OsGLIP1 and OsGLIP2 exhibit lipase activities.

<p>(A) Alignment of OsGLIP1 and OsGLIP2 amino acid sequences with functionally known homologues from <i>Arabidopsis</i> and <i>Tanacetum cinerariifolium</i>. Sequences were aligned using Genedoc. (B) Expression and purification of recombinant OsGLIP1-GST and OsGLIP2-GST proteins in <i>E</i>. <i>coli</i>. (C, D) Lipase activities of OsGLIP1 and OsGLIP2. OsGLIP1 and OsGLIP2 were incubated with <i>p</i>-nitrophenyl acetate (C) and <i>p</i>-nitrophenyl butyrate (D) at 30°C. The absorbance readings were collected every 5 minutes in a time course of 60 min or 120 min. The substrates were incubated with either GST or no protein as controls. Data are shown as means ± SD (<i>n</i> = 3).</p

GDSL lipases modulate immunity through lipid homeostasis in rice

<div><p>Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of <i>OsGLIP1</i> and <i>OsGLIP2</i> was suppressed by pathogen infection and salicylic acid (SA) treatment. <i>OsGLIP1</i> was mainly expressed in leaf and leaf sheath, while <i>OsGLIP2</i> showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of <i>OsGLIP1</i> and <i>OsGLIP2</i> increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in <i>OsGLIP1</i> and <i>OsGLIP2</i> overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of <i>OsGLIP</i> gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity.</p></div

Altered expression of pathogenesis-related (<i>PR</i>) genes in <i>OsGLIP1</i> transgenic plants.

<p>Eight-week-old transgenic and wild-type plants were inoculated with <i>Xoo</i> (strain PXO99A). The induction of the <i>PR</i> genes, <i>PR1a</i> (A), <i>PR1b</i> (B), <i>PR5</i> (C) and <i>PR10</i> (D), in response to pathogen infection was compromised in <i>OsGLIP1-OE</i> plants, while silencing of both <i>OsGLIP1</i> and <i>OsGLIP2</i> significantly promoted the induction of the <i>PR</i> genes. Data shown are means ± SD from three biological replicates. Asterisks indicate significant difference in comparison with the wild-type control (Student’s <i>t</i>-test, *<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Lipidomic profiling of <i>OsGLIP1/2-RNAi</i> and overexpression plants.

<p>Leaves from six individual plants (eight-week-old) were mixed as one sample from three representative transgenic lines of each transgene were used to normalize samples. Five leaf samples each genetic background were statistically analysed. (A) Total lipid composition in leaves of eight-week-old plants. (B-F) Abundance of individual lipid species, TAG (B), PA (C), MGDG (D), DAG (E) and DGDG (F). The lipid structures are presented as the number of carbon atoms: total double bonds in the fatty acyl groups. Data are shown as means ± SD (<i>n</i> = 5) of mixed leaf samples from three representative transgenic lines. *<i>P</i> < 0.05 or **<i>P</i> < 0.01, by Student’s <i>t</i>-test and Bonferroni correction for multiple (three comparisons) tests.</p

Exogenous feeding of MGDG and DGDG impairs rice disease resistance.

<p>(A) Two-week-old seedling leaves were cultured in liquid medium supplemented with MGDG or DGDG (100 μM with 0.1% Tween-20) for 24 hours. The treated plants were extensively washed and then inoculated with <i>Xoo</i> (strain PXO99A). Lesion lengths of MGDG and DGDG-fed plants were measured at 10 dpi with three biological replicates (> 10 plants each replicate). Data are shown as means ± SD from three biological replicates. (B) Bacterial growth in the MGDG and DGDG-fed plants at 0, 3 and 6 dpi, with mock treatment as control. Data are shown as means ± SD from three biological replicates. (C-E) Relative expression levels of <i>PR</i> genes <i>PR1a</i> (C), <i>PR5</i> (D) and <i>PR10</i> (E) in MGDG/DGDG treated leaves at 0 and 48 hpi. Data are shown as means ± SD from three biological replicates (<i>n</i> = 3). Student’s <i>t</i>-test, *<i>P</i><0.05, **<i>P</i> < 0.01 (A to E).</p

Proper intracellular localization is essential for OsGLIP1 function in rice defense responses.

