Tài liệu Báo cáo khoa học: DNA adenine methylation changes dramatically during establishment of

DNA adenine methylation changes dramatically during

establishment of symbiosis

Hiroyuki Ichida1,2, Tomoki Matsuyama3

, Tomoko Abe2 and Takato Koba1

1 Graduate School of Science and Technology, Chiba University, Matsudo, Japan

2 Accelerator Application Research Group, Nishina Center for Accelerator-Based Science, RIKEN, Hirosawa, Wako, Saitama, Japan

3 Cellular Biochemistry Laboratory, Discovery Research Institute, RIKEN, Hirosawa, Wako, Saitama, Japan

Restriction landmark genome scanning (RLGS) is a

method for the two dimensional display of end-labeled

DNA restriction fragments and is an unbiased method

for DNA methylation scanning in higher eukaryotes

[1]. Virtual image (Vi-) RLGS software simulates two

dimensional DNA electrophoresis patterns based on

whole-genome sequence data, allowing the rapid

matching of DNA spots to their sequences without the

time and effort of cloning [2]. Recent advances in

DNA sequencing technology have enabled the sequen￾cing of entire genomes in various organisms, and the

resulting data allow a comprehensive analysis of gen￾ome dynamics during host–microbe interactions.

Bradyrhizobium japonicum and Mesorhizobium loti

are symbiotic bacteria that perform nitrogen fixation

in host plant roots. Their principal hosts are soybean

and Lotus japonicus, which are an important grain

crop and a model legume, respectively. Biological

nitrogen fixation is an important source of nitrogen in

agricultural production, particularly for fabaceae

crops, including soybean; for example, 90% of

nitrogen is biologically fixed in well-nodulated soybean

plants [3]. Agrobacterium tumefaciens was originally

isolated as a causal agent of crown gall disease in

plants. It infects more than 90 families of dicotyledo￾nous plants, resulting in major agronomic losses

worldwide [4].

DNA methylation regulates critical functions in

prokaryotic and eukaryotic cells. C5-methyl-cytosine,

N4-methyl-cytosine, and N6-methyl-adenine are found

Keywords

cell cycle-regulated methyltransferase; DNA

adenine methylation; in silico restriction

landmark genome scanning; plant–microbe

interactions; specifically unmethylated

region

Correspondence

H. Ichida, Accelerator Application Research

Group, Nishina Center for Accelerator-Based

Science, RIKEN, 2-1,

Hirosawa, Wako, Saitama 351-0198, Japan

Fax: +81 48 4624674

Tel: +81 48 4621111 Ext. 5432

E-mail: [email protected]

(Received 31 October 2006, revised 7

December 2006, accepted 11 December

2006)

doi:10.1111/j.1742-4658.2007.05643.x

The DNA adenine methylation status on specific 5¢-GANTC-3¢ sites and

its change during the establishment of plant–microbe interactions was

demonstrated in several species of a-proteobacteria. Restriction landmark

genome scanning (RLGS), which is a high-resolution two dimensional

DNA electrophoresis method, was used to monitor the genomewide change

in methylation. In the case of Mesorhizobium loti MAFF303099, real

RLGS images obtained with the restriction enzyme MboI, which digests at

GATC sites, almost perfectly matched the virtual RLGS images generated

based on genome sequences. However, only a few spots were observed

when the restriction enzyme HinfI was used, suggesting that most GANTC

(HinfI) sites were tightly methylated and specific sites were unmethylated.

DNA gel blot analysis with the cloned specifically unmethylated regions

(SUMs) showed that some SUMs were methylated differentially in bacter￾oids compared to free-living bacteria. SUMs have also been identified in

other symbiotic and parasitic bacteria. These results suggest that DNA

adenine methylation may contribute to the establishment and ⁄ or mainten￾ance of symbiotic and parasitic relationships.

Abbreviations

CcrM, cell cycle-regulated methyltransferase; Dam, deoxyadenosine methyltransferase; RLGS, restriction landmark genome scanning; SUM,

specifically unmethylated region; Vi, virtual image.

FEBS Journal 274 (2007) 951–962 ª 2007 The Authors Journal compilation ª 2007 FEBS 951