Additional file 1 of The role of WRKY transcription factors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria × ananassa Duch.)

Additional file 1: Table S1. List of Botrytis resistant Arabidopsis T-DNA knockout lines

Additional file 2 of The role of WRKY transcription factors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria × ananassa Duch.)

Additional file 2: Table S2. Total reads number and mapped rate

Disease symptoms and bacterial growth after flood-inoculation with the host pathogen P. syringae pv. <i>maculicola</i>.

<p>Two-week-old plants grown in 1/2 strength MS under short-day conditions (8 hrs of daylight) were flood-inoculated with the host pathogen <i>P. syringae</i> pv. <i>maculicola</i> at 3×10⁶ CFU/ml. (a) Disease symptoms in wild-type (Col-0) and heterotrimeric G-protein mutants (<i>gpa1-4</i>, <i>agb1-2</i>, <i>gpa1-4 </i><i>agb1-2</i> , <i>agg1-2</i>, <i>agg2-2</i>, <i>agg1-1c </i><i>agg2-1</i> and <i>agg3-2</i>). Images were taken at 4 dpi. (b) Growth of <i>P. syringae</i> pv. <i>maculicola</i> in Col-0, <i>gpa1-4</i>, <i>agb1-2</i>, <i>gpa1-4 </i><i>agb1-2</i> , <i>agg1-2</i>, <i>agg2-2</i>, <i>agg1-1c </i><i>agg2-1</i> and <i>agg3-2</i>. Bacterial titers at 0 and 3 dpi were measured by taking leaf disks from four inoculated plants. All experiments were independently repeated three times, and each experiment was performed with four replications. Bars represent average and standard deviations from all experiments. Asterisks above bars represent statistically significant differences in comparison with wild-type plants using Student’s <i>t</i>-test (P<0.05). </p

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against<i>Pseudomonas syringae</i> Pathogens

<div><p>Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost <i>Pseudomonas</i> pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. <i>tabaci</i> whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. <i>maculicola</i>, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to <i>P. syringae</i>.</p> </div

Stomatal closure and number of bacterial cells that entered through stomata after inoculation of nonhost pathogen P. syringae pv. <i>tabaci</i>.

<p>(a) Determination of stomatal closure and (b) aperture size induced by P. syringae pv. <i>tabaci</i> in epidermal peels. Stomatal aperture size was examined 2 hrs after P. syringae pv. <i>tabaci</i> inoculation. The epidermal peels prepared from all wild-type (Col-0) and heterotrimeric G-protein mutants were incubated in stomata opening buffer (KCl-MES) for at least 3 hrs to assure fully open stomata. Images were taken under a light microscope. Approximately 150 stomata were examined with five epidermal peel samples for each experiment. (c) Detached Arabidopsis leaves were floated on nonhost pathogen P. syringae pv. <i>tabaci</i> and (d) host pathogen P. syringae pv. <i>maculicola</i> (1.2 × 10⁹ CFU/ml). Detached leaf samples were collected 2 and 4 hrs after incubation and treated with 10% bleach for surface sterilization. The number of bacterial cells in the apoplast was determined. Data shown are means ± standard deviation (error bars) from four replicates per each experiment. Two independent experiments were performed with similar results. Asterisks above bars represent statistically significant differences in comparison with wild-type using Student’s <i>t</i>-test (P<0.05).</p

Bacterial growth of P. syringae pv. <i>maculicola</i> and P. syringae pv. <i>tabaci</i> in wild-type and overexpression lines of GPA1 (GPA1OE) and AGB1 (AGB1OE).

<p>Four-week-old plants were flood-inoculated with both pathogens (5×10⁷ CFU/ml). Bacterial titers at 0 and 3 dpi were measured by taking leaf disks from four inoculated plants with three biological replications. All experiments were independently repeated two times. Images for disease phenotypes were taken at 4 dpi. Asterisks above bars represent statistically significant differences in comparison with wild-type plants using Student’s <i>t</i>-test (P<0.05).</p

Disease symptoms, bacterial growth and accumulation of avirulent bacterial strain P. syringae pv. <i>maculicola</i> (AvrRpm1) in G-protein mutants.

