Agrobacterium-mediated Cuscuta campestris transformation as a tool for understanding plant-plant interactions

Abstract

Cuscuta campestris, a stem parasitic plant, has served as a valuable model plant for the exploration of plant-plant interactions and molecular trafficking. However, a major barrier to C. campestris research is that a method to generate stable transgenic plants has not yet been developed. Here, we describe the development of a Cuscuta transformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes (CcWUS2 and CcGRF/GIF), leading to a robust protocol for Agrobacterium-mediated C. campestris transformation. The stably transformed and regenerated RUBY C. campestris plants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. The locations of T-DNA integration in the parasite genome were confirmed through TAIL-PCR. Transformed C. campestris also produced flowers and viable transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. Furthermore, RUBY is not only a useful selectable marker for the Agrobacterium-mediated transformation, but may also provide insight into the movement of molecules from C. campestris to the host during parasitism. Thus, the protocol for transformation of C. campestris reported here overcomes a major obstacle to Cuscuta research and opens new possibilities for studying parasitic plants and their interactions with hosts.

Keywords: Cuscuta; Agrobacterium‐mediated transformation; RUBY; mobile molecules; parasitic plant; plant–plant interactions.

Fig. 1

RUBY expression in explants. RUBY expression in cut explants (a–c) and bulk chopped explants (d–f) c. 30 d after inoculation.

Fig. 2

Process of Agrobacterium‐mediated Cuscuta transformation using A. tumefaciens EHA105 containing 35S:RUBY. Cuscuta seedlings were infected by EHA105‐35S:RUBY and incubated on co‐cultivation media (CCM) for 5 d (a). Cuscuta segments expressing RUBY were transferred on callus induction media (CIM) from CCM (b). One‐month‐old RUBY calli and were grown on CIM (c, d) and shoot induction media (e, f). Calli expressing RUBY were selected and transferred to shoot elongation media (g, h) and produced shoots (i). (d, f, h) are zoomed images of (c, e, g), respectively.

Fig. 3

Cuscuta campestris transformation efficiency with the RUBY reporter gene. (a) Transformation rate on various days following inoculation. Sixteen independent replicates were performed. (b) The percentage of RUBY calli growing on shoot elongation media, RUBY calli developing shoots, and RUBY Cuscuta coiled on a live host based on 50 RUBY segments (blue) and total segments from bulk chop method (purple) were quantified. Eight independent replicates were performed. Data are presented as the mean ± SD.

Fig. 4

Transgenic RUBY Cuscuta attaching to non‐living and living hosts. A shoot from transgenic callus (a) coiled on a toothpick (b, c). (c) Zoomed‐in image of the black box in (b) showing haustoria formation. RUBY Cuscuta coiled on (d) and parasitizing (e) the stem of a 2‐wk‐old tomato. (f) RUBY Cuscuta was successfully grown on Arabidopsis. Transgenic RUBY Cuscuta produced flowers on beets (g, h) and seeds (i). WT, wild‐type.

Fig. 5

Alignment of sequences from TAIL‐PCRs and the 35S:RUBY construct. (a) Diagram of 35S promoter and RUBY near RB region in 35S:RUBY. Red arrow, yellow arrow, and gray box are RUBY, 35S promoter, and right border, respectively. Black arrows (RB1 and RB2) are primers used for the TAIL‐PCRs. (b) Schematic designs of T‐DNA sequences and insertion points into the Cuscuta campestris genome. Yellow boxes indicate 35S promoter of 35S:RUBY and white boxes are C. campestris genome sequences.

Fig. 6

Cross sections of Cuscuta haustoria on tomato and Arabidopsis stems. Haustoria of wild‐type (WT) Cuscuta campestris successfully penetrated tomato stems and established a connection with the host vascular bundle (a, b). RUBY Cuscuta coiling around a host tomato stem but failing to penetrate (c, d). RUBY Cuscuta successfully attaching to tomato is shown at early (e, f) and mature (g, h) haustoria development stages. (b, d, f, h) Zoomed‐in images of the white box region of corresponding images in (a, c, e, g). RUBY Cuscuta penetrated and developed haustoria on Arabidopsis Col‐0 stem (i). Images of the white box in (i) before staining (j) and after toluidine staining (k). Vertical section of Cuscuta haustorium on Arabidopsis Col‐0 (l). Asterisks (*), white circles, and black arrows indicate the presence of Cuscuta haustorium, area of diffused RUBY pigment, and searching hyphae, respectively. cor, cortex; ixf, xylem fibers; ph, phloem; xy, xylem.

Fig. 7

Agrobacterium‐mediated Cuscuta transformation using chopped seedlings. Three‐day‐old seedlings (a) were chopped (b) and inoculated with Agrobacterium tumefaciens strain EHA105 harboring 35S:GUS‐pMAS:GFP. (c) Infected segments were incubated on co‐cultivation media. (d–f) Approximately 35 d after inoculation, segments infected by 35S:GUS‐pMAS:GFP, 35S:CcWUS2‐GUS, and 35S: CcGRF‐CcGIF‐GUS were stained with X‐gluc solution.

Fig. 8

Effect of CcWUS2 and CcGRF/GIF on Agrobacterium‐mediated Cuscuta transformation. (a) The transformation efficiencies of GUS (blue bar), CcWUS2‐GUS (purple bar), and CcGRF/GIF‐GUS (pink bar) were compared. (b) GUS expressions were classified into four groups according to the size of GUS expression. Thirty independent replicates were performed. Data are presented as the mean ± SD and two‐way ANOVA test with Dunnett's multiple comparisons tests was performed. GIF, GRF‐INTERACTING FACTOR; GRF, GROWTH‐REGULATING FACTOR; GUS, β‐glucuronidase; WUS2, WUSCHEL; ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001.