Study On DNA Methylation Patterns Of NB-LRR Family Encoding Genes In Arabidopsis

Nucleotide-binding siteleucine-rich repeat (NB-LRR) proteins in plants are encoded by the majority of disease resistance genes. NB-LRR proteins constitute a large family and play very important roles in disease resistance. The pivotal problems how NB-LRR genes in plants exist and how they are regulatedat transcriptional level remain to be solved. Cytosine DNA methylation is a pervasive and conservative epigenetic mark in eukaryotes, and it is important in regulation of genes.In this study, NB-LRR encoding genes in model plant Arabidopsis thaliana (Columbia ecotype) are selected for study on the DNA methylation patterns in the wild type and mutants whose original genes are key factors regulating DNA methylation. Based on the results, this study explores further the issues between the changes for gene expression at transcriptional levels and changes for DNA methylation patterns. The main results are as follows.1. The vast majority of NB-LRR encoding genes in Arabidopsis thaliana (Columbia) are modified by DNA methylation. DNA methylation can be found in the promoters as well as the gene bodies for the most NB-LRR encoding genes, and the main methylation typeis CG methylation. Interestingly, there are three members including Atlg58807, Atlg59124 and Atlg59218without DNA methylation modification.2. Loss of key regulators AGO4, MET1, CMT3, DRM1/2 and DDM1 function separately leads to decrease of the methylation counts of NB-LRR encoding genes. In ago4 plants, the methylation counts reduce by a fifth to a quarter or so in the promoters of different length of NB-LRR encoding genes, while the methylation counts reduce by about a tenth in the gene bodies. Loss of MET1 function results in reduction of methylation counts to less than half in the promoters and gene bodies of NB-LRR encoding genes.3. The methylation levels of gene bodies of NB-LRR family in Arabidopsis are higher than those of 500-bp and 1000-bp promoters. These are observed in the wild type and almost all the mutants, especially in the CG type methylation. The CG methylations in the promoters are always higher than those in the gene bodies of NB-LRR family, and the CHG methylation level are the highest.4. The average of methylation levels of NB-LRR encoding genes changes regularly according to the diverse structural regions of genes. Except met1, it is observed in the wild type and mutants that the CG methylation in the promoters where they are more far away the transcriptional start sitesare higher than that of the other parts of the promoters. While the non-CG methylation profile in the promoters is generally similar to the CG profile, but dynamic according to each genotype. In the gene bodies of NB-LRR encoding genes in wild type Arabidopsis, the average of methylation levels is highest in the introns, followed by coding sequences and untranslational regions, and the average of methylation levels is higher in the 3’-untranslational regions than in the 5’-untranslational regions; the CG methylation levels in the gene bodies are always higher than non-CG methylation level. Loss of AG04, CMT3, DRM1/2 function leads to the same methylation profile in the diverse regions of gene bodies of NB-LRR encoding genes as the wild type. Mutation of METlgives rise to high non-CG methylation levels of NB-LRR encoding genes and the methylation is higher in the intron regions than in the coding sequence regions. In ddml mutant, the CG methylation levels of coding sequences of NB-LRR encoding genes in Arabidopsis are higher than that of introns, which are followed by untranslational regions, while the average of CG methylation in the 3’-untranslational regions is higher than in the 5’-untranslational regions.5. Individual genes encoded by NB-LRR family in Arabidopsis are likely to be modulated by altered DNA methylation patterns. Analysis of transcriptome data from wild type and metl, cmt3, drml/2 and ddml mutants reveals that difference at transcription levels between wild type and mutants are significant for 63 of NB-LRR encoding genes. Of these,38 of NB-LRR encoding genes are up-regulated, and other 25 are down-regulated. There are two genes of NB-LRR family whose transcriptional levels are significantly higher in all the mutants than in the wild type Arabidopsis. While there is one of the family whose expression at transcription level is significantly lower in all the mutants than in the wild type Arabidopsis.The results in this study provide an important foundation for understanding the mechanisms of expression and regulation of NB-LRR encoding genes in Arabidopsis in the view of epigenetics especially DNA methylation.