Upregulated genes by PPA₁ and OPDA treatments and <i>B</i>. <i>cinerea</i> inoculation dependent on <i>TGA2/5/6</i> and <i>WRKY33</i>.

<p>Upregulated genes by PPA₁ and OPDA treatments and <i>B</i>. <i>cinerea</i> inoculation dependent on <i>TGA2/5/6</i> and <i>WRKY33</i>.</p

Identification of Arabidopsis Candidate Genes in Response to Biotic and Abiotic Stresses Using Comparative Microarrays

<div><p>Plants have evolved with intricate mechanisms to cope with multiple environmental stresses. To adapt with biotic and abiotic stresses, plant responses involve changes at the cellular and molecular levels. The current study was designed to investigate the effects of combinations of different environmental stresses on the transcriptome level of Arabidopsis genome using public microarray databases. We investigated the role of cyclopentenones in mediating plant responses to environmental stress through TGA (TGACG motif-binding factor) transcription factor, independently from jasmonic acid. Candidate genes were identified by comparing plants inoculated with <i>Botrytis cinerea</i> or treated with heat, salt or osmotic stress with non-inoculated or non-treated tissues. About 2.5% heat-, 19% salinity- and 41% osmotic stress-induced genes were commonly upregulated by <i>B</i>. <i>cinerea</i>-treatment; and 7.6%, 19% and 48% of genes were commonly downregulated by <i>B</i>. <i>cinerea</i>-treatment, respectively. Our results indicate that plant responses to biotic and abiotic stresses are mediated by several common regulatory genes. Comparisons between transcriptome data from Arabidopsis stressed-plants support our hypothesis that some molecular and biological processes involved in biotic and abiotic stress response are conserved. Thirteen of the common regulated genes to abiotic and biotic stresses were studied in detail to determine their role in plant resistance to <i>B</i>. <i>cinerea</i>. Moreover, a T-DNA insertion mutant of the <i>Responsive to Dehydration</i> gene (<i>rd20</i>), encoding for a member of the caleosin (lipid surface protein) family, showed an enhanced sensitivity to <i>B</i>. <i>cinerea</i> infection and drought. Overall, the overlapping of plant responses to abiotic and biotic stresses, coupled with the sensitivity of the <i>rd20</i> mutant, may provide new interesting programs for increased plant resistance to multiple environmental stresses, and ultimately increases its chances to survive. Future research directions towards a better dissection of the potential crosstalk between <i>B</i>. <i>cinerea</i>, <i>abiotic stress</i>, and oxylipin signaling are of our particular interest.</p></div

Expression of <i>OBIG</i>s/<i>PBIG</i>s in response to <i>B</i>. <i>cinerea</i>.

<p>Relative expression levels obtained through qRT-PCR for (A) <i>OBIG</i>s, <i>PBIG</i>s or <i>PBRG</i>s; and (B) <i>OBIG</i>s/<i>PBIG</i>s after infection with <i>B</i>. <i>cinerea</i> at 24 hpi. Expression of <i>B</i>. <i>cinerea</i>-inducible genes was quantitated relative to control conditions (no infection), and corrected for expression of the control gene (<i>AtActin2</i>). Error bars for qRT-PCR values are the standard deviations (<i>n</i> ≥ 3). Mean values followed by an asterisk is significantly different from wild-type at the tested time (<i>P</i> = 0.05). Experiments were repeated at least three times with similar results. hpi, hours post inoculation; <i>At Actin2</i>, <i>Arabidopsis thaliana Actin2</i> gene.</p

Regulation of genes by OPDA treatment and <i>B</i>. <i>cinerea</i> infection.

<p>Regulation of genes by OPDA treatment and <i>B</i>. <i>cinerea</i> infection.</p

Transcriptional reprogramming and scatter-plot comparisons of <i>DEG</i>s in <i>WRKY33</i> transgenic plants.

<p>(A) The numbers of <i>DEG</i>s (≥ 2-fold at <i>P</i> ≤ 0.05) between wild-type, <i>wrky33</i> and 35S:<i>WRKY33</i> at 0 or 24 hpi of inoculation with <i>B</i>. <i>cinerea</i>. Normalized expression value for each probe set in wild-type plants infected with <i>B</i>. <i>cinerea</i> at 24 hpi is plotted on the Y-axis; the value in <i>B</i>. <i>cinerea-</i>treated (B) <i>wrky33</i> mutant and (C) 35S:<i>WRKY33</i> plants infected with <i>B</i>. <i>cinerea</i> at 24 hpi is plotted on the X-axis. Venn diagram showing the overlapping numbers of <i>BIG</i>s and <i>BRG</i>s in wild-type and (D) <i>wrky33</i>; or (E) 35S:<i>WRKY33</i> plants at 24 hpi with <i>B</i>. <i>cinerea</i>. In (A, D and E), boxes represent total number, and arrows represent the number of <i>BIG</i>s and <i>BRG</i>s between the treatments and the genotypes tested. Wt, wild-type; <i>wrky33</i>, <i>wrky33</i> mutant; 35S:<i>WRKY33</i>, 35S:<i>WRKY33</i> overexpression transgenic line; hpi, hours post inoculation.</p

Scatter-plot comparison and functional classification of <i>DEG</i>s in the <i>B</i>. <i>cinerea</i>-Arabidopsis interaction network.

