Identification of potential host plant mimics of CLAVATA3/ESR (CLE)-like peptides from the plant-parasitic nematode Heterodera schachtii

Abstract

In this article, we present the cloning of two CLAVATA3/ESR (CLE)-like genes, HsCLE1 and HsCLE2, from the beet cyst nematode Heterodera schachtii, a plant-parasitic cyst nematode with a relatively broad host range that includes the model plant Arabidopsis. CLEs are small secreted peptide ligands that play important roles in plant growth and development. By secreting peptide mimics of plant CLEs, the nematode can developmentally reprogramme root cells for the formation of unique feeding sites within host roots for its own benefit. Both HsCLE1 and HsCLE2 encode small secreted polypeptides with a conserved C-terminal CLE domain sharing highest similarity to Arabidopsis CLEs 1-7. Moreover, HsCLE2 contains a 12-amino-acid CLE motif that is identical to AtCLE5 and AtCLE6. Like all other plant and nematode CLEs identified to date, HsCLEs caused wuschel-like phenotypes when overexpressed in Arabidopsis, and this activity was abolished when the proteins were expressed without the CLE motif. HsCLEs could also function in planta without a signal peptide, highlighting the unique, yet conserved function of nematode CLE variable domains in trafficking CLE peptides for secretion. In a direct comparison of HsCLE2 overexpression phenotypes with those of AtCLE5 and AtCLE6, similar shoot and root phenotypes were observed. Exogenous application of 12-amino-acid synthetic peptides corresponding to the CLE motifs of HsCLEs and AtCLE5/6 suggests that the function of this class of CLEs may be subject to complex endogenous regulation. When seedlings were grown on high concentrations of peptide (10 µm), root growth was suppressed; however, when seedlings were grown on low concentrations of peptide (0.1 µm), root growth was stimulated. Together, these findings indicate that AtCLEs1-7 may be the target peptides mimicked by HsCLEs to promote parasitism.

Figure 1

Amino acid sequence analysis of HsCLEs. (a) Putative protein sequence alignment of HsCLE1 and HsCLE2 generated using the T‐Coffee program (Notredame et al., 2000). The sequences highlighted in grey correspond to the signal peptide sequences predicted by the SignalP program (Emanuelsson et al., 2007). The 12‐amino‐acid CLAVATA3/ESR (CLE) motifs are indicated by a black line. (b) An unrooted neighbour‐joining tree of nematode and Arabidopsis dodeca‐CLE peptides generated using the paup program. Bootstrap values of 50% or higher are shown. The scale bar indicates the number of amino acid substitutions per site. At, Arabidopsis thaliana; Gr, Globodera rostochiensis; Hg, Heterodera glycines; Hs, Heterodera schachtii. (c) Amino acid sequence alignment of HsCLE2, AtCLE5 and AtCLE6 generated using the T‐Coffee program. Sequences highlighted in grey correspond to signal peptide sequences. The 12‐amino‐acid CLE motifs are indicated by a black line.

Figure 2

Overexpression of HsCLE1 and HsCLE2 caused a range of wuschel (wus)‐like phenotypes in Arabidopsis. (a, f) Representative of wild‐type seedlings. (b–e, g–i) Representative wus‐like phenotypes for HsCLE1 and HsCLE2 overexpression. (a) Wild‐type seedling 3 weeks post‐germination. (b) Three‐week‐old seedling exhibiting shoot apical meristem termination. (c, d) Ten‐week‐old seedling exhibiting inflorescence meristem termination. (e) A plant exhibiting floral meristem termination resulting in reduced silique production. The inset shows a close‐up view of a silique from a wild‐type plant (left) and HsCLE overexpression line (right). (f) Wild‐type flower. (g–i) Flowers showing reduced floral organ numbers.

Figure 3

Effect of CLAVATA3/ESR (CLE) peptides on Arabidopsis root growth. (a) Average root length of Arabidopsis seedlings grown on medium containing no peptide, or 0.1, 1 or 10 µm synthetic dodecapeptide corresponding to the indicated CLE motif. Data represent the mean ± SE (n≥ 16 except for 10 µm HsCLE1 which had only n= 7). Broken line indicates the average growth of roots after 9 days in the absence of peptide. Asterisks indicate statistically significant differences compared with no peptide treatment at a probability level of P≤ 0.05. Peptide assays were conducted three independent times with similar results. (b–d) Representative root tips of Arabidopsis seedlings grown on medium with or without synthetic CLE peptides for 14 days and visualized with differential interference microscopy. Scale bar represents 50 µm. (b) No peptide. (c) Stimulated root growth for 0.1 µm HsCLE1 and HsCLE2/AtCLE5/AtCLE6. (d) Terminated root growth for 0.1–10 µm AtCLE19p12, 1–10 µm HgCLEp12, 10 µm HsCLE1p12 and 10 µm HsCLE2p12.