An artificial host system enables the obligate parasite Cuscuta campestris to grow and reproduce in vitro

Abstract

Cuscuta campestris is an obligate parasitic plant that requires a host to complete its life cycle. Parasite-host connections occur via a haustorium, a unique organ that acts as a bridge for the uptake of water, nutrients, and macromolecules. Research on Cuscuta is often complicated by host influences, but comparable systems for growing the parasite in the absence of a host do not exist. We developed an axenic method to grow C. campestris on an artificial host system (AHS). We evaluated the effects of nutrients and phytohormones on parasite haustoria development and growth. Haustorium morphology and gene expression were also characterized. The AHS consists of an inert, fibrous stick that mimics a host stem, wicking water and nutrients to the parasite. It enables C. campestris to exhibit a parasitic habit and develop through all stages of its life cycle, including production of new shoots and viable seeds. The phytohormones 1-naphthaleneacetic acid and 6-benzylaminopurine affect haustoria morphology and increase parasite fresh weight and biomass. Unigene expression in AHS haustoria reflects processes similar to those in haustoria on living host plants. The AHS is a methodological improvement for studying Cuscuta biology by avoiding specific host effects on the parasite and giving researchers full control of the parasite environment.

Figure 1

Schematic depiction of the AHS and the key steps needed to grow Cuscuta campestris on the AH. dpi are counted from the day of assembly when the parasite was attached to the AH.

Figure 2

Cuscuta campestris grows without a plant host under the controlled conditions of the AHS. A–F, Cuscuta campestris 3 cm shoot tips were used as inoculum. A, 3 cm shoot tips attached to an AH at 0 dpi. B, Parasite coiled with holdfasts after 2.5 dpi. C, Parasite at 15 dpi, growth is evident as flower development (bottom arrow) in plant No. 1 and production of a new shoot (top arrow) in plant No. 2. D, New shoots elongated, 36 dpi. E, Parasite flowered and produced seeds (inset; scale is in millimeters). F, Cuscuta campestris attached to an AH without any media or water at 36 dpi. Parasite did not develop swollen holdfast or subsequent growth. G, Seedlings after scarification and germination of seeds obtained from C. campestris grown in the AHS. H, Seedling from seed obtained through AHS colonizing Arabidopsis 10 dpi. I, Seedling growing 36 dpi after being used as inoculum in a second AHS.

Figure 3

Cuscuta campestris growing in the AHS exposed to the fluorescent dye CFDA or DMSO solvent control from the AH. A, Haustorial region. Cells of the holdfast show fluorescence. Holdfast (Hf), Haustorium (H). B and C, Scale leaves protecting apical and lateral meristems. Scale bar in the bottom right panel refers to all panels.

Figure 4

Characterization of C. campestris coiling and Hf morphology in the AHS. A, Frequencies of coiling and Hf production evaluated at 36 dpi. The experiment was repeated three independent times, with an average of 15 replicates. Statistical differences were detected by ANOVA. A post hoc Tukey’s Honest Significant Difference (HSD) test was applied for frequency of Hf production. Differences with P < 0.05 are indicated with an asterisk. Graph presents means between the three experiments and ±se. B–G, Hf morphology as influenced by phytohormones in the media. Holdfasts of parasites growing with MMS media (B), MMS + BA (C), MMS + 1-NAA (D), MMS + NAA+BA (E) or just water (F). G, Parasite growing on an AH without any media, “Nothing.” H, Holdfasts (arrow heads) of C. campestris growing on N. benthamiana. Pictures were taken at 8 dpi.

