Broomrape (Phelipanche aegyptiaca Pers) is a parasitic plant known for inflicting significant economic losses in melon production. This holoparasite attaches itself to the root of the melon plants and establishes a connection with the vascular tissue to absorb nutrients. This parasitic interaction profoundly impairs host development and substantially diminishes melon yields (Bao et al., 2017). The attachment between broomrape and its host plant is facilitated through specialized structures called haustorium, which enable bidirectional movement of photosynthates and other essential macromolecules between the host and parasite, including microRNAs (miRNAs) (Bao et al., 2017). miRNAs, a class of endogenous single-stranded non-coding RNAs spanning 20–40 nt, are widespread in plants (Nizampatnam et al., 2015; Song et al., 2019; Cui et al., 2020). They play an important regulatory role in plant growth, development, hormone signal transduction, and stress response. Remarkably, miRNAs can even exert regulatory influence over gene expression in different species, highlighting their capacity for cross-species regulation (Guo et al., 2017, 2021). In addition, some miRNAs exhibit the ability to traverse between host and parasite plants, resulting in the reverse silencing of target genes, leading to the phenomenon of “trans-kingdom RNA silencing.” This intriguing process involves the transfer of miRNAs and subsequent reverse silencing of target genes, thus influencing gene expression across the plant-parasite interface (Zhang et al., 2016a,b).

The phenomenon of trans-kingdom RNA silencing was first discovered in the interaction between plants and fungi. Notably, Botrytis cinerea demonstrated the ability to transfer a repertoire of small RNAs into plant cells, thereby triggering the silencing of immune response genes across diverse hosts, including Arabidopsis and tomato (Weiberg et al., 2013). Subsequent investigations unveiled the mechanism by which pathogenic small RNAs, interfacing with Arabidopsis AGO1 and harnessing the host RNAi machinery, silence pivotal host genes such as MAPK kinases related to cell wall integrity and genes involved in reactive oxygen species accumulation (Weiberg et al., 2013). This strategic employment of small RNAs enabled the pathogen to seize control of host immune responses, thereby facilitating successful invasion (Zhang et al., 2016a,b, 2022). The trans-kingdom miRNAs represent an innovative class of effector molecules adept at quelling host immune response. Notably, the miRNA (Bc-siR37) from Bostalospora cinerea was significantly enriched in the host Arabidopsis during the early stage of invasion. Bc-siR37 targeted a minimum of 15 genes, including WRKY transcription factors and cell wall modifying enzymes in Arabidopsis (Wang B. et al., 2017). Trans-kingdom miRNAs regulate host target genes to help pathogens successfully invade and colonize. Similarly, in the context of wheat stripe rust, Pst-mi1R1 was identified as a key pathogenic factor of the pathogen, which perturbs the host by targeting the gene SM638 in the genome of the host wheat. Partial silencing of SM638 in wheat significantly reduced the resistance to wheat stripe rust in resistant varieties (Wang M. et al., 2017). Recent results show have extended the scope of trans-kingdom RNA silencing to parasitic plants, exemplified by Cuscuta campestris. Endogenous miRNAs from this parasitic plant can be transported to the host Arabidopsis thaliana, guiding and modulating the parasitization process by regulating the expression of different target genes in the host (Hudzik et al., 2023). This study underscore the intricate involvement of miRNAs in the host immune pathway, shedding light on a previously uncharted aspect of parasitic interactions.

The trans-kingdom RNAi constitutes a pivotal mechanism in bolstering plant defenses against pathogens, exhibiting bidirectional transmission capabilities between host and pathogen. Trans-kingdom RNAi vectors were constructed using Host-Induced Gene Silencing (HIGS) to silence pathogen virulence-associated target genes, thereby allowing the host to acquire resistance against the pathogen. Guo et al. (2017, 2021) used HIGS technology to produce genetically stable transgenic cotton. Herein, the VdH1 gene of Verticillium dahlia, pivotal for nuclear integrity, was effectively silenced, causing a reduction in virulence due to the loss of nucleus formation in the pathogen. This study is the first to demonstrate the potential of HIGS technology in plant disease resistance research (Zhang et al., 2016a,b). In a similar vein, Govindarajulu et al. (2015) harnessed HIGS technology to produce transgenic lettuce resistant to Bremia lactucae. By attenuating a key lettuce gene through RNAi, resistance against the pathogen was realized (Zhang et al., 2016a,b). Likewise, Chen et al. (2016) yielded transgenic wheat with enhanced resistance against Fusarium graminearum through HIGS-mediated silencing of the FcGls1 gene, impairing mycelial cell wall integrity. These remarkable instances offer valuable insights for crop breeding via inter-species RNAi applications.

