Transgenerational changes in the genome stability and methylation in pathogen-infected plants: (virus-induced plant genome instability)

Abstract

Previously, we reported the generation of a virus-induced systemic signal that increased the somatic and meiotic recombination rates in tobacco mosaic virus (TMV)-infected tobacco plants. Here, we analyzed the progeny of plants that received the signal and found that these plants also have a higher frequency of rearrangements in the loci carrying the homology to LRR region of the gene of resistance to TMV (N-gene). Analysis of the stability of repetitive elements from Nicotiana tabacum loci and 5.8S ribosomal RNA loci did not show any changes. Further analysis of the changes in the progeny of infected plants revealed that they had substantially hypermethylated genomes. At the same time, loci-specific methylation analysis showed: (1) profound hypomethylation in several LRR-containing loci; (2) substantial hypermethylation of actin loci and (3) no change in methylation in the loci of repetitive elements from N. tabacum or 5.8S ribosomal RNA. Global genome hypermethylation of the progeny is believed to be part of a general protection mechanism against stress, whereas locus-specific hypomethylation is associated with a higher frequency of rearrangements. Increased recombination events combined with the specific methylation pattern induced by pathogen attack could be a sign of an adaptive response by plants.

Figure 1.

Analysis of the progeny of infected and progeny of control plants. Schematic representation of the experimental set up. Briefly, in a previous experiment, single leaves of 10-week-old SR1 tobacco plants were inoculated with 300 ng of TMV RNA (21 plants) or mock treated (20 plants). About 24 h after inoculation, the upper, non-treated leaves (virus-free, checked as previously published, ref. 16) from these plants were grafted onto 10-week-old healthy plants (21 plants with leaves from virus-treated and 20 plants with leaves from mock-treated plants), from which the tops were previously removed (F₀). The seeds (F₁) derived from the newly emerged tissue were collected and named ‘progeny of infected’ (PI) or ‘progeny of control’ (PC). These seeds were used to analyze global and loci-specific methylation and RFLP of various loci.

Figure 2.

RFLP pattern of the N-gene-like R-genes; cloning and hybridization of the sequences with high degree of homology to the fourth exon of the N-gene. (A) RFLP analysis of N-gene-like loci was performed after digestion with three different enzymes, HindIII, EcoRI, DraI. (B) PCR amplification from the plasmid carrying the N-gene (3) or from SR1 plant genome (2), using primers annealing to the fourth exon of the N-gene; (1) is the marker. (C) Cloning of the PCR fragment (line 2 from Figure 2B) from SR1 plants. Digestion of the pGEM cloning vector with EcoRI shows the number of fragments of different sizes. The frame shows the band corresponding to the cloning vector; the bands of lower molecular weight represent the PCR fragments. (D) Hybridization of the colonies containing the PCR product (1—positive control, bacteria containing the vector; 2—negative control, empty bacteria; 3–10—clones with different degree of homology to the fourth exon. Clones 3–8 have the homology of >75%, whereas 9 and 10—only 60–65% (overexposed blot shown).

Figure 3.

RFLP showed increased instability of the R-gene loci, and similar stability of other loci. Examples of the polymorphisms in RFLP of N-gene-like loci from PI plants are shown in A–C. (A) RFLP in the sample digested with HindIII. (B) RFLP in the sample digested with EcoRI. (C) RFLP in the sample digested with DraI. Occasional differences (D) were found in hybridization with 5.8S probe (PI plant). Additional examples of the RFLP are shown in Figures 6S–8S.

Figure 4.

Global genome methylation was analyzed by ROPS assay after digestion with either HpaII or MspI. ‘Y’ axis shows radioactive incorporation (dpm/µg) in PC and PI lines. Each bar represents the average (with SD) from five individual assays, each representing the readings from five plants. Asterisks show the significance of the data, where one represents 95% and two represent at least 99% confidence interval.

Figure 5.

Analysis of methylation in LRR-containing loci. Representative gels show the DNA samples of PI and PC plants digested with HpaII (A) and MspI (B) and probed with the fourth exon of the N-gene. Black arrows show the fragment of substantially lower intensity (or complete disappearance), whereas the white arrows (the arrows are located just above the fragments) show the higher intensity (or appearance) of the two smaller fragments in PI samples.

Figure 6.

Analysis of methylation in actin loci. Representative gels show the DNA samples of PI and PC plants digested with HpaII (A and B) and MspI (C and D) and probed with Tob71 actin probe. In HpaII cut gels (A and B) white arrows show the absence or substantially lower intensity of the fragments in PI samples. Since there were no other fragments appearing on the gel, the only way to have the lower intensity of these fragments (white arrows in A and B) was the higher methylation in the larger (heavier) fragments. In MspI cut gels (C and D), white arrows show the higher intensity (or appearance) of the two larger fragments in PI samples. At the same time, there is a lower intensity of the smallest fragments, suggesting higher methylation in the two larger fragments (labeled with white arrow in C and D).

Figure 7.

Methylation status of actin, RENT and 5.8S loci in PI and PC plants performed using COBRA. ‘Bisulfite-treated genomic DNA from PI and PC lines was used for amplification of the actin, RENT and 5.8S loci. (A) Comparison of digestion patterns in actin loci of PI and PC plants after 16-h incubation with HpyCH4IV. The upper panel shows the undigested PCR product serving as a loading control. The lower panel shows the undigested fragment from the digested samples (see the entire gel in Figure 4S). Lines 1–6 represent individual PC plants; lines 7–11 represent individual PI plants (two samples from PI line #3, and one sample each from PI lines #8–10). (B) Comparison of digestion patterns in RENT loci of PI and PC plants after 16-h incubation with TaqI. Line ‘I’ is the undigested PCR product, lines ‘II’ and ‘III’ show 100- and 50-nt ladders, respectively. The upper panel shows the undigested PCR product serving as a loading control. The lower panel shows the undigested fragment from the digested samples (see the entire gel in Figure 4S). Coding is identical to that used for actin, except that there were six PI samples, lines 7–12. (C) Comparison of digestion patterns in 5.8S loci of PI and PC plants after 16-h incubation with HpyCH4IV or TaqI. The upper panel shows the undigested PCR product serving as a loading control. Two lower panels show the undigested fragments (completely absent) from the digested samples (see the entire gel in Figure 4S). Coding is identical to that used for actin. Heavy methylation of 5.8S loci result in complete digestion of the PCR product.