Once Cuscuta plants find their hosts and successfully coil around them, the next step is the start of haustorium initiation and formation of the prehaustorium, which is the immature haustorial structure seen prior to penetration of the host tissues (Yoshida et al., 2016). A group of cortical cells begin to accumulate starch-containing amyloplasts and enlarged nuclei. These cells are identified as the initial cells, which will then dedifferentiate and develop into haustorial meristem cells. The disc-like meristem that emerges in the inner cortex of the Cuscuta stem is the prehaustorium primordia, consisting of two types of cells: file and digitate cells ( Figure 1E ) (Lee, 2007). This primordium is also known as the “endophyte primordium” because it develops into an “endophyte.” An endophyte refers to the inner haustorial structure that grows into the host tissues during the penetration phase (Kuijt, 1977; Lee, 2007).

Based on previous studies, the two primary triggers for Cuscuta haustorium initiation are the far-red light signal and mechanical stimulation (Tada et al., 1996; Furuhashi et al., 2011) ( Figure 2 ). Upon receiving these light signals and physical contacts, the prehaustorium structure starts to develop. Cuscuta species probably employ phytochromes to sense the change in red light and far-red light ratio and adopt phototropism signaling transduction to control the initiation of haustoria. In Arabidopsis, phytochromes are transformed from their inactive form (Pr) to their active form (Pfr) in high red-light environments. Pfr then translocates from the cytoplasm into the nucleus, where Pfr interacts with phytochrome interaction factors (PIFs). As a result of their interaction, PIFs are phosphorylated and then degraded. PIFs are transcription factors that regulate the downstream gene expression involved in skotomorphogenesis and photomorphogenesis in Arabidopsis. Cuscuta species likely co-opt similar signaling pathways and use phytochromes to control the genes involved in hormone transport or biosynthesis ( Figure 2 ) (Furuhashi et al., 1997; Furuhashi et al., 2011; Furuhashi et al., 2021; Pan et al., 2022). A previous study indicates that strong far-red light or low red to far-red ratios (low red light and high far-red light mixture condition) promoted Cuscuta stem coiling and haustorium formation (Furuhashi et al., 1997; Haidar and Orr, 1999). The results of in vivo quantification of the percentage of phytochrome status also showed that a maximum number of prehaustoria were produced when a low percentage of phytochrome is in Pfr form (Haidar and Orr, 1999). This evidence supports the model that phytochromes are in the inactive (Pr) state under intense far-red light circumstances, which prevents phytochromes from entering the nucleus. PIFs are thus unrestricted from regulation and activate the genes, including those involved in auxin and cytokinin biosynthesis or transport, needed for haustorium initiation (Furuhashi et al., 2021; Pan et al., 2022) ( Figure 2 ).

Another component needed for forming the Cuscuta haustorium is physical contact with its host (Tada et al., 1996; Furuhashi et al., 2011). Several herbaceous and woody plant species have been described that can perceive mechanical cues from their environment and respond physiologically to change their growth or morphology by a process known as thigmomorphogenesis (Börnke and Rocksch, 2018). During the past decade, progress has been made in investigating the underlying molecular mechanisms of mechanoperception and thigmomorphogenesis in several plant species (Monshausen and Gilroy, 2009; Hamilton et al., 2015; Börnke and Rocksch, 2018; Mano and Hasebe, 2021), but how these thigmomorphogenesis signaling pathways are involved in Cuscuta haustorium organogenesis remains largely unknown. Here, we propose a hypothetical model based on our current understanding of non-parasitic plant models ( Figure 2 ). Ion channels and receptor-like kinases are examples of mechanosensory proteins (Monshausen and Gilroy, 2009; Hamilton et al., 2015; Börnke and Rocksch, 2018) that may be activated by a physical contact signal with hosts. Plasma membrane-localized mechanosensitive calcium channels, like Mid1-complementing activity (MCA), trigger Ca²⁺ influx, cytosolic Ca²⁺-dependent signaling, and activate the expression of downstream genes possibly through Ca²⁺ sensors, such as calmodulin or calcium-dependent protein kinases (Nakagawa et al., 2007; Kurusu et al., 2013; Börnke and Rocksch, 2018; Mano and Hasebe, 2021). Another potential candidate is the mechanosensitive channel of small conductance (MscS) -like (MSL) family proteins, which are reported to be stretch-activated anion channels for Cl⁻ and cause alteration in the membrane potential (Haswell et al., 2008; Maksaev and Haswell, 2012; Mano and Hasebe, 2021). Receptor-like kinases (RLKs) might also play a role in triggering protein kinase cascades and altering Ca²⁺ signaling (Humphrey et al., 2007; Shih et al., 2014; Börnke and Rocksch, 2018), which in turn may affect downstream gene transcription, change hormone status and induce haustorium formation ( Figure 2 ).

