
developmental processes. Analysis of single mutants and a doubleOstir1 Osafb2 mutant revealed OsTIR1 and OsAFB2 as crucial
mediators of the auxin signal, with only a minor prevalence of OsTIR1 in
the development of primary root and adventitious roots.
In rice, OsAUX1/LAX represent another class of carriers involved in
polar auxin transport. Before the CRISPR-Cas system started to be
commonly used in rice research, only one member of the OsAUX1/LAX
family, OsAUX1, was functionally characterized employing T-DNA
insertional mutagenesis, RNAi and overexpression. Later, two other auxin
carrier proteins, OsAUX3 and OsAUX4, were analyzed using the CRISPR-Cas
system. Two independent mutant lines were produced for both OsAUX3 and
OsAUX4 carriers. When targeting OsAUX3 , the capacity of
CRISPR-Cas9 to create different types of mutations was used. Two gRNAs
were designed to remove a protein domain precisely, which is unique
among the OsAUX1/LAX protein family members. Interestingly, the
phenotype of the Osaux3-1 mutant, lacking the specific domain,
was similar to the phenotype of the Osaux3-2 null mutant,
suggesting an essential role of this protein domain in determining the
function of OsAUX3. Opposite to OsAUX1, OsAUX3 and OsAUX4 positively
regulate primary root elongation while negatively affecting root hair
length.
As well as with other auxin signalling components, studying the
multigene AUX/IAA family can be relatively challenging. Surprisingly,
Jun et al. revealed the crucial role of OsIAA23 in root
development. An EMS-induced point mutation in the core region of domain
II of OsIAA23 protein resulted in a severe pleiotropic root phenotype
caused by the defect in quiescent centre maintenance. Therefore, to
reduce the severity of the phenotype, Jiang et al . decided to
produce different allelic versions of OsIAA23 while preserving
the core region of OsIAA23 domain II. To achieve that, CRISPR-Cas9 gRNA
was designed to obtain mutations just next to the core region of domain
II. Indeed, the Osiaa23 mutants still showed an extensive
reduction in lateral root number, but the overall effect on the
phenotype was reduced. Besides, this study demonstrates that studying
protein motifs and the function of specific protein domains with
CRISPR-Cas becomes more straightforward than before.
Cytokinin signalling pathway
It is generally accepted that auxin and cytokinin cooperate in dictating
the root developmental signals, but their phytohormone crosstalk’s
mechanism remains only partially understood. Apart from the modulation
of auxin signals, cytokinin signalling controls many developmental
processes of the root system. While the role of cytokinins in root
development in Arabidopsis was already well studied, the molecular
mechanisms involved in cytokinin signalling and its implication in
cereal root system development are still not fully understood. Genome
editing has already been utilized in the study of different components
of cytokinin signalling or metabolism. One of the first genome editing
targets involved in cytokinin regulation was HvCKX1 in barley.
The knockout of HvCKX1 exhibited inhibition of the root system in
2 weeks old seedlings.
In contrast, Gasparis et al . observed increased total root
length, fresh weight and total surface area of 10-day-old seedlings of
the barley ckx1 knockout line, which was characterized by
decreased CKX activity. The opposite effect on the root system
parameters was observed in the ckx3 line, which showed increased
CKX activity. A follow-up study would be necessary to clarify the role
of individual CKX genes in barley root system development.
To study the cytokinin histidine kinase receptors in rice, gRNA
sequences specific for both OsHK genes were used in tandem to
produce the hk5 hk6 double mutant. Assessing the phenotype of the
single mutants, only hk6 showed a slight increase in the primary
root length and the number of lateral roots compared to the wild type.
Oppositely, the double hk5 hk6 mutant displayed severe
pleiotropic defects in both roots and shoot parts.
