Abstract
Parasitic plants pose a significant threat to global agriculture, causing substantial crop losses and hampering food security. In recent years, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology has emerged as a promising tool for developing resistance against various plant pathogens. Its application in combating parasitic plants, however, remains largely unexplored. This review aims to summarise current knowledge and research gaps in utilising CRISPR to develop resistance against parasitic plants. First, we outline recent improvements in CRISPR gene editing tools, and what has been used to combat various plant pathogens. To realise the immense potential of CRISPR, a greater understanding of the genetic basis underlying parasitic plant-host interactions is critical to identify suitable target genes for modification. Therefore, we discuss the intricate interactions between parasitic plants and their hosts, highlighting essential genes and molecular mechanisms involved in defence response and multilayer resistance. These include host resistance responses directly repressing parasitic plant germination or growth and indirectly influencing parasitic plant development via manipulating environmental factors. Finally, we evaluate CRISPR-mediated effectiveness and long-term implications for host resistance and crop improvement, including inducible resistance response and tissue-specific activity. In conclusion, this review highlights the challenges and opportunities CRISPR technology provides to combat parasitic plants and provides insights for future research directions to safeguard global agricultural productivity.
1 Introduction
Plant pests and pathogens significantly threaten global food security, causing substantial yield losses (). Climate change exacerbates the issue by altering pathogen assemblages (). Efficient plant disease management is essential to sustainably meet global food demand. Current disease management methods include chemical control, which is efficient but can have adverse environmental impacts and promotes resistance (). On the other hand, biological control, while more environmentally friendly, often exhibits relatively limited consistency and cost-effectiveness (). However, successful examples of utilizing biological controls and natural resistance varieties have demonstrated their potential in effectively managing plant pests and diseases (; ). This indicates that leveraging host resistance could offer a promising and more sustainable alternative solution.
Therefore, harnessing knowledge about plant-pathogen interactions and defence responses is crucial for developing successful disease management strategies (). Developing disease-resistant crops relies on comprehending multi-dimensional defence mechanisms, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), to combat invading pathogens (). Introducing host resistance through conventional breeding is hindered by linkage drag and limited genetic diversity within elite germplasm (). Mutation breeding introduces variation but also genome-wide undesired mutations (). Genome editing, particularly CRISPR-Cas, enables precise gene modifications without off-target detrimental effects ().
In this review, we summarise the role of CRISPR in developing resistance against parasitic plants, outlining its improvements and applications against pathogens. Understanding the genetic basis of plant-host interactions is vital for targeted gene modification. We explore essential genes and mechanisms for defence and resistance, evaluating CRISPR’s effectiveness in enhancing crop resistance. We outline the challenges and opportunities of CRISPR technology for safeguarding agricultural productivity.
2 CRISPR editing tools and recent technological advances
Applications in plant biology have been no exception to the promise of targeted genome manipulation provided by CRISPR/Cas systems (). While some of the earliest examples of CRISPR/Cas utility in plant biology were gene knockouts in model organisms, the technology has now been expanded to a wide variety of applications including large-scale editing screens, base editing, targeted insertions, and transcriptomic and epigenomic modifications (; ; ; ). In parallel, improvements have been made in the delivery of CRISPR/Cas and other plant genome engineering reagents to plant cells, particularly for non-model and crop species (; ; ; ). Together, advancements in genome editing technology with efficient delivery of reagents provide great promise for gene discovery and functional genome modification.
RNA guided endonuclease systems, such as CRISPR/Cas, provide incredible precision for modifying specific targets in the genome. CRISPR systems utilize a guide RNA (gRNA) comprised of a constant repeat sequence and spacer sequence specific to a desired target site (). The only requirement for this target is an adjacent protospacer adjacent motif (PAM), which for S. pyogenes Cas9 (SpCas9) consists of a simple 5′-NGG-3′ sequence (). Minimal target sequence requirements, ease in reagent design, and robust cleavage has quickly established CRISPR as a highly effective tool for targeted genetic modification.
Many examples of CRISPR application in plants prioritize targeting protein coding sequences, using indels to induce a frameshift mutation (). This approach has been employed for large scale screens in which dozens to thousands of unique mutants are generated to uncover novel gene function and epistasis (). The adaptation of CRISPR systems from other species, such as CRISPR/Cas12 from Lachnospiraceae bacterium ND 2006 which recognizes TTTV (V = A, C, and G) PAM sequences, has provided greater flexibility in target site requirements (). Greater precision in modification type is provided by base editing via cytidine or adenine deaminases fused to Cas9 nickases which can be exploited for specific nucleotide or amino acid changes (). This precision is expanded by the recent development of prime editors for targeted sequence modification, deletion, or insertion (). In other applications, CRISPR is used to modify or disrupt noncoding or regulatory elements resulting in quantitative variation (). Modifications to gene regulation, however, are not limited to genetic changes. By using a catalytically inactive Cas protein tagged with transcriptional or epigenomic regulators, gene expression can be regulated in a target-specific manner without inducing double-stranded breaks (). We recommend a recent review for a more comprehensive discussion on recent developments in CRISPR/Cas plant genome engineering reagents ().
