Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct;67(19):5857-5868.
doi: 10.1093/jxb/erw353. Epub 2016 Oct 3.

Changes in the DNA Methylation Pattern of the Host Male Gametophyte of Viroid-Infected Cucumber Plants

Affiliations
Free PMC article

Changes in the DNA Methylation Pattern of the Host Male Gametophyte of Viroid-Infected Cucumber Plants

Mayte Castellano et al. J Exp Bot. .
Free PMC article

Abstract

Eukaryotic organisms exposed to adverse conditions are required to show a certain degree of transcriptional plasticity in order to cope successfully with stress. Epigenetic regulation of the genome is a key regulatory mechanism allowing dynamic changes of the transcriptional status of the plant in response to stress. The Hop stunt viroid (HSVd) induces the demethylation of ribosomal RNA (rRNA) in cucumber (Cucumis sativus) leaves, leading to increasing transcription rates of rRNA. In addition to the clear alterations observed in vegetative tissues, HSVd infection is also associated with drastic changes in gametophyte development. To examine the basis of viroid-induced alterations in reproductive tissues, we analysed the cellular and molecular consequences of HSVd infection in the male gametophyte of cucumber plants. Our results indicate that in the pollen grain, accumulation of HSVd RNA induces a decondensation of the generative nucleus that correlates with a dynamic demethylation of repetitive regions in the cucumber genome that include rRNA genes and transposable elements (TEs). We therefore propose that HSVd infection impairs the epigenetic control of rRNA genes and TEs in gametic cells of cucumber, a phenomenon thus far unknown to occur in this reproductive tissue as a consequence of pathogen infection.

Keywords: Cucumber; epigenetic inheritance; hop stunt viroid; viroid-induced pathogenesis; viroids and DNA methylation.; viroid–plant interactions.