Evidently, trans-kingdom miRNAs exhibit a propensity for horizontal transfer across several species, such as from humans to malignant malaria-causing insects (Plasmodium falciparum) (LaMonte et al., 2012); from bacteria to insects (LaMonte et al., 2012); from parasitic plants to host plants (Shahid et al., 2018; Hudzik et al., 2023); from plant to insect (Ibrahim et al., 2011); from fungal pathogens to plants (Weiberg et al., 2013), from plant-to-fungal pathogens (Cai et al., 2018); and even reciprocal transfers between plants and insects (Wang et al., 2016). However, a notable gap exists in the exploration of transferable miRNAs in melon plants resistante to the parasitic broomrape. This research aims to unravel the intriguing implications of this novel defense strategy within the interaction between melon and broomrape. By delving into the dynamics of trans-kingdom RNAi, it aspires to shed light on the broader landscape of cross-border RNAi application in plant disease management. Furthermore, this research holds the potential to guide the evolution of pathogenic bacteria as miRNA targets, thereby advancing innovative strategies for combatting broomrape in melon cultivation.

miRNAs represent a class of non-coding RNAs pivotal for regulatory functions that exert critical influences in almost all aspects of plant growth and development. Additionally, they play important regulatory roles in response to environmental variables such as light and nutrients, and both biotic and abiotic stresses (Budak et al., 2015; Shriram et al., 2016; Aslam et al., 2022; Kajla et al., 2023). The advent of small RNA sequencing, based on high-throughput sequencing, has effectively dismantled barriers in miRNA research, facilitating a more incisive exploration of their biological functions. While some reports have used small RNA sequencing to study melon miRNAs, the context of broomrape resistance in melon remains elusive. In this study, a total of 979 known miRNAs and 110 novel miRNAs were identified, belonging to 111 miRNA families. Intriguingly, the MIR156 family emerged as the most prolific, associated with plant resistance and accumulation of secondary metabolites (Cui et al., 2014; Wang, 2014; Rahimi et al., 2022). The results imply a potentially pivotal role for this miRNA family in the resistance of melon to broomrape infestation.

In this study, small RNA sequencing was performed to identify and quantify the expression of miRNAs in the root systems of susceptible and resistant melon lines before and after being infected by broomrape. The differential expression profiles were constructed by comparing the miRNA expression of resistant and susceptible strains. The Venn diagrams unveiled noteworthy distinctions, with cme-miR398a and cme-miR408 standing out as significantly divergent across all examined combinations. It is worth stating that while we identified the differentially expressed miRNAs in resistant and susceptible lines by small RNA sequencing, the target genes of these miRNAs and their regulatory functions remain unknown, which can be well addressed by degradome sequencing. In comparison, predictive target genes based on sequence complimentarity often entail high rates of false positives. Alternately, experimental approaches, though comprehensive, suffer from limitations of expense and time. In this context, degradome sequencing presents an elegant solution. The degradome sequencing technology is based on the principle that plant miRNAs can shear target mRNAs. The intersection of the sheared mRNA fragments are collected and sequenced by high-throughput sequencing technology, thereby allowing the prediction of miRNA target genes by Target finder software. The degradome sequencing effectively overcome the drawbacks and defects of the previous methods for identifying miRNA targets. The target genes of these two miRNAs encode cysteine-rich repeat secretory protein and copper-transporting ATPase. A recent study showed that both proteins are associated with the defense response of the organisms (Tao et al., 2021). Therefore, we speculate that these two miRNAs and their target genes play an important role in the resistance of melon to broomrape infestation.

It is well documented that there is a complex transfer of substances between the host and the parasitic organism, including miRNA, and that these substances have essential effects on both parties after the transfer. miRNAs can be transferred through the host to the parasitic plant and improves the host resistance by RNA interference. It has been shown that Cuscuta campestris transfers a large number of miRNAs to the host Arabidopsis and Benthamiana, which may function as virulence factors during parasitization as cross-species regulators of host gene expression. However, the extent of long-distance miRNA movement during broomrape parasitization has remained a question. The results of this study show that certain miRNAs are transferred from resistant melon varieties to broomrape. One of the screened transfer miRNAs, cme-miR156a, is a very conserved small non-coding RNAs in plants, and its target gene family is the SPL (SQUAOSA promoter binding like) family (Matthew et al., 2002; Bonnet et al., 2010). The miR156-SPL pathway plays a regulatory role in several plant growth and developmental processes, including phytohormone signaling and stress response (Wang, 2014; Lei et al., 2016; Miao et al., 2019). Therefore, it is hypothesized that the melon can initiate defense response through the overexpression and transfer of miR156 to broomrape, thus affecting the infestation process of broomrape. In addition, miR398a is mainly involved in the negative regulation of dismutase genes (Raichur et al., 2007; Zidenga et al., 2012). When melon was infected by broomrape, the expression of cme-miR398a in the roots was elevated, indicating that cme-miR398a may increase ROS content by inhibiting the activity of reactive oxygen species (ROS) enzymes in the cells at the infected site, which eventually resulted in an allergic necrotic response and prevented broomrape infestation.

In this study, a combination of small RNA sequencing and degradome sequencing was used to identify the differentially expressed miRNAs and their target genes between susceptible and resistant lines when infested by broomrape and to explore the defense response of melon against broomrape. In addition, 4 transferable miRNAs were identified in the analysis of melon and broomrape interactions. This comprehensive investigation into the novel defense strategy that underpins melon’s interaction with broomrape furnishes valuable reference insights, with implications extending to the broader application of cross-border RNA interference in the realm of plant disease control.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

J-CM: Writing – original draft, Writing – review & editing. MY: Writing – review & editing. J-HL: Funding acquisition, Writing – review & editing. J-YY: Writing – original draft. H-JW: Funding acquisition, Writing – original draft, Writing – review & editing.