3 CRISPR applications in disease and parasite resistance
Recent advancements in genome editing technology provide powerful tools to address various agricultural challenges, including creating disease and pest-resistant crop lines (; ). CRISPR/Cas systems have demonstrated remarkable efficiency in combatting virus infections, as well as fungal and bacterial diseases across diverse plant species (). This versatile technology holds immense promise for revolutionising agricultural practices and bolstering crop resilience against pathogenic threats.
Engineering host resistance in plants has long been anchored in the classical “gene for gene” hypothesis. This principle revolves around the interaction between host R (resistance) genes and pathogen Avr (avirulence) genes, determining the outcome of resistance or disease occurrence. One approach for broad-spectrum resistance is through the modification of R genes by CRISPR/Cas reagents (). Precisely mutating the leucine-rich repeat (LRR) domain within R genes enables alterations in elicitor recognition specificity and confers resistance against diverse pathogens. However, relying solely on a single R gene for resistance may prove inadequate due to pathogen mutations that might enable them to circumvent specific resistance mechanisms, necessitating the exploration of alternative strategies. Concurrently, host susceptibility (S) genes are potential targets for engineering host resistance (). CRISPR/Cas editing of S genes results in durable, broad-spectrum resistance against fungal and bacterial pathogens.
In summary, the transformative potential of CRISPR/Cas tools in engineering disease resistance in plants presents exciting opportunities in agricultural research. While several review articles have discussed the application of CRISPR in plant disease resistance (; ; ), it is crucial to recognise that plant pathogens, such as viruses, bacteria, and fungi, are not the sole threats to food security. Parasitic plants also significantly impact agricultural productivity worldwide (). According to a stochastic model that has been published, it is projected that the yearly economic losses attributed to all parasitic weeds will likely reach approximately US $200 million, with an annual rise of roughly US $30 million (). Compared to abundant studies on plant pathogens, research and discussion on host resistance mechanisms to combat parasitic plants are relatively limited. The application of CRISPR technologies to improve crops’ defence against parasitic plants is still in its early stages and lacks a systematic review. Therefore, this review will focus on the importance and significance of utilising CRISPR to resist parasitic plants, highlighting past successful examples and proposing potential future research directions to foster resilient and sustainable crop protection measures.
4 Notorious parasitic weeds and global food security
Parasitic plants pose a significant risk to food security globally, approximately affecting millions of hectares of croplands and targeting vital cereal crops and vegetables (; ). These parasitic weeds develop specialised organs, haustoria, to invade host vascular systems and hijack water and nutrients (), leading to substantial reductions in agricultural productivity and, in some cases, complete crop failure (; ). Based on the host tissue invaded, parasitic weeds can be classified as stem or root parasites (). Host-dependence further categorises them into obligate hemiparasitic, facultative hemiparasitic, or holoparasitic. A well-known example of stem holoparasitic plants is the Cuscuta species (dodders) (Figure 1), which parasitises numerous critical vegetable and fruit crops. Conversely, root hemiparasitic plants, like the Striga species, commonly known as witchweed (Figure 1), parasitise a broad spectrum of cereal crops. More detailed classification descriptions have been well discussed in previous review articles (). These diverse classifications highlight the complexity of parasitic weed interactions with host plants and ecosystems. Controlling parasitic plants is challenging due to their well-adapted life cycles, high seed production, and genetic diversity. For example, Striga can produce up to 0.5 million seeds per plant, with seeds remaining viable in the soil for extended periods (). Their ability to disperse seeds widely and adapt to various environments makes eradication problematic.