Figures

Fig. 1.
Fig. 1.
Effects of HSVd infection on cucumber reproductive tissues. (A) Male and female flowers obtained from HSVd-infected and mock-inoculated cucumber plants were measured as indicated by the dashed lines shown on the representative images of male flowers. The graph shows mean values of 750 (HSVd-infected) and 650 (control) flowers. Statistical significance was tested using a paired t-test. (B) Micrographs of representative pollen grains recovered from HSVd-infected and mock-inoculated cucumber plants. The graph shows the mean diameter of 200 pollen grains recovered from infected and control plants. No significant differences were observed (paired t-test). (C) Representative images of DAPI-stained pollen grains from non-infected and HSVd-infected plants and magnifications of the respective generative nuclei (GN) of the cells. Scale bars = 30 µm. The box-plots show the distribution of the measured areas of the GN of more than 100 pollen grains. Statistical significance was tested using a paired t-test with Welch’s correction. (D) Representative images of DAPI-stained generative nuclei of cells. The area corresponding to the nucleolus is highlighted. Scale bars = 150 µm. The box-plots show the distribution of the measured areas of the nucleolus in 32 pollen grains. Statistical significance was tested using a paired t-test. (E) Representative images of germinated pollen grains recovered from mock- and HSVd-infected plants. The graph shows the germination rate of infected pollen grains in comparison with control samples. A total of 1200 pollen grains were analysed for each sample in six independent replicates. Statistical significance was tested using a paired t-test. In all the figure parts the error bars represent the standard error. Only significant P values are shown in the figure. (This figure is available in colour at JXB online.)
Fig. 2.
Fig. 2.
HSVd accumulates in pollen grains of infected cucumber plants. (A) Detection of HSVd RNA by RT-PCR in pollen grains (P) recovered from infected plants (right). Total RNA extracted from successive washing of pollen grains (w3 and w4) was analysed by RT-PCR in order to discard contamination with HSVd RNA of non-pollen-specific plant tissue. Total RNAs extracted from infected (+) and mock-inoculated (-) cucumber leaves were used as RT-PCR controls (left). (B) Size distribution and polarity of canonical (20 to 25 nts) fully homologous total viroid-derived sRNAs (vd-sRNAs) recovered from the infected pollen library. The values on the y-axis represent the abundance of vd-sRNAs in the library. (C) The vd-sRNAs were plotted onto a circular sequence of the HSVd RNA, in either sense (plus) or antisense (minus) configuration. The arrow indicates the position of nucleotides 1 and 297 in the circular HSVd RNA. (This figure is available in colour at JXB online.)
Fig. 3.
Fig. 3.
Characterization of the small RNAs recovered from cucumber pollen grains by deep sequencing. (A) Whole-genome distribution of sRNA density in control and HSVd-infected pollen grains. The outermost to innermost tracks depicti: C. sativus chromosomes (Chr 1 to 7); heat map of gene density (light = low density, dark = high density); heat map of transposable element density (light = low density, dark = high density); sRNA density in mock-infected pollen grains; sRNA density in HSVd-infected pollen grains. (B) The differential accumulation and distribution of the total reads of endogenous cucumber sRNAs ranging between 21 and 25 nt recovered from the mock- and HSVd-infected samples. (C) Relative increase of ribosomal-derived sRNAs (rb-sRNAs) and TE-derived sRNAs (te-sRNAs) in infected pollen: the ratio of reads obtained in infected pollen compared to the control library is shown. sRNAs derived from coding transcripts (cRNA) and centromeric regions (CRR) exhibit no significant alterations in the samples analysed. (D) Relative increase of rb-sRNAs (left) and te-sRNAs (right) in infected pollen compared to non-infected pollen based on sRNA-length. (This figure is available in colour at JXB online.)
Fig. 4.
Fig. 4.
HSVd infection affects the methylation patterns of rRNA genes and TE in pollen grains. (A) The relative (HSVd/Mock) total rDNA methylation levels. Total methylation means are 0.40 (mock) and 0.37 (HSVd). Statistical differences were determined using a paired t-test. (B) Analysis of symmetric and asymmetric cytosine methylation levels in analysed samples of rDNA. Symmetric methylation means are 0.89 (mock) and 0.83 (HSVd). Asymmetric methylation means are 0.06 (mock) and 0.05 (HSVd). Statistical differences were determined using a paired t-test. (C) Position-specific relative methylation levels in CG and CHG contexts in the analysed samples of rDNA. (D) Relative (HSVd/Mock) total TE methylation. Total methylation means are 0.055 (mock) and 0.022 (HSVd). Statistical differences were determined using a paired t-test. (E) Analysis of symmetric and asymmetric cytosine methylation in the analysed samples of TEs. Symmetric methylation means are 0.084 (mock) and 0.041 (HSVd). Asymmetric methylation means are 0.037 (mock) and 0.018 (HSVd). Statistical differences were determined using a paired t-test. (F) Position-specific relative methylation levels in CG and CHG contexts in the analysed TEs. (This figure is available in colour at JXB online.)
Fig. 5.
Fig. 5.
Differential accumulation of the precursor for rRNAs (pre-rRNAs) and TE-derived transcripts in infected pollen. (A) Representative RT-PCR analysis of the pre-rRNA (left) and TE (right) expression in serial dilutions (500, 100, and 20ng) of HSVd-infected and control total RNAs. RT-PCR amplification of ubiquitin mRNA served as a normalization control. (B) The relative accumulation (in relation to ubiquitin expression) of pre-rRNA (left) and TE-derived transcripts (right) in the serial dilutions shown in (A), determined by measurement of the band intensity, which was measured using the Image-J application (https://imagej.nih.gov/ij/). Error bars represent the standard error. (C) Comparison of relative pre-RNA and TE transcript accumulation (estimated from the sum of the intensity of the RT-PCR products) in control and infected pollen grains. The data are mean values obtained for pre-rRNA and TE amplification relative to the ubiquitin normalization control. Statistical significance was tested using a paired t-test. (D) Comparison of relative pre-RNA and TE transcript accumulation in control (and HSVd-infected pollen grains estimated by RT-qPCR analysis relative to the ubiquitin normalization control. The results shown are the means of three replicates. Error bars represent the standard error. Statistical significance was tested using a paired t-test. (This figure is available in colour at JXB online.)

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Adkar-Purushothama CR, Brosseau C, Giguere T, Sano T, Moffett P, Perreault JP. 2015. Small RNA derived from the virulence modulating region of the potato spindle tuber viroid silences callose synthase genes of tomato plants. Plant Cell 27, 2178–2194. - PMC - PubMed
    1. Agorio A, Vera P. 2007. ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis. Plant Cell 19, 3778–3790. - PMC - PubMed
    1. Alvarez ME, Nota F, Cambiagno DA. 2010. Epigenetic control of plant immunity. Molecular Plant Pathology 11, 563–576. - PMC - PubMed
    1. Aparicio F, Sanchez-Pina MA, Sanchez-Navarro JA, Pallas V. 1999. Location of prunus necrotic ringspot ilarvirus within pollen grains of infected nectarine trees: evidence from RT-PCR, dot-blot and in situ hybridisation. European Journal of Plant Pathology 105, 623–627.
    1. Aufsatz W, Mette MF, van der Winden J, Matzke M, Matzke AJM. 2002. HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA. Embo Journal 21, 6832–6841. - PMC - PubMed

Publication types

MeSH terms

Feedback