Overexpression of an AP2/ERF Type Transcription Factor <i>OsEREBP1</i> Confers Biotic and Abiotic Stress Tolerance in Rice

<div><p>AP2/ERF–type transcription factors regulate important functions of plant growth and development as well as responses to environmental stimuli. A rice AP2/ERF transcription factor, <i>OsEREBP1</i> is a downstream component of a signal transduction pathway in a specific interaction between rice (<i>Oryza sativa</i>) and its bacterial pathogen, Xoo (<i>Xanthomonas oryzae</i> pv. <i>oryzae</i>). Constitutive expression of <i>OsEREBP1</i> in rice driven by maize <i>ubiquitin</i> promoter did not affect normal plant growth. Microarray analysis revealed that over expression of <i>OsEREBP1</i> caused increased expression of lipid metabolism related genes such as lipase and chloroplastic lipoxygenase as well as several genes related to jasmonate and abscisic acid biosynthesis. PR genes, transcription regulators and <i>Aldhs</i> (alcohol dehydrogenases) implicated in abiotic stress and submergence tolerance were also upregulated in transgenic plants. Transgenic plants showed increase in endogenous levels of α-linolenate, several jasmonate derivatives and abscisic acid but not salicylic acid. Soluble modified GFP (SmGFP)-tagged OsEREBP1 was localized to plastid nucleoids. Comparative analysis of non-transgenic and <i>OsEREBP1</i> overexpressing genotypes revealed that <i>OsEREBP1</i> attenuates disease caused by Xoo and confers drought and submergence tolerance in transgenic rice. Our results suggest that constitutive expression of <i>OsEREBP1</i> activates the jasmonate and abscisic acid signalling pathways thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions. <i>OsEREBP1</i> is thus, a good candidate gene for engineering plants for multiple stress tolerance.</p></div

<i>OsEREBP1-ox</i> plants show reduced susceptibility to bacterial pathogen Xoo.

<p>(A) Leaf lesion development: Six week old plants of Kitaake (control), 4–3 and 2–4 (T3 progeny of transgenic lines) were inoculated by clipping the leaves with a scissor dipped in the Xoo inoculum. Photos of leaves showing lesions 14 days post inoculation. (B) Leaf lesion length: Lesion lengths (in cms) were measured 7, 14 and 21 days post inoculation from 10 inoculated leaves. Values are means ± SD of three replicates. (C) Bacterial growth curves: Three leaves for each of the cultivars 3, 6, 9 and 12 days post inoculation were individually ground in water and plated at various dilutions to get suitable bacterial counts. Each data point represents average and standard deviation of three independent experiments.</p

Subcellular localization of OsEREBP1-SmGFP and Xb22a-SmGFP fusion proteins.

<p>Confocal micrographs of individual rice protoplast transformed with Ubi:<i>SmGFP</i> (SmGFP), Ubi:<i>Xb22a</i>-<i>SmGFP</i> (Xb22a) or Ubi:<i>OsEREBP1</i>-<i>SmGFP</i> (OsEREBP1) plasmids. The columns from left are: GFP-flourescence in green (false color), chlorophyll fluorescence in red (false color), differential interference contrast transmission (DIC) and the merged image.</p

Transcript levels of genes showing upregulation in <i>OsEREBP1-ox</i> plants.

<p>Total RNA was extracted from 4 week old non-transformed (Kit) and transgenic (4–3 and 2–4) plants and qPCR was performed with primers for genes which include (A) <i>NAC6</i>, <i>AP59</i>, <i>EREBP1 & WRKY</i> (transcription factors); (B) <i>Jmt</i>, <i>RERJ & OsLis</i> (Jasmonate biosynthesis and regulation); (C) <i>PBZ</i>, <i>PR10</i>, <i>Lox</i> (Pathogenicity related); (D) <i>CatB</i>, <i>HLH148 & Lea5</i> (ABA pathway). <i>Actin</i> was used as internal standard. Primer details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127831#pone.0127831.s005" target="_blank">S1 Table</a>. Data representing mean±SE from three independent biological replicates was subjected to further statistical analysis by one way ANOVA using SigmaPlot Version 11.0. The asterix indicate <i>p</i> value of 0.05.</p

Fatty acid composition of total lipids extracted from the leaves of 2–3 week old non-transgenic (KIT) and <i>OsEREBP1-ox</i> (2–4 and 4–3) plants.