Genome editing was employed to specifically knockout members of a class
of cytokinin signal regulators, type B response regulators (RR). In this
case, the CRISPR-Cas9 cassette consisted of a tandem array of four
unique gRNA sequences targeting RR21 , RR22 , RR23 ,
and RR24 genes in rice. In this way, a triple mutant rr21
rr22 rr23 was isolated and analyzed. Apart from the defects associated
with floral development, the triple mutant also had shorter seminal
roots and decreased lateral root density, likely linked to reduced cell
proliferation.
CRISPR represents a handy tool that allows us to broaden our knowledge
of the different regulatory components involved in root development and
bring us closer to understanding the complexity of the underlying root
development mechanisms.
7. Enhancing tolerance to abiotic stress conditions by
CRISPR-Cas-induced modulation ofroot system architecture
Modifying specific root system architecture characteristics proved
beneficial to enhance tolerance to unfavourable conditions of the
environment. Interestingly, the most recent genome editing strategies
for changing the cereal root system architecture alter the growth angle
of the roots. Kitomi et al . introduced a promising approach to
enhancing salinity tolerance by modulating root system architecture in
rice. In high saline conditions, the soil surface root phenotype was
proposed to offer plants the possibility of avoiding unfavourable
conditions. The loss-of-function allele of DEEPER ROOTING1(DRO1 )-like (DRL1 ) was identified and shown to be
responsible for the soil surface root phenotype in rice while being
negatively regulated by auxin. Analysis of CRISPR-Cas9 mutants revealed
that DRO1, DRL1, and another DRO1 -homolog, DRL2 ,
control the root growth angle in rice, with DRO1 and DRL1being more important in the gravitropic response. More recently, a
mutant with a steep seminal root and lateral root growth angle was
discovered in a mutagenized population of barley. The steeper root
system architecture can potentially be more advantageous in drought
conditions. It was shown that the root angle is regulated byENHANCED GRAVITROPISM2 (EGT2 ) gene, which is conserved in
barley and wheat, and that EGT2 acts in an auxin-independent
manner.
Moreover, the authors suggest that more genes with function in the
regulation of root growth angle could be identified in the future. AsDRO1 -family genes and the EGT2 gene have a specified role
in RGA, without adverse effect on other morphological traits, they could
be very promising genome editing targets for improving the tolerance to
various abiotic stress factors. It would be interesting to transfer the
knowledge to other cereal crops and to test their response to different
stress factors.
While auxin and cytokinin predominantly control the developmental
processes, other plant hormones play essential roles in regulating the
plant ability to adapt to a changing environment. Abscisic acid
responses are generally involved in drought stress. It was shown that
targeting genes involved in ABA catabolism are a promising strategy to
enhance tolerance to abiotic stress conditions. CRISPR-Cas9 mediated
knockout of rice ABA 8’hydroxylase (OsABA8ox2 ), an enzyme
involved in ABA catabolism, resulted in elevated ABA and IAA levels in
rice. Changes in the phytohormone status were reflected in the high
root-to-shoot ratio and lateral root density under drought stress
conditions, leading to an increased survival rate after drought stress
and subsequent rewatering. Another study focused on one of the factors
regulating ABA responses, ENHANCED RESPONSE TO ABA1(ERA1 ), which is encoding β-subunit of the protein
farnesyltransferase. The role of this posttranslational modification
enzyme was partially revealed by analysis of CRISPR-Cas9 mutants. Three
gRNA sequences targeting three different exons of OsERA1 were
separately cloned in a binary vector and used for rice transformation.
Homozygous mutations in two of these gRNA target sites in theOsERA1 gene were lethal already in early development. Only
mutants with 1-bp insertion in the first exon of OsERA1 could be
further analyzed. Osera1 mutants exhibited increased primary root
growth under nonstressed conditions and enhanced drought responses,
resulting from the increased sensitivity to ABA. These findings imply
that the natural ability of ABA to modulate root system architecture
could be utilized in novel strategies for improving drought tolerance in
cereal plants.