BABA-Induced DNA Methylome Adjustment to Intergenerational Defense Priming in Potato to Phytophthora infestans

Abstract

We provide evidence that alterations in DNA methylation patterns contribute to the regulation of stress-responsive gene expression for an intergenerational resistance of β-aminobutyric acid (BABA)-primed potato to Phytophthora infestans. Plants exposed to BABA rapidly modified their methylation capacity toward genome-wide DNA hypermethylation. De novo induced DNA methylation (5-mC) correlated with the up-regulation of Chromomethylase 3 (CMT3), Domains rearranged methyltransferase 2 (DRM2), and Repressor of silencing 1 (ROS1) genes in potato. BABA transiently activated DNA hypermethylation in the promoter region of the R3a resistance gene triggering its downregulation in the absence of the oomycete pathogen. However, in the successive stages of priming, an excessive DNA methylation state changed into demethylation with the active involvement of potato DNA glycosylases. Interestingly, the 5-mC-mediated changes were transmitted into the next generation in the form of intergenerational stress memory. Descendants of the primed potato, which derived from tubers or seeds carrying the less methylated R3a promoter, showed a higher transcription of R3a that associated with an augmented intergenerational resistance to virulent P. infestans when compared to the inoculated progeny of unprimed plants. Furthermore, our study revealed that enhanced transcription of some SA-dependent genes (NPR1, StWRKY1, and PR1) was not directly linked with DNA methylation changes in the promoter region of these genes, but was a consequence of methylation-dependent alterations in the transcriptional network.

Keywords: DNA methylation/demethylation; Phytophthora infestans; intergenerational resistance; potato acquired resistance; stress-responsive genes.

FIGURE 1

Intergenerational resistance of potato against P. infestans. BABA-induced resistance against P. infestans in the parental line (F₀); intergenerational resistance in the vegetative F₁ progeny and the generative F₁ progeny. Three days after BABA treatment potato leaves were challenged with P. infestans and Pitef1 gene expression at 48 hpi and trypan-blue stained leaves at day 5 (dpi) were analyzed. Values represent means of at least three independent experiments, each with at least three biological replicates. Asterisks (^∗) indicate values that differ significantly from unprimed and vr P. infestans-inoculated potato leaves at α<0.05, respectively.

FIGURE 2

Expression patterns of SAMS (A) and SAHH (B) contributing to the potato cellular methylation potential in response to BABA treatment and challenge inoculation with P. infestans MP977 (3 days after BABA treatment). Analyses were performed at 1–48 h after 5 mM BABA exposure (white background) and 1–48 hpi after challenge inoculation with vr P. infestans (gray background). Light lines refer to unprimed and dark lines to primed plants. Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from unprimed (water treated) or unprimed and P. infestans inoculated potato leaves at α<0.05, respectively.

FIGURE 3

Transcriptional changes in potato DNA methylating genes MET1 (A), CMT3 (B), DRM2 (C); and DNA demethylating genes StDML2 (D), and ROS1 (E) in response to BABA and challenge inoculation with vr P. infestans MP977 (3 days after BABA treatment). Analyses were performed at 1–48 h after 5 mM BABA treatment (white background) and 1–48 hpi after challenge inoculation with P. infestans (gray background). Light lines and dark lines refer to unprimed and primed plants, respectively. Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from unprimed (water-treated) or unprimed and P. infestans inoculated potato leaves at α<0.05 (^∗), respectively.

