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.