FIGURE 1
Various methods have been attempted to manage parasitic plant infestations, including agricultural practices, chemical or bioinoculant applications, and host resistances (
5 CRISPR applications in enhancing resistance against parasitic plants
5.1 Identifying targets for CRISPR: pre-attachment and post-attachment resistance
Understanding how host plants defend against parasitic plants is crucial for effectively utilizing gene editing to enhance host resistance. Recent research has highlighted similar host-parasitic plant defence response to interactions seen in other host-pathogen relationships (
Host resistance mechanisms can be divided into pre-attachment and post-attachment categories based on whether these defences occur before or after parasitic plants establish themselves on hosts (
5.2 CRISPR applications in enhancing pre-attachment resistance
Pre-attachment resistance encompasses a range of strategies employed by host plants to prevent the attachment and invasion of parasitic plants before direct contact occurs. These mechanisms include inhibiting the germination of parasitic plant seeds. Strigolactones (SLs), a class of plant hormones, play a crucial role in triggering the germination of root parasitic plants in the family Orobanchaceae (
In addition to inhibiting parasite seed germination, some host plants release toxic compounds through their root exudates, hampering the development of parasitic plant seedlings (
In recent studies, genetic manipulation techniques such as CRISPR-Cas9 have been employed to target genes responsible for strigolactone biosynthesis and parasitism, resulting in resistance against parasitic plants in crops respectively (
However, these alterations in SLs have broader effects. Recent CRISPR/Cas9 edited sorghum experiments emphasize that the benefits of LGS1-based resistance are influenced by parasite genotype and environmental conditions (
Similarly, SL biosynthesis is also a target for CRISPR/Cas mediated resistance. SLs are produced through the carotenoid pathway involving Carotenoid Cleavage Dioxygenase (CCD) 7, CCD8, and More Axillary Growth 1 (MAX1) genes (
The intricate mechanisms underlying pre-attachment resistances remain largely unexplored. The identification of Striga resistance genes plays a vital role in the development of genotypes boasting lasting resistance. Fortunately, recent studies have leveraged population structure analysis and genome-wide association studies (GWAS) to pinpoint promising candidates (
5.3 CRISPR applications in enhancing post-attachment resistance
Following attachment, post-attachment resistance unfolds as a plant’s defensive strategy, activated upon detection of parasitic plants affixed to the host. This defence repertoire encompasses various mechanisms, such as hypersensitive responses (HRs), hormone-driven signalling pathways, fortification of cell walls, and accumulation of defensive secondary metabolites (
Among these post-attachment resistance responses, modifying cell walls has been prominently observed and reported in prior research as a crucial strategy. Various host plants resistant to root and stem parasitic plants have harnessed this mechanism (
Further exploration of different mechanisms in post-attachment resistances and their integration into plant genetic engineering is essential. While identifying pivotal elements within defence mechanisms marks progress, it is evident that this information alone falls short. It is imperative to delve into the facilitators of inducible responses and strategically integrate these systems—encompassing potential promoters, regulators, and receptors—into plant genetic engineering (
6 Discussions and future perspectives
Many current and future applications of CRISPR-mediated editing prioritize induction of genomic modifications followed by segregating away lines with active CRISPR systems. This is valuable when considering near-term agricultural incorporation and public acceptance (
6.1 Precise modification of amino acid sequence
Constitutive resistance responses can be engineered through gene knockout of negative regulators, though it may entail a growth trade-off. On the other hand, targeted defence necessitates precise modification of amino acid sequences on specific receptor-ligand binding sites or protein-protein interaction sites. Recognition of parasitic plant signals and effectors is the critical first step in host immunity. CRISPR base editors or prime editors offer a promising strategy to modify peptide sequences responsible for detecting pathogenic effectors while preserving signal transduction motifs (
The vulnerability of specific host plants to parasitic plants results from the failure to recognize signals or effectors, impeding effective immune responses (
FIGURE 2

Enhancing Parasitic Plant Resistance using new CRISPR Technologies. (A) Protein engineering of receptors or transcription factors via CRISPR base and prime editing modifies parasite perception and protein binding affinity. Susceptibility of certain host plants to parasitic plants results from signal or effector non-recognition, hampering immune responses. CRISPR base and prime editing on receptors allows pathogen/effector perception, initiating defence signalling. In parallel, susceptibility in some host plants arises from the inability to activate downstream resistance due to a missing link in transcriptional activation. CRISPR base and prime editing adjusts transcription factor binding affinity, bridging connections and promoting downstream defence responses. (B) Conditional immunity with inducible or cell/tissue-specific activation via CRISPR-mediated transcriptional regulation. Inducible defence responses against parasitic plants are achieved through tailored promoters that express Cas enzymes and single-guide (sg) RNAs upon sensing parasitic signals or effectors. Inactive dCas enzymes are unable to cleave DNA but can still bind specific sequences via guide RNAs. dCas proteins fused with transcriptional activators (TA) trigger resistance-associated gene expression. Cell and tissue-type-specific promoters driving dCas enzymes and sgRNA expression can confer localized defence responses. Therefore, the activation of particular target genes can be directed with CRISPR-based synthetic transcription factor complexes. This CRISPR-mediated transcriptional regulation strategy offers conditionally activated transcription for parasitic plant resistance. (C) Hypothetical illustration of synthetic mobile CRISPR application for enhancing host resistance against parasitic plants. Based on previous studies, parasitic plants haustorium not only can transport water and nutrients but can also transport miRNA, mRNA, and small peptides bidirectionally, and these mobile C. campestris molecules might act as trans-species regulators of host-gene expression and may act as effectors or virulence factors to promote parasitism. CRISPR can be applied in plant host resistance by directly targeting genes of parasitic plants. Recent advancements offer compact CRISPR-Cas variants like CasΦ and CasMINI, under half the size of traditional Cas9. These compact forms could serve as candidates transported through haustoria to directly modulate parasitic plant genes. Leveraging CRISPR KO for targeted mutation and Cas13 for highly precise transcriptional regulation. This figure was created with https://www.biorender.com/.