In the adhesion phase, prehaustoria continue to grow toward the host and form adhesive disks (also known as holdfasts or upper haustorium). The Cuscuta haustorial epidermal cells in contact with the host proliferate anticlinally, elongate, and differentiate into secretory holdfast cells. These holdfast cells can secrete adhesive glue containing de-esterified pectins to secure the haustorium on their host’s surface and seal the gap between themselves and their host ( Figure 1F ) (Vaughn, 2002).

Once a haustorium securely attaches to its host, the haustorium development enters the penetration phase (also known as intrusive phase), and the file and digitate cells in endophyte primordium continue to divide and form inner haustorial cells. These cortex-originated inner haustorial cells then penetrate through the epidermis and cortex of their host tissues. At the tip of the penetrating haustorium, the inner haustorial cells elongate and differentiate into searching hyphae (Vaughn, 2003). Searching hyphae continue invading through the apoplastic region of host cortex tissues and searching for host vasculature via tip growth ( Figure 1G ).

The final stage of developing a functional haustorium is establishing a successful vascular connection between host and parasite. The searching hyphae continue tip growth to seek host vascular tissues. Once the searching hyphae reach the host vascular cells, they form interspecies plasmodesmata connections at the tip of the searching hyphae. These searching hyphae then convert their cell identity depending on the type of cells they contact. For instance, the searching hyphae that make contact with host xylem cells will obtain xylem identity and become xylic hyphae (Vaughn, 2006). The xylic hyphae differentiate into xylem conductive elements and form xylem bridges connecting the xylem system between the host and the parasite ( Figure 1H ). On the other hand, the searching hyphae that make contact with host phloem sieve elements will obtain phloem identity and become phloic hyphae (also known as absorbing hyphae) (Vaughn, 2006). The phloic hyphae differentiate into phloem-conductive elements with finger-like protrusions connecting the phloem system between the host and the parasite. The mechanisms involved in the transition of cell identity are currently unknown. However, based on the discovery of mRNA, small RNA, and small peptides exchanged between host and parasite via haustorial connection (Kim et al., 2014; Kim and Westwood, 2015; Shahid et al., 2018), we propose a potential hypothesis that these exchanged signals likely function as signals to facilitate cell identity conversion, which will be of interest for future investigation.

A stem parasitic plant-specific model system is required because the mechanisms involved in haustorium induction differ between root and stem parasitic plants. For example, many studies indicate that detecting quinones or phenolics from the hosts, known as haustorium-inducing factors (HIFs), is a critical factor in inducing haustorium development for root parasitic plants, like those seen in the Orobanchaceae (Chang and Lynn, 1986; Goyet et al., 2019). On the other hand, stem parasites use tactile stimuli and light signals for haustorium induction (Tada et al., 1996; Furuhashi et al., 2011). These different factors required for triggering haustorium formation show the distinct strategies needed to adapt to underground and aboveground parasitism. For underground parasitism, physical contact and light signals would not be effective searching mechanisms for host root systems surrounded by soil. Consequently, the ability to detect host-specific HIFs would be the primary criteria for root parasitic plant haustorium induction. Therefore, using Cuscuta species for studying stem parasitic plant haustorium organogenesis could help us understand the unique mechanisms deployed in aboveground parasitism.

Besides serving as a sound system for studying the distinctive mechanisms involved in haustorium initiation, Cuscuta species also perform some unique parasitizing behaviours that are currently underinvestigated, including hyperparasitism (Wilson and Calvin, 2017), autoparasitism (Krasylenko et al., 2021), and cross-organ parasitism (Jhu and Sinha, 2022). These unique parasitizing behaviours among multiple parasitic plants have been previously reviewed (Krasylenko et al., 2021; Jhu and Sinha, 2022). Therefore, here, we focus on providing clear definitions with concise discussion and then propose hypotheses to understand why Cuscuta species evolved these unique parasitizing behaviours.

Hyperparasitism indicates the phenomenon of a parasitic plant parasitizing another parasitic plant. For stem parasites that attach to the aerial part of their host, this phenomenon is also known as epiparasitism because they grow “on top of” another parasite (Wilson and Calvin, 2017). Several previous studies have reported Cuscuta hyperparasitism on other parasitic plants. Hyperparasitism can happen between species or within the same species (Hawksworth, 1996). When a parasitic plant parasitizes the same parasitic plant species, this is known as autoparasitism. One specific type of autoparasitism is when the parasitic plants form functional haustoria on themselves, which is also known as self-parasitism (Fineran, 1965). In Cuscuta species, both autoparasitism and self-parasitism are commonly observed (Krasylenko et al., 2021). This is a distinctive parasitism strategy compared with several facultative or obligate root parasitic plants, which have self- and kin-recognition mechanisms to avoid forming haustoria on themselves or their similar relatives.

