Performance of pigmented and anthocyanin-deficient Mimulus guttatus plants in a common garden

Measures of vigour and seed set made in an experimental common garden site in Syracuse

Performance of pigmented and anthocyanin-deficient Mimulus guttatus plants in a drought experiment

Performance of pigmented and anthocyanin-deficient Mimulus guttatus plants in a drought experimen

Appendix B. Root frequencies and measures of shoot biomass for two Yellowstone National Park grasslands.

Root frequencies and measures of shoot biomass for two Yellowstone National Park grasslands

Appendix A. The fragment length polymorphism method used in the study to identify root fragments in soil cores, with a table of the species-diagnostic fragment lengths.

The fragment length polymorphism method used in the study to identify root fragments in soil cores, with a table of the species-diagnostic fragment lengths

Defining the Metabolic Functions and Roles in Virulence of the <i>rpoN1</i> and <i>rpoN2</i> Genes in <i>Ralstonia solanacearum</i> GMI1000

<div><p>The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as <i>Escherichia coli</i>. <i>Bacillus subtilis</i> and <i>Pseudomonas putida</i>. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal <i>rpoN</i> gene to meet the cellular demands of RpoN regulation. The bacterium <i>Ralstonia solanacearum</i> is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual <i>rpoN</i> genes. To determine if the <i>rpoN</i> genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized Δ<i>rpoN1</i>, Δ<i>rpoN2</i> and Δ<i>rpoN1</i> Δ<i>rpoN2</i> mutants of <i>R</i>. <i>solanacearum</i> GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the <i>rpoN1</i> gene. Furthermore, the <i>rpoN1</i> gene was required for wilt disease on tomato whereas <i>rpoN2</i> had no observable role in virulence or metabolism in <i>R</i>. <i>solanacearum</i> GMI1000. Interestingly, plasmid-based expression of <i>rpoN2</i> did not fully rescue the metabolic deficiencies of the Δ<i>rpoN1</i> mutants; full recovery was specific to <i>rpoN1</i>. In comparison, only <i>rpoN2</i> was able to genetically complement a Δ<i>rpoN E</i>. <i>coli</i> mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in <i>R</i>. <i>solanacearum</i> GMI1000.</p></div

The <i>rpoN1</i> gene was required for wilt disease on tomato.

<p>(A) cv. Bonny Best at 4 dpi. (B) cv. Hawaii 7996 at 7 dpi. Tomato plants infected with the Δ<i>rpoN1</i> and Δ<i>rpoN1</i> Δ<i>rpoN2</i> mutants did not show signs of wilting disease compared to the Δ<i>rpoN2</i> mutant and wild-type <i>R</i>. <i>solanacearum</i> GMI1000.</p

<i>In planta</i> growth of wild type, Δ<i>rpoN1</i>, Δ<i>rpoN2</i> and Δ<i>rpoN1</i> Δ<i>rpoN2 R</i>. <i>solanacearum</i> GMI1000 in tomato.

<p>Bacterial growth (CFUs per gram of fresh tissue) was determined at 4 dpi in cv. Bonny Best and 7 dpi in cv. Hawaii 7996. For both tomato cultivars, the Δ<i>rpoN1</i> and Δ<i>rpoN1</i> Δ<i>rpoN2</i> mutants yielded lower CFUs (reduced growth) compared to the Δ<i>rpoN2</i> mutant and wild-type <i>R</i>. <i>solanacearum</i> GMI1000. [Data points represent mean values (n = 8) ± SD. Analysis was done using Student t-test to identify significant changes (<i>P</i> < 0.005), which are marked with double asterisk].</p

Quantification of wilt disease caused by wild-type, Δ<i>rpoN1</i>, Δ<i>rpoN2</i> and Δ<i>rpoN1</i> Δ<i>rpoN2 R</i>. <i>solanacearum</i> GMI1000 on tomato.

<p>(A) cv. Bonny Best. (B) cv. Hawaii 7996. Wilt symptoms were rated daily on a disease index scale, and the values were used to calculate a disease-wilting index (dwi). The dwi for both the Δ<i>rpoN1</i> and Δ<i>rpoN1</i> Δ<i>rpoN2</i> mutant was significantly reduced compared to the Δ<i>rpoN2</i> mutant and wild-type <i>R</i>. <i>solanacearum</i> GMI1000. Note that nine plants were used for each treatment.</p

Alignment of regions I (residues 1–50) and II (residues 51–100) of <i>E</i>. <i>coli</i> RpoN with RpoN1 and RpoN2 of <i>R</i>. <i>solanacearum</i> GMI1000.

<p>(A) <i>E</i>. <i>coli</i> RpoN (EcRpoN) and RpoN1 of <i>R</i>. <i>solanacearum</i> GMI1000 (RsRpoN1) (B) EcRpoN and <i>R</i>. <i>solanacearum</i> GMI1000 RpoN2 (RsRpoN2). (C) RsRpoN1 and RsRpoN2. <i>E</i>. <i>coli</i> RpoN and RpoN2 have homology in region II, which might enable RpoN2 to interact with EBPs of <i>E</i>. <i>coli</i>. Alignments were generated using EMBOSS.</p