<p>(A) Normalized expression value for each probe set in wild-type plants infected with <i>B</i>. <i>cinerea</i> at 24 hpi (Wt-24) is plotted on the Y-axis; the value in wild-type plants sampled before <i>B</i>. <i>cinerea</i> treatment (0 hpi; WT-0) is plotted on the X-axis. Functional classes of (B) <i>BIG</i>s; and (C) <i>BRG</i>s at 24 hpi compared with 0 hpi in wild-type. Gene identifications for 1660 <i>BIG</i>s and 1054 <i>BRG</i>s were entered for this analysis. Error bars are SD. GO categories that a significantly over or underrepresented at <i>P</i> < 0.05 are in black text. Normalized frequency of genes to the number of genes on the microarray chip was determined as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172343#pone.0172343.ref050" target="_blank">50</a>].</p

Regulation of genes by OPDA and/or PPA₁ treatments and <i>B</i>. <i>cinerea</i> infection.

<p>(A) Number; and (B) percentage of OPDA- and PPA₁-regulated genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172343#pone.0172343.ref033" target="_blank">33</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172343#pone.0172343.ref054" target="_blank">54</a>] that are differentially expressed in response to <i>B</i>. <i>cinerea</i>. Bars in red, yellow or blue show whether the <i>B</i>. <i>cinerea</i>-<i>DEG</i>s are dependent on <i>WRKY33</i>, <i>TGA2/5/6</i>, or both, respectively. Not <i>DEG</i>s, not <i>B</i>. <i>cinerea</i>-<i>DEG</i>s; <i>DEG</i>s, <i>B</i>. <i>cinerea</i>-<i>DEG</i>s; <i>WRKY33</i>-dependent, <i>B</i>. <i>cinerea</i>-<i>DEG</i>s dependent on <i>WRKY33</i>; <i>TGA</i>-dependent, <i>B</i>. <i>cinerea</i>-<i>DEG</i>s dependent on <i>TGA2/5/6</i>; <i>WRKY33/TGA</i>-dependent, <i>B</i>. <i>cinerea</i>-<i>DEG</i>s dependent on <i>WRKY33</i> and <i>TGA2/5/6</i>.</p

Microarray analysis of Arabidopsis <i>WRKY33</i> mutants in response to the necrotrophic fungus <i>Botrytis cinerea</i>

<div><p>The WRKY33 transcription factor was reported for resistance to the necrotrophic fungus <i>Botrytis cinerea</i>. Using microarray-based analysis, we compared Arabidopsis <i>WRKY33</i> overexpressing lines and <i>wrky33</i> mutant that showed altered susceptibility to <i>B</i>. <i>cinerea</i> with their corresponding wild-type plants. In the wild-type, about 1660 genes (7% of the transcriptome) were induced and 1054 genes (5% of the transcriptome) were repressed at least twofold at early stages of inoculation with <i>B</i>. <i>cinerea</i>, confirming previous data of the contribution of these genes in <i>B</i>. <i>cinerea</i> resistance. In Arabidopsis wild-type plant infected with <i>B</i>. <i>cinerea</i>, the expressions of the differentially expressed genes encoding for proteins and metabolites involved in pathogen defense and non-defense responses, seem to be dependent on a functional <i>WRKY33</i> gene. The expression profile of 12-oxo-phytodienoic acid- and phytoprostane A₁-treated Arabidopsis plants in response to <i>B</i>. <i>cinerea</i> revealed that cyclopentenones can also modulate <i>WRKY33</i> regulation upon inoculation with <i>B</i>. <i>cinerea</i>. These results support the role of electrophilic oxylipins in mediating plant responses to <i>B</i>. <i>cinerea</i> infection through the TGA transcription factor. Future directions toward the identification of the molecular components in cyclopentenone signaling will elucidate the novel oxylipin signal transduction pathways in plant defense.</p></div

Phenotypic analysis of T-DNA insertion alleles of common-regulated genes in response to <i>B</i>. <i>cinerea</i>.

<p>^<b>a</b> Expression of common up-/down-regulated genes data were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125666#pone.0125666.t002" target="_blank">Table 2</a> of this study and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125666#pone.0125666.ref020" target="_blank">20</a>].</p><p>^b Wt, disease response comparable to wild-type plants; S, susceptible. SAIL_737_G01 plants show increased local susceptibility to <i>B</i>. <i>cinerea</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125666#pone.0125666.g004" target="_blank">Fig 4</a>).</p><p>T-DNA insertion mutants were assayed for their disease responses at least three times.</p><p>Phenotypic analysis of T-DNA insertion alleles of common-regulated genes in response to <i>B</i>. <i>cinerea</i>.</p

Regulation of genes by PPA₁ treatment and <i>B</i>. <i>cinerea</i> infection.

<p>Regulation of genes by PPA₁ treatment and <i>B</i>. <i>cinerea</i> infection.</p