Haustorium initiation in the obligate parasitic plant Phelipanche ramosa involves a host-exudated cytokinin signal

Abstract

The heterotrophic lifestyle of parasitic plants relies on the development of the haustorium, a specific infectious organ required for attachment to host roots. While haustorium development is initiated upon chemodetection of host-derived molecules in hemiparasitic plants, the induction of haustorium formation remains largely unknown in holoparasitic species such as Phelipanche ramosa. This work demonstrates that the root exudates of the host plant Brassica napus contain allelochemicals displaying haustorium-inducing activity on P. ramosa germinating seeds, which increases the parasite aggressiveness. A de novo assembled transcriptome and microarray approach with P. ramosa during early haustorium formation upon treatment with B. napus root exudates allowed the identification of differentially expressed genes involved in hormone signaling. Bioassays using exogenous cytokinins and the specific cytokinin receptor inhibitor PI-55 showed that cytokinins induced haustorium formation and increased parasite aggressiveness. Root exudates triggered the expression of cytokinin-responsive genes during early haustorium development in germinated seeds, and bio-guided UPLC-ESI(+)-/MS/MS analysis showed that these exudates contain a cytokinin with dihydrozeatin characteristics. These results suggest that cytokinins constitutively exudated from host roots play a major role in haustorium formation and aggressiveness in P. ramosa.

Keywords: Brassica napus; Phelipanche ramosa; broomrape; cytokinins; haustorium; microarray; parasitic plant; root development; root exudates.

Fig. 1.

Brassica napus root exudates can trigger the formation of early haustorial structures in P. ramosa. (A–D) Confocal laser scanning microscope observation of P. ramosa germinated seeds stained with acridine orange. (E–H) Light microscope observation of thin sections of P. ramosa germinated seeds stained with toluidine blue. Open squares, embryo; filled squares, albumen; open circles, radicle; filled circles, apex; open triangles, testa; filled triangles, papillae. (A, E) Seeds before treatment. (B, F) Seeds incubated for 72 h in control buffer. (C, G) Seeds incubated for 72 h in half-diluted B. napus root exudates. Sale bars=100 µm. (D, H) Close-up view of the root apex of seeds incubated for 72 h in B. napus root exudates. Scale bars=30 µm. (I) Assessment of radicle length of P. ramosa germinated seeds upon treatment with B. napus root exudates during 72 h. Exudates were half diluted in buffer solution (0.5 mM HEPES, 0.05% PPM). The control condition corresponds to a treatment with the buffer solution alone. Means are values ±SE (n=6). Means annotated with asterisks are significantly different from the control points (t-test, *P<0.05, **P<0.01, and ***P<0.001). (J) Time course assessment of EHS development upon continuous treatment with B. napus root exudates for 6, 12, 24, 48, and 72 h, respectively. At the end of each treatment time, germinated seeds were rinsed, and exudates were discarded and replaced by buffer solution. Means are values ±SE (n=6).

Fig. 2.

Induction of EHSs at the apex of P. ramosa germinated seeds enhances parasite aggressiveness. A 20 mg aliquot of P. ramosa germinated seeds was incubated for 48 h in B. napus root exudates or control solution before placing them in contact with B. napus roots. Five plantlets per plate and five plates per condition were used. The number of developing tubercles was assessed using the phloem-mobile dye 6-carboxyfluorescein and the results were expressed as a percentage of the maximum number of tubercles observed in control conditions. Exudates were half diluted in buffer solution (0.5 mM HEPES, 0.05% PPM). The control condition corresponds to a treatment with the buffer solution alone. Means are values ±SE (n=5 plates). Means annotated with asterisks are significantly different from the control points (t-test, *P<0.05 and **P<0.01).

Fig. 3.

Gene Ontology analyses of all contigs. Pram contigs were assigned to GO slim terms for biological processes (A), molecular functions (B), and cellular components (C) using the PANTHER algorithm (http://www.pantherdb.org) (Mi et al., 2016). Numbers indicate percentages of each GO slim term within main ontologies.

Fig. 4.

Analysis of differentially expressed genes (DEGs) during haustorial development. (A) Number of DEGs with log₂ (FC) (fold change between B. napus exudate-treated/control P. ramosa germinated seeds) ≥1.5 or ≥ –0.75 at 12 h and 24 h. (B) Selected significant GO terms for pooled annotated DEGs. GO term enrichment analysis was carried out on the pooled DEGs using the AGRIGO algorithm. Fisher’s exact test (Hochberg FDR <0.05).

