Analysis of reference gene stability by GeNorm.

<p>Low stability values (M-values) indicate stable gene expression. Genes are ordered left to right in order of decreasing stability.</p

Summary of reference gene ranking by BestKeeper, NormFinder and GeNorm analysis.

<p>*Indicates the most stably expressed gene/s and + the least stably expressed gene as determined by each program.</p

NormFinder estimation of the optimum number of reference genes.

<p>Lowest accumulated standard deviation (Acc. SD) values indicate the optimal number of reference genes. The recommended upper limit for Acc. SD is set at 0.15.</p

Descriptive statistics of reference gene expression by BestKeeper.

<p>Based on inspection of standard deviations (std dev; SD) genes can be ranked from the most stably expressed (lowest SD: <i>gyrA</i>), to the least stably expressed (highest SD: <i>gapB</i>). Genes with std dev [±CP]>1 are considered to be inconsistent. CP = crossing point or threshold cycle (Ct).</p

Expression levels of reference gene candidates.

<p>A: Expression of reference genes across <i>A. brasilense</i> strains. B: Expression of reference genes across different culture conditions. Gene expression levels are represented by average Ct values. NB: nutrient broth; Flocc: Flocculation medium; N-free: Nitrogen free medium. Each bar represents the mean Ct values from three independent experiments. Error bars indicate standard error.</p

Specificity of qRT-PCR amplification.

<p>A: Melt curves for candidate reference genes. B: Agarose gel (2%) showing amplification of a single product of expected size for each candidate reference gene.</p

Strains and plasmids used in this study.

<p>Amp^r, Km^r, Tet^r indicate resistance to ampicillin, kanamycin, and tetracycline, respectively; CR, Congo red binding; Floc, flocculation.</p><p>Strains and plasmids used in this study.</p

qRT-PCR primer sets used in this study.

<p>qRT-PCR primer sets used in this study.</p

Plant-root binding abilities of <i>A. brasilense</i> strain.

<p>Colonization of wheat root by Sp7 (1 and 4), Sp7-flcAΔ (2 and 5) and Sp72001 (3 and 6). <i>A</i>. <i>brasilense</i> strains harbor the reporter plasmid pLA-lacZ, containing a constitutively expressed <i>lacZ</i> gene and were stained with X-gal. Sp7 (1, 4) has strong binding ability to wheat roots and can be found all over the root surface; Sp7-flcAΔ (2, 5) and Sp72001 (3, 6) lost the ability to bind to wheat roots and could only be found in lateral root emergence areas. Scale bars indicate 50 µm (group A) and 5 µm (group B) (Magnification ×100 in group A and ×1000 in group B).</p

Differential protein expression in Sp7 and Sp7-flcAΔ analyzed by 2-DE.

<p>The left section shows the normalized expression volume of the spot in wild type (Sp7) and <i>flcA</i> knock out strain (Sp7-flcAΔ) under flocculation conditions; the relative fold change is shown above each column (Nx indicates that relative fold change could not be calculated as the protein was only detected in either Sp7 or Sp7-flcAΔ). The right section is a 3D representation of the area of interest as provided by PDquest software. Arrows indicate spots with relatively higher expression.</p