6.2 Inducible defence responses
Inducible defence responses are an adaptive mechanism triggered by plants upon detecting threats such as pathogens, herbivores, or parasites. This mechanism optimizes resource allocation, thereby bolstering survival and reproductive success (
CRISPR technologies are well poised to enable inducible defence response. Expression of Cas enzymes by inducible promoters enables genome manipulation only in response to specific stimuli including pathogens and parasites (
6.3 Cell-type or tissue-type specific defence mechanisms
Cell-type-specific barriers and defence mechanisms at the host and parasite interface constitute a pivotal aspect of plants’ repertoire to counteract parasitic plant incursions (
Similar to inducible defence response, cell-type-specific promoters can limit CRISPR activity to desired cell types (
6.4 Direct targeting of parasitic plant genes and miRNAs
Based on prior research, haustoria of parasitic plants serve not only as conduits for water and nutrients but also facilitate the bidirectional transport of miRNA, mRNA, and small peptides (
7 Conclusion
In harnessing the potential of CRISPR technologies for enhanced crop protection, the intricate balance between modifying defence responses and preserving crop yield becomes apparent. Through high-throughput gene editing, targeted nucleotide modifications, and synthetic gene regulation, CRISPR systems have been shown to provide immense power in gene discovery and crop improvement. CRISPR knockout in bolstering pre-attachment resistance by targeting strigolactone pathways and enhancing post-attachment defences through cell wall fortification offers promising avenues for combating parasitic plants. However, the trade-offs of genetic modifications impacting plant growth and physiology, underline the need for precise regulatory approaches. Inducible defence responses through innovative synthetic transcriptional regulation offer adaptive immunity, while cell-type specificity empowers localized defences. The precise modification of amino acid sequences using CRISPR base and prime editing presents a future of tailored immunity. The convergence of these strategies embodies a promising avenue for bolstering crop productivity and resilience, underpinning a transformative shift in agricultural practices towards more robust and sustainable solutions.
Statements
Author contributions
M-YJ: Conceptualization, Funding acquisition, Investigation, Project administration, Validation, Visualization, Writing–original draft, Writing–review and editing. EE: Conceptualization, Investigation, Validation, Writing–original draft, Writing–review and editing. NS: Conceptualization, Funding acquisition, Supervision, Validation, Writing–review and editing.
Funding
The authors declare financial support was received for the research, authorship, and/or publication of this article. M-YJ is supported by a grant made to the University of Cambridge by the Bill and Melinda Gates Foundation and the UK Foreign, Commonwealth and Development Office (OPP1028264) known as the Enabling Nutrient Symbioses in Agriculture (ENSA) project. EE is supported by the National Science Foundation Postdoctoral Research Fellowship in Biology under Grant No. 2305688. Research on parasitic plants in the NRS lab is funded by the California Tomato Research Institute (CTRI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments
We thank Eli Marable for feedback on the early draft of this manuscript.
Conflict of interest
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.
The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
Publisher’s note
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Summary
Keywords
CRISPR, parasitic plants, haustorium, resistance, defence, gene editing, inducible defence responses, cell-type specific
Citation
Jhu M-Y, Ellison EE and Sinha NR (2023) CRISPR gene editing to improve crop resistance to parasitic plants. Front. Genome Ed. 5:1289416. doi: 10.3389/fgeed.2023.1289416
Received
05 September 2023
Accepted
16 October 2023
Published
25 October 2023
Volume
5 - 2023
Edited by
Bing Yang, University of Missouri, United States
Reviewed by
Jonas De Saeger, Ghent University Global Campus, Republic of Korea
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© 2023 Jhu, Ellison and Sinha.
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*Correspondence: Min-Yao Jhu, myj23@cam.ac.uk; Neelima R. Sinha, nrsinha@ucdavis.edu
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