Growth of <i>Methylobacterium</i>-inoculated barley.

<p>The data are presented as mean ± standard deviation, and analyzed with one-way ANOVA and Dunnett’s test. Statistical significance was indicated with</p><p>* (<i>p</i> < 0.05),</p><p>** (<i>p</i> < 0.01),</p><p>*** (<i>p</i> < 0.001),</p><p>**** (<i>p</i> < 0.0001).</p><p>Growth of <i>Methylobacterium</i>-inoculated barley.</p

Maximum-likelihood phylogenetic tree of <i>Methylobacterium</i> isolates and related taxa, based on 16S rRNA gene sequences.

<p>Isolates from rice are colored in green and those from barley are in blue. Numbers in parentheses indicate isolates belonging to the same species, estimated by WC-MS analysis. For isolates from rice, the inoculation effect is not taken into account in the figure. <i>M aquaticum</i> strain 22A is taken as a representative strain for the <i>M</i>. <i>platani/aquaticum</i> cluster shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129509#pone.0129509.g001" target="_blank">Fig 1</a>. Bootstrap percentages based on 1000 replicates are shown if greater than 80%. <i>Microvirga flocculans</i> TFB (AB098515) was used as an outgroup. Bar, 0.1 changes per nucleotide position.</p

Identification and composition of <i>Methylobacterium</i> species isolated from rice plants subjected to <i>Methylobacterium</i> inoculation.

<p>Total number of <i>Methylobacterium</i> isolates is shown in parentheses. Identification was performed by WC-MS and 16S RNA sequencing, and details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129509#pone.0129509.g001" target="_blank">Fig 1</a>.</p

MSP dendrogram based on WC-MS analysis of the isolates from leaves of inoculated rice seed.

<p>The isolates were named with the treated strain name and isolate numbers. The inoculated strains are colored in red. The representatives selected from each cluster are colored in blue with the closest type strain name and percentage identity of 16S RNA gene in parentheses. Isolate 22A-9 was pink-pigmented fungus and was identified by ITS region sequencing, as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129509#pone.0129509.ref019" target="_blank">19</a>].</p

MSP dendrogram based on WC-MS analysis of the isolates from barley of different cultivars.

<p>The representatives are shown in blue with the closest species name and percentage identity of the 16S RNA gene in parentheses. Strains shown in gray were not pink-pigmented.</p

Isolates’ genotypes and characteristics of barley.

<p>Isolates’ genotypes and characteristics of barley.</p

Growth of <i>Methylobacterium</i>-inoculated rice.

<p>The data are presented as mean ± standard deviation, and analyzed with one-way ANOVA and Dunnett’s test. Statistical significance was indicated with</p><p>* (<i>p</i> < 0.05),</p><p>** (<i>p</i> < 0.01),</p><p>*** (<i>p</i> < 0.001),</p><p>**** (<i>p</i> < 0.0001).</p><p>Growth of <i>Methylobacterium</i>-inoculated rice.</p

Table_2_Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.xlsx

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.</p

Table_1_Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.xlsx

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.</p

Data_Sheet_1_Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.pdf

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.</p