Establishment and maintenance of DNA methylation in nematode feeding sites

Abstract

A growing body of evidence indicates that epigenetic mechanisms, particularly DNA methylation, play key regulatory roles in plant-nematode interactions. Nevertheless, the transcriptional activity of key genes mediating DNA methylation and active demethylation in the nematode feeding sites remains largely unknown. Here, we profiled the promoter activity of 12 genes involved in maintenance and de novo establishment of DNA methylation and active demethylation in the syncytia and galls induced respectively by the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita in Arabidopsis roots. The promoter activity assays revealed that expression of the CG-context methyltransferases is restricted to feeding site formation and development stages. Chromomethylase1 (CMT1), CMT2, and CMT3 and Domains Rearranged Methyltransferase2 (DRM2) and DRM3, which mediate non-CG methylation, showed similar and distinct expression patterns in the syncytia and galls at various time points. Notably, the promoters of various DNA demethylases were more active in galls as compared with the syncytia, particularly during the early stage of infection. Mutants impaired in CG or CHH methylation similarly enhanced plant susceptibility to H. schachtii and M. incognita, whereas mutants impaired in CHG methylation reduced plant susceptibility only to M. incognita. Interestingly, hypermethylated mutants defective in active DNA demethylation exhibited contrasting responses to infection by H. schachtii and M. incognita, a finding most likely associated with differential regulation of defense-related genes in these mutants upon nematode infection. Our results point to methylation-dependent mechanisms regulating plant responses to infection by cyst and root-knot nematodes.

Keywords: Arabidopsis; DNA methylation; Heterodera schachtii; Meloidogyne incognita; active DNA demethylation; gall; promoter activity; syncytium.

Figure 1

Promoter activity of DNA methyltransferases and demethylases in the syncytia induced by (H) schachtii on Arabidopsis roots. Time-course experiments showing GUS activity controlled by the CG methyltransferases MET1, METb, and MET3 (A), the non-CG methyltransferases CMT1, CMT2, CMT3, DRM2, and DRM3 (B), and the DNA demethylases ROS1, DME, DML2, and DML3 (C) at 3, 7, 10, and 14 dpi. Red arrows point to syncytia. Scale bar = 200 µm.

Figure 2

Mutations of DNA methyltransferases and demethylases enhance plant susceptibility to H. schachtii. (A–E): H. schachtii infection assays of mutant alleles of MET2b (SALK_102231C and SALK_093835C), MET3 (SALK_024049C and SALK_099592C) (A), CMT1 (SALK_138685C and SALK_030404C) and CMT2 (SALK_012874C and CS879822) (B), CMT3 (CS6365) (C), DRM2 (CS16386 and SALK_129477C) and DRM3 (SALK_136439C and SALK_024820C) (D), ROS1 (CS66099) (E), ROS1 (SALK_045303C), DML2 (SALK_131712C), and DML3 (SALK_056440C) (F). The number of J4 female nematodes per root system was counted 3 weeks post inoculation. Data are presented as means ± SE (n = 20). Asterisks denote statistically significant differences from the wild-type Col-0, Ler or C24 as determined by t tests (P < 0.05).

Figure 3

Promoter activity of DNA methyltransferases and demethylases in the galls induced by M. incognita on Arabidopsis roots. Time-course experiments showing GUS activity controlled by the CG methyltransferases MET1, MET2b, and MET3 (A), the non-CG methyltransferases CMT1, CMT2, CMT3, DRM2, and DRM3 (B), and the DNA demethylases ROS1, DME, DML2, and DML3 (C) in the galls formed by M. incognita at 4, 7, and 14 dpi. Red arrows point to galls. Scale bar = 200 µm.

Figure 4

Mutations of DNA methyltransferases and demethylases alter plant susceptibility to M. incognita. (A–E): M. incognita infection assays of mutant alleles of MET2b (SALK_102231C and SALK_093835C), MET3 (SALK_024049C and SALK_099592C) (A), CMT1 (SALK_138685C and SALK_030404C) and CMT2 (SALK_012874C and CS879822) (B), CMT3 (CS6365) (C), DRM2 (CS16386 and SALK_129477C) and DRM3 (SALK_136439C and SALK_024820C) (D), ROS1 (CS66099) (E), ROS1 (SALK_045303C), DML2 (SALK_131712C) and DML3 (SALK_056440C) (F). The number of galls per root system was counted 3 weeks post inoculation. Data are presented as means ± SE (n = 20). Asterisks denote statistically significant differences from the wild-type Col-0, Ler or C24 as determined by t tests (P < 0.05).

Figure 5

Expression levels of PR genes in DNA demethylase mutants in response to infection by cyst and root-knot nematodes. (A–C): RT-qPCR quantification of the expression of PR1 (A), PR5 ) and PDF1.2 (C) in roots tissues of mutant alleles of ROS1 (CS66099 and SALK_045303C), DML2 (SALK_131712C), and DML3 (SALK_056440C) under non-infected conditions relative to wild-type plants. (D-F): Expression of PR1 (D), PR5 (E) and PDF1.2 (F) in the (H) schachtii-infected roots tissues of wild-type (Col-0 and C24) and mutant alleles of ROS1 (CS66099 and SALK_045303C), DML2 (SALK_131712C), and DML3 (SALK_056440C) relative to non-infected root tissues. (G–I): Expression of PR1 (G), PR5 (H) and PDF1.2 (I) in the M. incognita-infected roots tissues of wild-type (Col-0 and C24) and mutant alleles of ROS1 (CS66099 and SALK_045303C), DML2 (SALK_131712C), and DML3 (SALK_056440C) relative to non-infected root tissues. Relative gene expression levels were obtained from three biological samples and presented as mean ± SE. PP2AA3 and actin8 were used as internal reference genes to normalize gene expression. Asterisks indicate statistically significance differences from control treatments at P < 0.05.