<p>Leaves from 6-week-old plants were syringe-infiltrated with the avirulent pathogen <i>P. syringae</i> pv. <i>maculicola</i> (AvrRpm1) at 2.8 x 10⁶ CFU/ml. (a) Disease symptoms in wild-type (Col-0) and heterotrimeric G-protein mutants (<i>gpa1-4</i>, <i>agb1-2</i>, <i>gpa1-4 </i><i>agb1-2</i> , <i>agg1-2</i>, <i>agg2-2</i>, <i>agg1-1c </i><i>agg2-1</i> and <i>agg3-2</i>). Images were taken three days after inoculation. (b) Growth of the avirulent pathogen <i>P. syringae</i> pv. <i>maculicola</i> (AvrRpm1) in Arabidopsis mutants. Leaf samples for bacterial quantification were taken at 0 and 3dpi. Bars represent mean and standard deviation for four biological replicates from each experiment. Two independent experiments were performed. Asterisks above bars represent statistically significant differences in comparison with wild-type plants using Student’s <i>t</i>-test (P<0.05). </p

Measurement of bacterial growth after inoculation with nonhost pathogens P. syringae pv. <i>tabaci</i> and P. syringae pv. phaseolicola in Col-0, <i>gpa1-4</i>, <i>agb1-2</i>, <i>gpa1-4</i><i>agb1-2</i> , <i>agg1-2</i>, <i>agg2-2</i>, <i>agg1-1c</i><i>agg2-1</i> and <i>agg3-2</i>.

<p>Bacterial growth after flood-inoculation of P. syringae pv. <i>tabaci</i> (a) and P. syringae pv. phaseolicola (b) Two-week-old plants grown in 1/2 strength MS under short day conditions (8 hrs of daylight) were flood-inoculated with the nonhost pathogen <i>P. syringae</i> pv. <i>tabaci</i> at 3×10⁶ CFU/ml and P. syringae pv. phaseolicola at 7×10⁶ CFU/ml. Bacterial titers at 0 and 3 dpi were measured by taking leaf disks from four inoculated plants. All experiments were independently repeated three times, and each experiment was performed with four replications. Bars represent average and standard deviations from all experiments. Asterisks above bars represent statistically significant differences in comparison with wild-type plants using Student’s <i>t</i>-test (P<0.05).</p

Heat map of transcript accumulation of heterotrimeric G-protein and defense related genes (SA, JA and FLS2 signaling pathways) regulated in response to P. syringae pv. <i>maculicola</i> and P. syringae pv. phaseolicola infections.

<p>(a) Expression of GPA1, AGB1 AGG1, AGG2 and AGG3 in wild-type upon P. syringae infection. Seedlings grown on 1/2 strength MS medium were inoculated with bacterial pathogens. The gene expression levels were determined 12 and 24 hrs after host, P. syringae pv. <i>maculicola</i> (Psm), and nonhost pathogen, P. syringae pv. phaseolicola (Psp), inoculations. (b) Gene expression profiling of various defense-related genes in G-protein mutants in comparison to wild-type (Col-0) plants. The expression of SA and JA defense-related genes and genes involved in <i>FLS2</i>-mediated defense signaling were examined after 12 and 24 hrs (hpi) in response to host, P. syringae pv. <i>maculicola</i>, and nonhost pathogen, P. syringae pv. phaseolicola. Each column is the fold change of gene expression as determined by qRT-PCR at 12 and 24 hpi in pathogen-inoculated samples. The relative gene expression values normalized by <i>Ubiquitin5</i> (UBQ5) and <i>Elongation </i><i>factor 1 </i><i>alpha</i> (EF1α) were represented as n-fold compared to the mock-treated plants. Red and blue indicate up-regulated and down-regulated expression levels, respectively.</p

Proposed model for the plant innate immunity signaling network regulated by heterotrimeric G-proteins in Arabidopsis.

<p>Proposed model for the plant innate immunity signaling network regulated by heterotrimeric G-proteins in Arabidopsis.</p