Functional analyses of chitinases in the moss <i>Physcomitrella patens</i>: chitin oligosaccharide-induced gene expression and enzymatic characterization

<p>Plant chitinases play diverse roles including defense against pathogenic fungi. Using reverse-transcription quantitative PCR analysis, we found that six chitinase (PpChi) genes and two genes for chitin elicitor receptor kinases (PpCERKs) are expressed at considerable levels in the moss <i>Physcomitrella patens</i> subsp. <i>patens</i>. The expressed PpChis belonged to glycoside hydrolase family 19 (class I: PpChi-Ia and -Ib; class II: PpChi-IIa and -IIc; and class IV: PpChi-IV) and to glycoside hydrolase family 18 (class V: PpChi-Vb). Treatment with chitin tetramer or hexamer increased the expression of class I and IV PpChi genes and decreased that of class II PpChi genes. Recombinant PpChi-Ia, PpChi-IV, and PpChi-Vb were characterized. PpChi-IV exhibited higher activity against chitin tetramer and pentamer than PpChi-Ia did. PpChi-Vb showed transglycosylation activity and PpChi-Ia inhibited fungal growth. These results suggest that chitinases of different classes play different roles in defense mechanism of moss plant against fungal pathogens.</p> <p>Possible roles of each class of chitinase and chitin elicitor receptor kinase (CERK) for self-defense against pathogenic fungi in moss plant.</p

Comparative genomic analysis among <i>Bradyrhizobium</i> sp. DOA9, <i>B</i>. <i>japonicum</i> USDA110, and <i>Bradyrhizobium</i> sp. BTAi1.

<p>Each genome is represented by a circle, and the numbers of shared and unique genes are shown by the overlapping and nonoverlapping regions. The proportion of total genes represented by each area of the diagram is shown in parentheses. The total number of genes in each genome is shown in square brackets.</p

Pulse-field gel electrophoresis of <i>Bradyrhizobium</i> sp. DOA9 genomic DNA.

<p>DOA9 cells were digested in 1% pulse field grade (PFG) agarose plugs with proteinase K, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117392#sec002" target="_blank">Materials and Methods</a>, and run 0.8% certified megabase agarose in TAE buffer to separate fragments of 225–6,000 kb (A), or 1% certified megabase agarose in 0.5 x TBE buffer (B) to separate fragments of 225–2,200 kb, respectively. Closed arrowheads and open arrowheads indicate the putative chromosome and the plasmid, respectively. Lane M1: PFGE marker, 3.5–5.7 Mb, <i>Saccharomyces pombe</i> chromosomal DNA. Lane M2: low-range (2.03–194 kb) PFG marker DNA ladder. DOA9: DOA9 genomic DNA.</p

Phylogenetic relationships of <i>Bradyrhizobium</i> sp. DOA9 and related bacteria based on 16S rRNA gene sequences.

<p>Bootstrap values are expressed as percentages of 1,000 replications. Evolutionary distances were computed using the Kimura two-parameter method. The bar represents one estimated substitution per 100-nucleotide positions. Strains capable of Nod factor-dependent and -independent nodulation are marked with (ND) and (NI), respectively. Photosynthetic strains are highlighted in gray.</p

Phylogenetic relationships of pDOA9 and related plasmids.

<p>Bootstrap values are expressed as percentages of 1,000 replications. Evolutionary distances were computed using the Kimura two-parameter method. The bar represents one estimated substitution per 100-nucleotide positions.</p

Comparison of nodulation gene clusters in <i>Bradyrhizobium</i> sp. DOA9 and related bacteria.

<p>Double slash marks represent DNA regions that are not shown. Colored strips represent the conserved gene regions between the compared strains, and the color indicates the percentage similarity, as detailed in the key. T: region where the transposase genes were located.</p

Genome Analysis of a Novel <i>Bradyrhizobium</i> sp. DOA9 Carrying a Symbiotic Plasmid