Fig. 5.

Effect of treatments with cytokinins on EHS induction. (A) Maximum EHS induction at the apex of P. ramosa germinated seeds after 72 h of treatment with an optimal concentration of tZOG (10^–7 M), TDZ (10^–8 M), K (10^–4 M), BAP (10^–5 M), c/tZ (10^–7 M), DHZ (10^–7 M), IP (10^–5 M), tZ (10^–7 M), DHZOG (10^–6 M), tZR (10^–6 M), DHZR (10^–5 M), DHZROG (10^–5 M), mT (10^–5 M), IPR (10^–5 M), and tZROG (10^–6 M). Means are values ±SE (n=6). Values with the same letter are not significantly different from the control points (ANOVA P<0.01). (B) Concentration-dependent stimulation by tZOG, TDZ, K, BAP, c/tZ, DHZ, IP, tZ, DHZOG, tZR DHZR DHZROG, mT, and tZROG with concentrations ranging from 10^–4 M to 10⁻¹⁰ M. EC₅₀=half-maximal effective concentration. Means are values ±SE (n=6). Values with the same letter are not significantly different from the control points (ANOVA P<0.01).

Fig. 6.

Induction of EHSs and parasite aggressiveness are controlled by cytokinins. (A) P. ramosa germinated seeds were co-treated with BAP (10⁻⁵ M) and PI-55 at concentrations ranging from 10^–6 M to 10^–9 M. The control condition corresponds to a treatment with BAP (10^–5 M) alone. Means are values ±SE (n=6). Means annotated with different letters are significantly different from the control point (t-test, **P<0.001). (B) A 20 mg aliquot of P. ramosa germinated seeds was incubated for 48 h in c/tZ (10^–7 M) or control solution before placing them in contact with B. napus roots. Five plantlets per plate and five plates per condition were used. The number of developing tubercles was assessed using the phloem-mobile dye 6-carboxyfluorescein and the results were expressed as a percentage of the maximum number of tubercles observed in control conditions. The control condition corresponds to a treatment with the buffer solution alone (0.5 mM HEPES, 0.05% PPM). Means are values ±SE (n=5). Means annotated with asterisks are significantly different from the control points (t-test, *P<0.05 and **P<0.01). (C) A 20 mg aliquot of P. ramosa germinated seeds was incubated for 48 h in BAP (10^–5 M), BAP (10^–5 M)/PI-55 (10^–6 M), or control solution before placing the seeds in contact with B. napus roots. Five plantlets per plate and five plates per condition were used. The control condition corresponds to a treatment with the buffer solution alone (0.5 mM HEPES, 0.05% PPM). Means are values ±SE (n=5). Means annotated with astericks are significantly different from the control points (t-test, **P<0.01). Means annotated with plus signs are significantly different from the BAP alone points (t-test, ⁺P<0.05 and ⁺⁺P<0.01, respectively).

Fig. 7.

Brassica napus root exudates trigger the expression of cytokinin-responsive genes. (A–C) Expression levels of cytokinin-responsive genes (PrRR5, PrCKX2, PrCKX4, and PrZFP6) in P. ramosa germinated seeds treated for 24 h with either B. napus root exudates, BAP (10^–5 M), c/tZ (10^–7 M), or purified fraction 9 measured by RT-qPCR. The values correspond to the fold change compared with the control treated germinated seeds. The control condition corresponds to a treatment with buffer solution (0.5 mM HEPES, 0.05% PPM) supplemented with the corresponding solvent. Exudates and fraction 9 were half-diluted and 130-fold diluted in buffer solution (0.5 mM HEPES, 0.05% PPM), respectively. Two biological replicates were performed, each in three technical replicates. Means are values ±SD (n=6). Means annotated with asterisks are significantly different from the control points (t-test, ***P<0.001).

Fig. 8.

Fractionation of B. napus root exudates. (A) Distribution of EHS induction activity after RP-HPLC fractionation of SPE concentrated B. napus root exudates. Half-diluted exudates and SPE extracts (1000-fold diluted) were used as positive controls, Coïc medium was used as negative control. HPLC-purified fractions were 100-fold diluted. All dilutions were performed in buffer solution (0.5 mM HEPES, 0.05% PPM). Means are values ±SE (n=3). Means annotated with asterisks are significantly different from the negative control (t-test, ***P<0.001). (B). UPLC-ESI(+)-MS/MS analysis of fraction 9 detected in the MRN channel of DHZ.