Deploying hyperparasitism and autoparasitism strategies might be the evolutionary consequence of adapting to aboveground parasitism. One hypothesis proposed by McLuckie is that self-parasitic haustoria in Cassytha or Cuscuta species might facilitate water conduction and long-distance transport (McLuckie, 1924). In addition, based on the observation that Cuscuta species also often form attachments to non-biological materials (Bernal-Galeano et al., 2022), we propose that the formation of these non-conductive haustoria or self-parasitic haustoria might both serve as physical support for Cuscuta to spread over a wider area and reach longer distances, which increases the possibility of finding new hosts and is therefore, beneficial for parasite survival.

Cross-organ parasitism refers to the phenomenon of a parasitic plant forming nonconventional haustorial attachments with other host organs (Jhu and Sinha, 2022). For example, C. campestris is known as a parasitic stem plant but has been reported to be able to form haustoria on tomato seedling roots (Jhu and Sinha, 2022). The reason why Cuscuta species evolved to have the ability to parasitize different organs of their host is still a mystery. However, the mechanisms of haustorium induction and host-specificity might contribute to these characteristics. Previous studies indicate that parasitic plants that can form self-parasitic haustoria, like Cuscuta, Cassytha and some hemiparasitic Orobanchaceae, can form haustoria without depending on detecting host-specific HIFs and are also more likely to show hyperparasitism (Krasylenko et al., 2021). These parasites usually also have a wider host range. To parasitize different host species, which might have different anatomical structures, having a higher degree of haustorium plasticity and HIF-independent haustorium induction might be criteria to adapt to different host structures and form successful haustorial connections with them. Therefore, this higher degree of haustorium plasticity might also confer on Cuscuta the ability to form haustoria on different organs of their hosts.

Another advantage of using Cuscuta species as a model organism is the availability of whole genome information and different types of transcriptome data. The genomes of C. campestris and C. australis were both published in 2018 (Sun et al., 2018; Vogel et al., 2018). The Cuscuta transcriptome profiles in different developmental stages and tissue types are readily available (Ranjan et al., 2014; Jhu et al., 2021; Jhu et al., 2022; Bawin et al., 2022). Cuscuta mobile mRNAs and miRNAs that can transfer into the host plants are also reported (Kim et al., 2014; Shahid et al., 2018). These resources will facilitate research on Cuscuta development and the interaction between hosts and Cuscuta plants.

Another key criterion that makes an organism a good model organism is the ability to propagate or reproduce easily in vast numbers. Cuscuta species can quickly propagate through vines and produce many fruits and seeds, which can remain viable for more than ten years (Lanini and Kogan, 2005; Goldwasser et al., 2012; Masanga et al., 2022). These characteristics make them excellent model organisms. In addition, as mentioned in previous sections, many studies have shown that far-red light signals and physical contacts can trigger Cuscuta haustorium formation (Furuhashi et al., 1995; Tada et al., 1996; Furuhashi et al., 1997; Haidar and Orr, 1999). Therefore, several in vitro methods of growing Cuscuta or inducing haustorium formation without hosts have also been developed simultaneously (Kaga et al., 2020; Jhu et al., 2021; Bernal-Galeano et al., 2022). These methods allow us to investigate the function of genes in haustorium organogenesis more effectively and remove the influence or variations created by host conditions.

To study the function of genes, generating mutants or transgenic plants with specific gene knockouts or gene overexpression is one critical approach. Unfortunately, the current major limitation of using Cuscuta species as a model is lack of efficient transformation methods to obtain stable transgenic lines. Several methods for producing successful transformation events have been published in different Cuscuta species, including Cuscuta trifolii, Cuscuta reflexa, and Cuscuta europaea (Borsics et al., 2002; Švubová and Blehová, 2013; Lachner et al., 2020). However, regenerating the transformed cells into calli and whole plants is still an unsolved issue. Developing a transformation system to generate stable transgenic Cuscuta plants will be a significant breakthrough technology.

At the same time, scientists have been developing different tools and methods for functional gene studies to bypass and overcome the current transformation obstacles. Based on previous studies, Cuscuta haustoria not only transport water and nutrients but also can transport small RNAs, messenger RNAs and small peptides (Kim et al., 2014; Kim and Westwood, 2015; Shahid et al., 2018; Wu, 2018; Johnson and Axtell, 2019). Therefore, host-induced gene silencing (HIGS) is a commonly used method to knock-down specific gene expressions in Cuscuta by attaching them to transgenic hosts that carry RNA interference (RNAi) constructs targeting Cuscuta genes (Albert et al., 2006; Alakonya et al., 2012; Jhu et al., 2021; Jhu et al., 2022). This method has been used widely in the Cuscuta research field to conduct functional analysis on genes of interest.