FIGURE 4

Global potato genome methylation in response to BABA treatment and challenge inoculation with P. infestans MP977 (at 72 hrs after BABA treatment). ELISA 5-mC assay (A); Immunodetection of 5-mC estimated as Alexa Fluor⁴⁸⁸ fluorescence intensity (B). Analyses were performed at 1–48 h after BABA (5 mM) treatment (white background) and 1–48 hpi (gray background) after challenge inoculation; the immunostaining pattern of representative nuclei at 0–48 h after BABA treatment (C). The red color: DAPI staining (indicating lack of methylation); the green color: methylated DNA immunodetected with anti-5-mC antibody and Alexa Fluor⁴⁸⁸ goat anti-mouse IgG. The fluorescence signals for nuclei and 5-mC were pseudocolored red and green, respectively, using the ImageJ program. Scale bar = 5 μm. Light columns and dark columns refer to unprimed and primed plants, respectively. The negative control was provided by immunostaining without the secondary or primary antibody. In both cases, we observed no fluorescence of Alexa Fluor⁴⁸⁸ on the slide of leaf nuclei, which means that the antibodies were specific and showed no unspecific binding. Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from unprimed (water treated) or unprimed and P. infestans inoculated potato leaves at α<0.05, respectively.

FIGURE 5

Gene expression and methylation level of NPR1 promoter after BABA treatment in potato. Melting curves of the MS-HRM assay at 3 h (A) and 48 h after BABA treatment (B). It is noted that higher fluorescence levels in BABA-treated plants compared to the unprimed showed enhanced DNA methylation of the NPR1 promoter at 3 h after BABA treatment. The RT-qPCR analysis of NPR1 expression in primed leaves of potato plants at 1–48 h after BABA treatment (C). Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from unprimed (water-treated) potato leaves at α<0.05, respectively.

FIGURE 6

Gene expression and methylation level of StWRKY1 promoter after BABA treatment in potato. Melting curves of the MS-HRM assay at 6 h after BABA treatment (A). Details of the MS-HRM assay are given in the Materials and Methods. Higher fluorescence levels in BABA-treated plants compared to the unprimed showed enhanced DNA methylation of the StWRKY1 promoter at 6 h. RT-qPCR analysis of StWRKY1 expression in primed leaves of potato plants at 1–48 h after BABA treatment (B). Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from unprimed (water-treated) potato leaves at α<0.05, respectively.

FIGURE 7

Gene expression and methylation level of PR1 promoter after BABA treatment in potato. Melting curves of the MS-HRM assay at 6 h after BABA treatment (A). Details of the MS-HRM assay are given in the Materials and Methods section. Fluorescence levels in BABA-treated plants compared to the unprimed showed no significant differences in the PR1 promoter methylation upon BABA exposure. RT-qPCR analysis of PR1 expression in primed leaves of potato plant at 1–48 h after BABA treatment (B). Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from unprimed (water treated) potato leaves at α<0.05, respectively.

FIGURE 8

Gene expression and methylation level of R3a promoter after BABA treatment in potato. MS-HRM curves of the R3a promoter at 3 h after BABA or water treatment (unprimed) indicating its DNA methylated status, at 3 h after sequential treatment of BABA/vr P. infestans MP977 (primed-challenged), at 3 h after vr P. infestans MP977 inoculated (PTI-type of resistance) or Avr3a-encoding P. infestans MP946 inoculated (ETI-type of resistance) (A), respectively. Values represent means of data ± SD of at least three independent experiments. RT-qPCR analysis of R3a gene expression in BABA-primed potato leaves followed by challenge inoculation with vr P. infestans MP977 or in unprimed potato only subjected to virulent P. infestans MP977 or Avr3a-encoding P. infestans MP946 inoculation (B). Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) indicate values that differ significantly from those of unprimed (water treated) plants.

FIGURE 9

DNA methylation of the R3a promoter region involved in intergenerational priming for defense against vr P. infestans in potato. The MS-HRM melting curve of the R3a promoter region showed hypomethylation in the vegetative progeny of BABA-primed parents (A) and less methylated promoter of this gene in the generative progeny obtained from seeds (B). Values represent means of data ± SD of at least three independent experiments. The RT-qPCR analysis of R3a gene expression after vr P. infestans inoculation in the offspring of BABA-primed plants obtained from tubers (C) or seeds (D). Values represent mean ± SD of at least three independent experiments. Asterisks (^∗) or (^∗∗∗) indicate values that differ significantly from the progeny of unprimed (water-treated) potato leaves at α<0.05 and α<0.01, respectively, at each time point.