A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

Abstract

The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium‐mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T‐DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T‐DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk’s epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA.

A host‐free transcriptome for haustoriogenesis in Cuscuta campestris: Signature gene expression identifies markers of successive development stages

“…Because a host is lacking, there is no further incentive for the haustoria to differentiate beyond the penetrating stage, although feeding‐hyphae‐like structures can sometimes be observed when the haustorium is prevented from drying out (data not shown). Recent observations that a fluorescent dye was taken up with amazing speed by the haustoria and the surrounding adhesive ring (Lachner et al, 2020), support the notion that some form of uptake competence may already exist at this point. More investigations are needed in the future to explore this possibility.…”

“…In addition, the assignment of the harvested tissue samples to a development stage may differ, depending on the experimenter's judgment and morphological observation skills. The publication of the genome sequence of Cuscuta campestris (Vogel et al, 2018) and Cuscuta australis (Sun et al, 2018), the possibility to study fluorescent fusion proteins in calli (Švubová & Blehová, 2013) and the adhesive disks of the infection organs (Lachner et al, 2020), and the development of an advanced artificial host system providing control of the parasite environment throughout its life cycle (Bernal‐Galeano & Westwood, 2021) are just some of the recent milestones in parasitic plant research that helped to ascertain Cuscuta a role as a shoot parasitic model. In order to be able to draw conclusions across datasets from past (Ranjan et al, 2014), present (Jhu et al, 2021; this study) and future transcriptomic studies, it is highly desirable to overcome potential shortcomings in morphological sample assignment and to identify robust molecular markers that are highly specific for the different developmental stages.…”

“…The possibility to replace one highly variable factor (the host) can provide points of reference for future studies and may help to decipher how different aspects of haustorium development are controlled. The most commonly used way of host‐free haustorium induction is a combination of far‐red (FR) light and tactile stimuli (Lachner et al, 2020; Olsen et al, 2016; Tada et al, 1996), although also blue light was shown to induce haustoria (Haidar, 2003; Kaga et al, 2020). Host‐free induced haustoria are easy to monitor, and they exhibit a more uniform and predictable development compared to host‐induced haustoria.…”

Cuscuta species: Model organisms for haustorium development in stem holoparasitic plants

“…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.…”

The Enigma of Interspecific Plasmodesmata: Insight From Parasitic Plants

“…On the parasite side, the first genome sequences have been published for Cuscuta (Sun et al, 2018 ; Vogel et al, 2018 ). Although transgenic parasitic plants cannot yet be produced efficiently, recent progress gives reason to believe that genetic manipulation of these parasites will soon be a standard (Lachner et al, 2020 ).…”