Natural genetic variation in Arabidopsis for responsiveness to plant growth-promoting rhizobacteria

The plant growth-promoting rhizobacterium (PGPR) Pseudomonas simiae WCS417r stimulates lateral root formation and increases shoot growth in Arabidopsis thaliana (Arabidopsis). These plant growth-stimulating effects are partly caused by volatile organic compounds (VOCs) produced by the bacterium. Here, we performed a genome-wide association (GWA) study on natural genetic variation in Arabidopsis for the ability to profit from rhizobacteria-mediated plant growth-promotion. To this end, 302 Arabidopsis accessions were tested for root architecture characteristics and shoot fresh weight in response to exposure to WCS417r. Although virtually all Arabidopsis accessions tested responded positively to WCS417r, there was a large variation between accessions in the increase in shoot fresh weight, the extra number of lateral roots formed, and the effect on primary root length. Correlation analyses revealed that the bacterially-mediated increase in shoot fresh weight is related to alterations in root architecture. GWA mapping for WCS417r-stimulated changes in root and shoot growth characteristics revealed 10 genetic loci highly associated with the responsiveness of Arabidopsis to the plant growth-promoting activity of WCS417r. Several of the underlying candidate genes have been implicated in important plant growth-related processes. These results demonstrate that plants possess natural genetic variation for the capacity to profit from the plant growth-promoting function of a beneficial rhizobacterium in their rhizosphere. This knowledge is a promising starting point for sustainable breeding strategies for future crops that are better able to maximize profitable functions from their root microbiome

The rhizosphere revisited: root microbiomics

The rhizosphere was defined over 100 years ago as the zone around the root where microorganisms and processes important for plant growth and health are located. Recent studies show that the diversity of microorganisms associated with the root system is enormous. This rhizosphere microbiome extends the functional repertoire of the plant beyond imagination. The rhizosphere microbiome of Arabidopsis thaliana is currently being studied for the obvious reason that it allows the use of the extensive toolbox that comes with this model plant. Deciphering plant traits that drive selection and activities of the microbiome is now a major challenge in which Arabidopsis will undoubtedly be a major research object. Here we review recent microbiome studies and discuss future research directions and applicability of the generated knowledge

Functional Analysis of Hyaloperonospora arabidopsidis RXLR Effectors

The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae DCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by th

Functional Analysis of Hyaloperonospora arabidopsidis RXLR Effectors

The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors

Multifactorial analysis and clustering of Waco9 RXLRs.

<p>(A) Heatmap of the relative effects of the 13 Waco9 RXLRs on flg22-mediated growth reduction and the level of resistance to <i>H. arabidopsidis, P. capsici,</i> and <i>P. syringae</i> infection. For this heatmap, the data from each experiment were converted into a score where 1 represents the maximum value, −1 represents the minimal value and the score for Col-0 was set at 0. The RXLR overexpressing lines were clustered based on the scores from the experimental results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110624#pone-0110624-g003" target="_blank">Figures 3</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110624#pone-0110624-g006" target="_blank">6</a> using hierarchical clustering. The red-dotted line shows the threshold for significant difference between groups (<i>p</i><0.05), resulting in five significantly different groups. (B) Ordination biplot generated by RDA. The statistically significant RDA axes (RDA1 and RDA2; <i>p</i><0.05) are plotted and the five different groups based on hierarchical clustering are indicated. The dendogram based on the clustering method is projected in the ordination plot (gray lines). (C) The eigenvectors derived from the RDA. For three out of five RDA axes the amount of variation explained is shown in percentages and the contribution of each phenotype score to each RDA axes is indicated with different shades of gray. RDA4 and RDA5 are not shown since they explain only a very small part of the variation (3% and 1% respectively). The <i>p</i>-values of the RDA’s are RDA1 <i>p</i>≤0.001, RDA2 <i>p</i>≤0.001, RDA3 <i>p</i> = 0.56, RDA4 <i>p</i> = 0.95 and RDA5 <i>p</i> = 1.0.</p

Sequence features of successfully cloned <i>RXLR</i> genes of <i>H. arabidopsidis</i> isolate Waco9.

1<p>Gene ID of RXLRs from Emoy2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110624#pone.0110624-Baxter1" target="_blank">[18]</a>).</p>2<p>Number of amino acid to signal peptide cleavage site.</p>3<p>When present, number of amino acid to signal peptide cleavage site.</p>4<p>Homologs present in this group, adapted from Cabral <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110624#pone.0110624-Baxter1" target="_blank">[18]</a>.</p><p>Sequence features of successfully cloned <i>RXLR</i> genes of <i>H. arabidopsidis</i> isolate Waco9.</p

Effect of ectopic expression of Waco9 <i>RXLR</i> genes on the level of resistance against <i>H. arabidopsidis</i>.

<p>In four experiments, two independent overexpressing lines of each of the 13 Waco9 <i>RXLR</i> genes was tested twice for the level of resistance against <i>H. arabidopsidis</i> Waco9. Two-week-old plants were spray inoculated and 6 days later the number of conidiophores per plant was determined. In each experiment the number of conidiophores on Col-0 is set at 100%. Subsequently, the number of conidiophores in all other lines is given relative to Col-0 in the same experiment. The enhanced susceptible mutant <i>eds1-2</i> was included as a positive control. Results represent mean ± SEM (<i>n</i> = 18) and asterisks indicate significant differences (ANOVA and Fisher’s LSD corrected for type I errors; <i>p</i><0.05).</p

Effects of Waco9 RXLRs on flg22-induced reduction of seedling growth.

<p>In four independent experiments seedlings of Col-0, the flagellin receptor mutant <i>fls2</i>, and two independent lines of each of the 13 Waco9 RXLR overexpressors were grown on liquid MS medium in the presence or absence of 50 (A), (C) and (E) or 500 nM (B), (D) and (F) of flg22. After 10 days of growth, the fresh weight (FW) of a pool of 10 plants per line was determined. In (A) and (B) the relative FWs of Col-0 and <i>fls2</i> are depicted for the 4 independent experiments, in which the FW of the untreated plants was set at 100%. Results represent mean ± SEM (<i>n</i> = 3) and asterisks indicate significant differences between treated and non-treated plants (Students <i>t</i>-test; <i>p</i><0.05). In (C) and (D) the relative FWs of the flg22-treated RXLR overexpressors are depicted for the 4 experiments. These relative FWs are normalized to the relative FW of untreated Col-0 (set at 100%). The dotted line shows the average relative FW of flg22-treated Col-0. Results represent mean ± SEM (<i>n</i> = 3) and asterisks indicate significant differences in relative FW compared to Col-0 (ANOVA and Fisher’s LSD corrected for type I errors; <i>p</i><0.05). In (E) and (F) the averages of the relative FWs of the flg22-treated RXLR overexpressors are depicted (i.e. the averages of the results in (C) and (D)), again the dotted line represents the average relative FW of flg22-treated Col-0).</p

Effect of EDV-mediated delivery of Waco9 RXLR9 on <i>P. syringae</i> ΔCEL-induced callose deposition.

<p>Five-week-old Col-0 plants were infiltrated with <i>P. syringae</i> ΔCEL carrying YFP, RXLR9 or ATR13. After 12 h, leaves were harvested and stained with analine blue for the detection of callose deposition. Pictures were taken (A) and the number of callose spots was quantified (B). The combined results of three independent experiments are shown. Results represent mean ± SEM (<i>n</i> = 17–23) and asterisks indicate significant differences (ANOVA and Fisher’s LSD corrected for type I errors; <i>p</i><0.05).</p