Figure 5

Anatomy of parasitic organs produced by C. campestris growing on an AH with MMS media with NAA and BA. A, Haustorial region inner face view. Haustoria (H) are observed in the center of the parasitic organ surrounded by the adhesion disk or holdfast (Hf). Upon removal from the AH, paper fibers (P) remain attached to the haustorial region. B, Lateral view of haustorial region with visible holdfast and protruding haustoria. Dotted line circle indicates a parasitic organ. C, Haustorial region attached to an AH. Orange dotted line indicates the sagittal plane of the haustorial region, sectioning direction used for (D–H). Dotted blue line indicates the transverse plane of the haustorial region, sectioning direction used for (I–L). D and G, Sections of C. campestris (Cc) growing on stems of N. benthamiana (Nb), with orange dotted lines in (G) indicating the epidermis of the host. E, F, and H, Cuscuta campestris growing on AHs. (D–F) were stained with phloroglucinol–HCl and fuchsia color indicates components of lignin. (G, H, and I–L) were stained with toluidine blue-O. H–L, Organized growth of haustorial cells on AH. H, Prehaustorial growth attached to AH, with star indicating outer layer of spindle. For (I–L), C. campestris was detached from the AH before sectioning, with the star indicating the face of C. campestris haustoria that had been in contact with the AH. I, Parasitic organ in an early developmental stage. Holdfast epidermal cells on the face were in contact with the host, so represent the adhesion disk. Yellow dotted line circles meristematic cells (Mc). J, A more developed parasitic organ showing File cells (Fc) and Digitate cells (Dc) positioned toward the side of the parasite facing the AH. K, Mature parasitic organ with elongated cells protruding through the middle of the Hf toward the AH. Searching hyphae are visible in the surface of the haustorium. L, Higher magnification of section of C. campestris in (K). Segments of tracheary elements (ring structures, indicated by arrows) are observed pointing in the direction of the haustorium tip that faces the AH. Sections in (E) and (F) were 20 dpi, (H) was 16 dpi and (I–L) were 36 dpi. Different stages of parasitic organ development were observed at 36 dpi in the same haustorial regions.

Figure 6

Cuscuta campestris growth in the AHS supplied with different media. Parasite fresh weight (A), parasite biomass (B), and parasite total length (C) were measured at 36 dpi. Different media were tested: MMS media, MMS with BA, MMS with NAA, MMS with NAA and BA, and water alone. Statistical differences were detected by ANOVA with post hoc Tukey’s HSD test. Differences were considered statistically significant at P < 0.05 and indicated with different letters. Data are pooled from three independent experiments. Analysis included only plants that coiled and developed healthy holdfasts. Number of samples ranged from 39 to 51 per treatment. The center line in the boxes of the boxplot shows the median. The bottom and top of the boxes correspond to the 25th and 75th quantiles, respectively. Whiskers extend 1.5*IQR (interquartile range). Dots are observations, with those above or below the end of the whiskers being outliers.

Figure 7

Effect of AHS media composition on the ability of C. campestris to generate parasitically competent shoots, flowers, and fruits. A, The percentage of plants with active shoots at 36 dpi. Active shoots were defined as those with tips of at least 3 cm of healthy, turgid shoot, which are characteristic of shoots capable of forming new host connections. B, The percentage of plants with flowers only or with fruit production. Analyses included only plants that coiled and developed a healthy holdfasts. Graphs present means ± se between three independent experiments, each experiment with 6–19 plants per treatment. Statistical differences were detected by ANOVA with post hoc Tukey’s HSD test. Differences were considered statistically significant at P < 0.05 and indicated with different letters.

Figure 8

DEUs found in the haustorial region of C. campestris growing in the AHS, and comparison with upregulated unigenes reported in other haustoria developmental conditions. Left: Volcano plot showing the distribution of upregulated and downregulated DEUs (20,749 DEU, P-adj < 0.05) found in the haustorial region of C. campestris growing in the AHS. The red line corresponds to a P-adj of 0.01. Colors of data points correspond to those of the Venn diagram at right. Right: Venn diagram showing the intersection of DEUs reported for three categories of haustoria developmental conditions. First category includes upregulated and downregulated unigenes in functional haustorial regions of parasites growing in AHS (18,565 DEU with P-adj < 0.01 were included). The second category corresponds to up regulated unigenes in immature haustorial tissue, 87 h after induction of haustoria triggered by contact with a host leaf (Kaga et al., 2020). The third category includes upregulated unigenes expressed in functional haustorial regions of C. campestris growing on plant hosts (Kim et al., 2014; Ranjan et al., 2014).