<div><p><i>Bradyrhizobium</i> sp. DOA9 isolated from the legume <i>Aeschynomene americana</i> exhibited a broad host range and divergent nodulation (<i>nod</i>) genes compared with other members of the <i>Bradyrhizobiaceae</i>. Genome analysis of DOA9 revealed that its genome comprised a single chromosome of 7.1 Mbp and a plasmid of 0.7 Mbp. The chromosome showed highest similarity with that of the <i>nod</i> gene-harboring soybean symbiont <i>B. japonicum</i> USDA110, whereas the plasmid showed highest similarity with pBBta01 of the <i>nod</i> gene-lacking photosynthetic strain BTAi1, which nodulates <i>Aeschynomene</i> species. Unlike in other bradyrhizobia, the plasmid of DOA9 encodes genes related to symbiotic functions including nodulation, nitrogen fixation, and type III/IV protein secretion systems. The plasmid has also a lower GC content (60.1%) than the chromosome (64.4%). These features suggest that the plasmid could be the origin of the symbiosis island that is found in the genome of other bradyrhizobia. The <i>nod</i> genes of DOA9 exhibited low similarity with those of other strains. The <i>nif</i> gene cluster of DOA9 showed greatest similarity to those of photosynthetic bradyrhizobia. The type III/IV protein secretion systems of DOA9 are similar to those of <i>nod</i> gene-harboring <i>B. elkanii</i> and photosynthetic BTAi1. The DOA9 genome exhibited intermediate characteristics between <i>nod</i> gene-harboring bradyrhizobia and <i>nod</i> gene-lacking photosynthetic bradyrhizobia, thus providing the evidence for the evolution of the <i>Bradyrhizobiaceae</i> during ecological adaptation. <i>Bradyrhizobium</i> sp. DOA9 isolated from the legume <i>Aeschynomene americana</i> exhibited a broad host range and divergent nodulation (<i>nod</i>) genes compared with other members of the <i>Bradyrhizobiaceae</i>. Genome analysis of DOA9 revealed that its genome comprised a single chromosome of 7.1 Mbp and a plasmid of 0.7 Mbp. The chromosome showed highest similarity with that of the <i>nod</i> gene-harboring soybean symbiont <i>B. japonicum</i> USDA110, whereas the plasmid showed highest similarity with pBBta01 of the <i>nod</i> gene-lacking photosynthetic strain BTAi1, which nodulates <i>Aeschynomene</i> species. Unlike in other bradyrhizobia, the plasmid of DOA9 encodes genes related to symbiotic functions including nodulation, nitrogen fixation, and type III/IV protein secretion systems. The plasmid has also a lower GC content (60.1%) than the chromosome (64.4%). These features suggest that the plasmid could be the origin of the symbiosis island that is found in the genome of other bradyrhizobia. The <i>nod</i> genes of DOA9 exhibited low similarity with those of other strains. The <i>nif</i> gene cluster of DOA9 showed greatest similarity to those of photosynthetic bradyrhizobia. The type III/IV protein secretion systems of DOA9 are similar to those of <i>nod</i> gene-harboring <i>B. elkanii</i> and photosynthetic BTAi1. The DOA9 genome exhibited intermediate characteristics between <i>nod</i> gene-harboring bradyrhizobia and <i>nod</i> gene-lacking photosynthetic bradyrhizobia, thus providing the evidence for the evolution of the <i>Bradyrhizobiaceae</i> during ecological adaptation.</p></div

General genome features of DOA9 and related bradyrhizobiaceae

<p>^a The values of plasmid is shown in parentheses.</p><p>^b The estimated values with gaps.</p><p>General genome features of DOA9 and related bradyrhizobiaceae</p

The genome structure of <i>Bradyrhizobium</i> sp. DOA9.

<p>(A) Circular representation of the chromosome of <i>Bradyrhizobium</i> sp. DOA9. The outermost, second, and third circles represent BLASTN comparisons with <i>B</i>. <i>japonicum</i> USDA110, <i>Bradyrhizobium</i> sp. ORS278, and <i>Bradyrhizobium</i> sp. ORS28, respectively (e-value < 10⁻¹⁰). The innermost and second-innermost circles show the GC skew and the GC content, respectively. The GC content circle shows the deviation from the average GC content of the entire sequence (higher than average GC content is represented in green, and lower than average content is represented in purple). The markings inside the innermost circle represent genome positions (in Mb). The positions of the putative replication origin, putative replication terminus, and nitrogen-fixation genes are shown outside of the outermost circle. (B) Circular representation of the plasmid (pDOA9) of DOA9. The outermost, second, and third circles represent BLASTN comparisons with the plasmid pBBta01 of <i>Bradyrhizobium</i> sp. BTAi1, the draft genome of <i>Bradyrhizobium elkanii</i> 587 (GenBank accession number AJJK00000000), and <i>B</i>. <i>japonicum</i> USDA110, respectively (e-value < 10⁻¹⁰). The innermost and second-innermost circles show the GC skew and the GC content, respectively. The GC content circle shows the deviation from the average GC content of the entire sequence (higher than average GC content in represented in green, and lower than average is represented in purple). The markings inside the innermost circle represent genome positions (in Mb). The positions of the <i>repABC</i> operon, T3/T4SS, <i>nod</i> genes, <i>nif-fix</i> gene clusters, and <i>hup</i> cluster are shown outside of the outermost circle.</p

Nodulation genes detected in the genome of <i>Bradyrhizobium</i> sp. DOA9 and higher BLASTP similarity with other microorganisms.

<p>Nodulation genes detected in the genome of <i>Bradyrhizobium</i> sp. DOA9 and higher BLASTP similarity with other microorganisms.</p