Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation

Abstract

Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular procambium/cambium are regulated by CLE-receptor kinase-WOX signaling modules. Previous data showed that cyst nematode CLE-like effector proteins delivered into host cells through a stylet, act as ligand mimics of plant A-type CLE peptides and are pivotal for successful parasitism. Here we report the identification of a new class of CLE peptides from cyst nematodes with functional similarity to the B-type CLE peptide TDIF (tracheary element differentiation inhibitory factor) encoded by the CLE41 and CLE44 genes in Arabidopsis. We further demonstrate that the TDIF-TDR (TDIF receptor)-WOX4 pathway, which promotes procambial meristem cell proliferation, is involved in beet cyst nematode Heterodera schachtii parasitism. We observed activation of the TDIF pathway in developing feeding sites, reduced nematode infection in cle41 and tdr-1 wox4-1 mutants, and compromised syncytium size in cle41, tdr-1, wox4-1 and tdr-1 wox4-1 mutants. By qRT-PCR and promoter:GUS analyses, we showed that the expression of WOX4 is decreased in a clv1-101 clv2-101 rpk2-5 mutant, suggesting that WOX4 is a potential downstream target of nematode CLEs. Exogenous treatment with both nematode A-type and B-type CLE peptides induced massive cell proliferation in wild type roots, suggesting that the two types of CLEs may regulate cell proliferation during feeding site formation. These findings highlight an important role of the procambial cell proliferation pathway in cyst nematode feeding site formation.

Fig 1. Identification of B-type CLE mimics from cyst nematodes.

(A) 12-aa alignment of nematode B-type CLE peptide with Arabidopsis TDIF and Selaginella kraussiana TDIF (SkCLE1) (B) Suppression effects of Arabidopsis TDIF and HsCLEB on tracheary element differentiation. Leaf disks from wild-type and TDRp:GUS plants were precultured with bikinin and then were transferred to media without bikinin or with Arabidopsis TDIF or HsCLEB. (C) Relative expression level of WOX4 and AtHB8 in Col-0 and tdr-1 seedlings that were treated with Arabidopsis TDIF P9A, Arabidopsis TDIF, HsCLEB P9A, and HsCLEB for 7 days. AtUBC21 gene (At5g25760) was used as internal control. Error bars represent SD of the means (n = 3). Asterisks indicate statistically significant differences according to Student’s t-test (P < 0.01). The experiments were repeated three times with similar results.

Fig 2. Expression pattern of TDIF-TDR-WOX4 pathway upon nematode infection.

Three Arabidopsis promoter:GUS lines including (A) TDRp:GUS. (B) WOX4p:GUS. (C) CLE41p:GUS were infected with H. schachtii. GUS staining was performed at different time points (days) after inoculation (dpi). For cross-sectioning, roots at 3 dpi were used. N = nematode; S = syncytium.

Fig 3. Expression of TDR at early stages of nematode infection.

TDRp:GFP expression was evaluated at nematode feeding sites after 1 dpi (A) and 2 dpi (B-D). (A) and (B) pictures the same nematode feeding site. (D) is a higher magnification of the feeding site shown in (C). N = nematode; AC = adjacent cell; ISC = initial syncytial cell.

Fig 4. Involvement of TDIF-TDR-WOX4 in syncytium formation.

(A) Nematode infection is decreased in tdr-1 wox4-1 mutant. Average number of females per plant was counted at 14 d and 30 d post inoculation. (B) Syncytium size is reduced in tdr-1, wox4-1, and tdr-1 wox4-1 mutants. Syncytium area was measured at 14 d post inoculation. Error bars represent SE of the means (n = 36). Asterisks indicate statistically significant differences compared with Col-0 by Student’s t-test (*P < 0.05 and **P < 0.01). The experiments were performed three times with similar results.

Fig 5. Reduced WOX4 gene expression in clv1-101clv2-101rpk2-5 mutant roots.

(A) WOX4 gene expression under nematode infection. Small root pieces containing nematode infection sites were cut from wild type and clv1-101 clv2-101 rpk2-5 mutant plants at 5 d after nematode inoculation. (B) WOX4 gene expression under HsCLE2 peptide treatment. Wild type and clv1-101 clv2-101 rpk2-5 plants were grown on vertical plates without and with HsCLE2 peptide for 5 days and whole roots were cut for qRT-PCR analysis. For qRT-PCR, AtUBC21 gene (At5g25760) was used as internal control. Error bars represent SD of the means (n = 3). Asterisks indicate statistically significant differences using Student’s t-test (**P < 0.01). The experiments were carried out three times with similar results. (C)-(D) WOX4p:GUS expression at nematode feeding sites in wild type and clv1-101 clv2-101 rpk2-5 backgrounds. GUS staining was performed at 3 d post inoculation. N = nematode; S = syncytium.

Fig 6. Decreased CLE41 gene expression in clv1-101 clv2-101 rpk2-5 mutant and reduced nematode development on the cle41 mutant.

(A)-(B) CLE41p:GUS expression in nematode feeding sites of wild type and clv1-101 clv2-101 rpk2-5 background. (C) qRT-PCR analysis of CLE41 gene expression in wild type and clv1-101 clv2-101 rpk2-5 mutant at 5 dpi. Nematode development (D) and syncytium size (E) is reduced in the cle41 mutant. Error bars represent SD of the means (n = 3). Asterisks indicate statistically significant differences by Student’s t-test (*P < 0.05 and **P < 0.01). The experiments were repeated three times with similar results.

Fig 7. Combined effect of nematode HsCLE2 and HsCLEB peptides on vascular cell proliferation.

Seedlings including Col-0, clv1-101 clv2-101 rpk2-5, tdr-1, tdr-1 clv1-101 clv2-101, and wox4-1 were grown for 10 d in liquid medium supplemented without (A-E) or with HsCLE2 and HsCLEB peptides (5 μM each) (F-J). Hypocotyls were sectioned and stained with toluidine blue. (K) Vascular diameter measurement of hypocotyl sections (n = 5 to 8). Asterisk indicates P < 0.05 (*) or P < 0.01 (**) by using Student’s t-test.