Laser Irradiation-Induced DNA Methylation Changes Are Heritable and Accompanied with Transpositional Activation of mPing in Rice.

DNA methylation is an integral component of the epigenetic code in most higher eukaryotes. Exploring the extent to which DNA methylation can be altered under a specific condition and its heritability is important for elucidating the biological functions of this epigenetic modification. Here, we conducted MSAP analysis of rice plants with altered phenotypes subsequent to a low-dose Nd3+YAG laser irradiation. We found that all four methylation patterns at the 5'-CCGG sites that are analyzable by MSAP showed substantial changes in the immediately treated M0 plants. Interestingly, the frequencies of hypo- and hypermethylation were of similar extents, which largely offset each other and render the total methylation levels unchanged. Further analysis revealed that the altered methylation patterns were meiotically heritable to at least the M2 generation but accompanied with further changes in each generation. The methylation changes and their heritability of the metastable epigenetic state were verified by bisulfite sequencing of portion of the retrotranspon, Tos17, an established locus for assessing DNA methylation liability in rice. Real-time PCR assay indicated that the expression of various methylation-related chromatin genes was perturbed, and a Pearson correlation analysis showed that many of these genes, especially two AGOs (AGO4-1 and AGO4-2), were significantly correlated with the methylation pattern alterations. In addition, excisions of a MITE transposon, mPing, occurred rampantly in the laser irradiated plants and their progenies. Together, our results indicate that heritable DNA methylation changes can be readily induced by low-dose laser irradiation, and which can be accompanied by transpostional activation of transposable elements.

Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice.

Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site (NBS) and leucine-rich repeat (LRR) superfamily. Expression of Pib was low under non-challenged conditions, but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea, thereby conferring resistance to the pathogen. It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question. We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied. Surprisingly, induced expression of Pib by M. grisea infection did not entail its promoter demethylation, and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wild-type plants. Accordingly, the blast disease-resistance was compromised in the 5'-azaC-treated plants relative to wild-type. In contrast, the disease susceptibility was not affected by the 5'-azaC treatment in another two rice cultivars that did not contain the Pib gene, ruling out effects of other R genes and non-specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance. Taken together, our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M. grisea infection, and its conferred resistance to the pathogen.

Heritable alteration in DNA methylation pattern occurred specifically at mobile elements in rice plants following hydrostatic pressurization.

Intrinsic DNA methylation pattern is an integral component of the epigenetic network in many eukaryotes. Exploring the extent to which DNA methylation patterns can be altered under a specific condition is important for elucidating the biological functions of this epigenetic modification. This is of added significance in plants wherein the newly acquired methylation patterns can be inherited through organismal generations. We report here that DNA methylation patterns of mobile elements but not of cellular genes were specifically altered in rice plants following hydrostatic pressurization. This was evidenced by methylation-sensitive gel-blot analysis, which showed that 10 out of 10 studied low-copy transposons and retrotransposons manifested methylation alteration in at least one of the 8 randomly chosen pressure-treated plants, whereas none of the 16 studied low-copy cellular genes showed any change. Both gel-blotting and genome-wide fingerprinting indicated that the methylation alteration in mobile elements was not accompanied by a general genetic instability. Progeny analysis indicated retention of the altered methylation patterns in most progeny plants, underscoring early occurrence of the alterations, and their faithful epigenetic inheritance.