<p>Values are mol%± S.D (n = 3)</p><p>16:0, hexadecanoic acid (palmitic acid);</p><p>18:0, octadecanoic acid (stearic acid);</p><p>18:2, ∆ 9,12- octadecadienoic acid (linoleic acid);</p><p>18:3, ∆ 9,12,15-octadecatrienoic acid (α-linolenic acid)</p><p>Fatty acid composition of total lipids extracted from the leaves of 2–3 week old non-transgenic (KIT) and <i>OsEREBP1-ox</i> (2–4 and 4–3) plants.</p

Phenotypes and genotypes of transgenic rice plants.

<p>A) Photographs were taken of six week-old seedlings grown under greenhouse conditions. B) Pictures were taken of plants after the panicle initiation stage. <b>C</b>) Southern blot hybridization of <i>BamH</i>I-digested genomic DNA showed single independent insertions in progenies of 2–4 and 4–3 (transgenic lines), whereas no band was observed in non-transformed Kitaake (control) when probed with hygromycin gene obtained by PCR amplification of pC1300 plasmid using <i>HygS/AS</i> primer pair. D) PCR analysis of genomic DNA using a vector-specific (<i>UbiS</i>) and insert-specific (<i>AP2AS</i>) primer pair showed presence of transgene in the progenies of 2–4 and 4–3 and not in Kitaake. Ap2/pNC1300 plasmid DNA was used as positive control. E) RT-PCR of cDNA using <i>OsEREBP1</i> primers showed increased transcript in 2–4 and 4–3 as compared to Kitaake. <i>EF1a</i> was used to normalize the cDNA. All primer details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127831#pone.0127831.s005" target="_blank">S1 Table</a>.</p

<i>OsEREBP1</i> confers submergence tolerance in rice.

<p>(A) Two-week old plants were removed from water after 7 days of submergence and photographed. (B) Plant height after submergence treatment. Two-week old plants were submerged for 7 days and plant height of 10 plants was measured at 0 and 7 days after submergence. The error bars represent means ± SD (n = 3) and asterisk indicates that the differences in length were significant (P<0.01) as analyzed by one way ANOVA using SigmaPlot Version 11.0. (C) Plant viability after submergence treatment. Two-week old seedlings of transgenic lines (4–3, 2–4) and Kitaake control were submerged for 14 days and allowed to recover under normal conditions. The plants were scored as viable if they produced new leaves. (D) Accumulation of reactive oxygen species upon submergence. Two-week old seedlings were submerged for 7 days and immediately after desubmergence, the leaves were stained with DAB or NBT to detect hydrogen peroxide or superoxide radicals, respectively.</p

<i>OsEREBP1-ox</i> plants show increased tolerance to drought.

<p>(A) Photos of transgenic rice plants (4–3 and 2–4) and non-transgenic control (Kit) plants subjected to drought stress for 26 days followed by 7 days of watering. (B) Drought tolerance at the reproductive stage. Drought was imposed by withdrawing water at panicle initiation stage till plants were completely brown before they were re-watered. Recovery was indicated by new tillers which formed panicles and grains following rewatering. (C) Percentage recovery of drought stress plants (DS1 and DS2) compared to well-watered ones (WW). Drought was imposed at four leaf stage (DS1) or panicle initiation or booting stage (DS2) till the plants showed complete browning of leaves. Pots were rewatered and percentage recovery was calculated based on the number of plants that produced new leaves out of the total number of plants per pot. Error bars represent mean of three independent experiments.</p

Model for role of <i>OsEREBP1</i> in biotic and abiotic stress responses.

<p>Increased expression of <i>OsEREBP1</i> results in accumulation of jasmonates and ABA and induction of specific subsets of defense genes leading to biotic and abiotic stress tolerance. JA regulates transcription factor <i>RERJ1</i> and induces expression of JA-responsive genes resulting in increased tolerance to pathogen infection and drought stress. A cross talk between ABA and JA signalling mediated by OsHLH148/OsJaz1 interaction results in upregulation of transcripts encoding ERF’s such as <i>AP59</i> associated with drought acclimation. ABA independently enhances recovery from submergence and provides protection against drought during desubmergence by inducing expression of <i>Lea5</i> and <i>CatB</i> genes.</p

Phytohormone levels in the leaves of OsEREBP1-ox plants v/s Non-transgenic controls.

<p>Values are ng phytohormones g^-1FW ±S.D (n = 3)</p><p>SA, Salicylic acid; JA, Jasmonic acid; ABA, Abscisic acid; <i>cis</i>-OPDA, <i>cis</i>-12-oxo-phytodienoic acid; JA-Ile, (+)-7-iso-jasmonic acid-L-Ile; COOH-JA-IL.</p><p>Phytohormone levels in the leaves of OsEREBP1-ox plants v/s Non-transgenic controls.</p