CRISPR gene editing to improve crop resistance to parasitic plants

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.

Keywords: CRISPR; cell-type specific; defence; gene editing; haustorium; inducible defence responses; parasitic plants; resistance.

FIGURE 1

Utilizing CRISPR Techniques to Enhance Pre-attachment and Post-attachment Defence Mechanisms against Parasitic Plants. (A) Overview of a CRISPR-based approach to reinforce the host plant’s resistance mechanisms against stem parasitic Cuscuta species during and after attachment. The cellular receptor CUSCUTA RECEPTOR 1 (CuRe1) is a leucine-rich repeat (LRR) receptor-like protein (RLP) responsible for recognizing Cuscuta-derived factors at the cell surface (Hegenauer et al., 2016; Hegenauer et al., 2020). Teaming up with the coreceptor SlSOBIR1, this recognition event initiates downstream defensive reactions, including hypersensitive responses. In resistant tomato cultivars, the Cuscuta R-gene for lignin-based resistance 1 (CuRLR1) is an N-terminal coiled-coil (CC)-nucleotide-binding site (NBS)-LRR protein (Jhu et al., 2022a). CuRLR1 might be involved in sensing specific signalling pathways or even function as a receptor for identifying unknown signals or effectors produced by Cuscuta. Activation of CuRLR1 sets off subsequent signalling sequences, leading to the activation of genes participating in the lignin biosynthesis pathway. Consequently, there is a buildup of lignin in the cortex region of the tomato stem, acting as a physical barrier to hinder haustorium penetration. Transcription factors like Lignin Induction Factor 1 (LIF1; an AP2-like transcription factor) and MYB55 positively regulate enhanced resistance based on host lignin. Conversely, WRKY16, which experiences upregulation upon infestation by Cuscuta campestris, plays a critical role as a negative regulator of lignin production and the function of LIF1. Based on previous research, one hypothesis suggests that WRKY16 acts as a connecting link (indicated by a dashed arrow) between CuRe1 and the lignification response. By employing CRISPR technology to target and knockout WRKY16 precisely, a sustained accumulation of lignin is achieved, thereby reinforcing the plant’s resilience against C. campestris. (B) Overview of CRISPR Applications for Reinforcing Pre-attachment Resistance by Impeding Seed Germination of Root Parasitic Plants. The biosynthesis of strigolactones (SLs), orchestrated by the carotenoid pathway involving genes like More Axillary Growth 1 (MAX1), is a pivotal mechanism explored for enhancing pre-attachment resistance. The MAX1 genes encode cytochrome P450 monooxygenases of the CYP711A subfamily, acting as carlactone (CL) oxidases responsible for converting CL into carlactonoic acid. CRISPR-based knockout generated max1 mutant lines demonstrate heightened resilience against the root parasitic plant Phelipanche aegyptiaca. This resilience is attributed to reduced SL levels due to max1 mutant. LOW GERMINATION STIMULANT 1 (LGS1), encoding a sulfotransferase enzyme, is pivotal in SL biosynthesis. In susceptible sorghum host plants, the principal SL in root exudates is 5-deoxystrigol, a potent stimulant for root parasitic plant Striga seed germination. In contrast, orobanchol, an SL with an opposing stereochemistry to 5-deoxystrigol, fails to induce Striga seed germination. By leveraging CRISPR technology, targeted mutations in LGS1 facilitate a shift in the dominant SL composition within host plant root exudates. This composition changes from 5-deoxystrigol to orobanchol, significantly reducing parasite seed germination rates. Consequently, these altered root exudates enhance pre-attachment resistance in the host plants. The three-dimensional structural representations of carlactone, carlactonoic acid, orobanchol, and 5-deoxystrigol are from PubChem. Text highlighted in red indicates the key reinforced resistance responses, while text highlighted in blue signifies the potential trade-off side effects associated with constitutively activated resistance responses. This figure was created with https://www.biorender.com/.

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/.