<p>(A) Schematic diagram shows full-length OsGLIP1 with its signal peptide (SP) and the truncated protein without SP (OsGLIP1^ΔSP-GFP) or SP alone (OsGLIP1^SP-GFP) fused with GFP. (B-D) Subcellular localization of OsGLIP1 -GFP (B) OsGLIP1^ΔSP-GFP (C) and OsGLIP1^SP-GFP (D) proteins in root cells of transgenic plants. Note that removing of the signal peptide (SP) abolished OsGLIP1-GFP ER and lipid body targeting, while the SP alone was sufficient for the subcellular compartment targeting. Scale bars = 20 μm. (E, F) Deletion of the signal peptide attenuated OsGLIP1 action in suppressing plant immunity. Lesions (E) and lesion lengths (F) of representative <i>OsGLIP1-GFP</i> and <i>OsGLIP1</i>^<i>ΔSP</i><i>-GFP</i> transgenic plants inoculated with <i>Xoo</i>. Note that the OsGLIP1-GFP fusion protein also suppressed rice defense, while OsGLIP1^ΔSP-GFP lost its immune inhibition capacity. Arrows indicate bottoms of lesions. Data are shown as means ± SD (<i>n</i> > 10). Scale bar in (E) = 1cm. Student’s <i>t</i>-test, **<i>P</i> < 0.01.</p

Subcellular localization of OsGLIP1 and OsGLIP2.

<p>(A) Localization of OsGLIP1-GFP and OsGLIP2-GFP in the root cells of transgenic plants. OsGLIP1 and OsGLIP2 were fused with GFP and expressed under the control of the maize <i>Ubiquitin</i> (<i>Ubi1</i>) promoter in the stable transgenic rice plants. Scale bars = 20 μm. (B) A 3D projection of OsGLIP1-GFP fluorescence signals. Scale bar = 20 μm. (C) Images of OsGLIP1-GFP labelled vesicle-like structures (Type I and Type II). Scale bar = 2 μm. (D) Size distribution of OsGLIP1-GFP labelled vesicles. The size of vesicles is represented by the diameter. (E) The localization of OsGLIP1-GFP in response to BFA treatment, in comparison with mock treatment. Scale bar = 20 μm. (F) Co-localization of OsGLIP1-GFP with Nile Red that labels lipid droplets. Scale bars = 20 μm (left) or 5 μm (right). (G) Distribution of OsGLIP1-GFP and OsGLIP2-GFP proteins in fractionated membranes of two-week-old rice seedlings. Microsomal membranes were fractionated on linear 20% to 55% (w/v) sucrose gradients. Equal volumes of protein samples were separated on SDS-PAGE gel and analysed by immunoblot using antibodies specific for GFP (GLIP1/2-GFP), BiP2 (ER), HSP70 (cytoplasm) and ACTIN. Note that OsGLIP1 and OsGLIP2 mainly localize to the lipid bodies (cytoplasm) and the ER.</p

Expression of <i>OsGLIP1</i> and <i>OsGLIP2</i> was suppressed in responses to pathogen infection and chemical treatments.

<p>(A) Down-regulation of <i>OsGLIP1</i> and <i>OsGLIP2</i> expression in eight-week-old plants infected with <i>Xoo</i> (strain PXO99A) in a time course of 48 hours. (B, C) Down-regulation of <i>OsGLIP1</i> and <i>OsGLIP2</i> expression in two-week-old seedlings sprayed with 1 mM SA (B) or 300 μM BTH (C). All treatments were repeated for three times with similar results (A-C). The rice <i>Actin1</i> gene was used as an internal control. Data are shown as means ± SD from three biological replicates (Student’s <i>t</i>-test, *<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Disease resistance to bacterial blight in <i>OsGLIP1-OE</i>, <i>OsGLIP2-OE</i> and <i>OsGLIP1/2</i>-<i>RNAi</i> plants.

<p>(A-F) Lesions and statistical analysis of lesion lengths of representative <i>OsGLIP1-OE</i> (A and D), <i>OsGLIP2-OE</i> (B and E) and <i>OsGLIP1/2</i>-<i>RNAi</i> (C and F) lines (eight-week-old) inoculated with bacterial pathogen <i>Xoo</i> at 14 dpi, with the wild type (TP309, WT) as control. Arrows indicate the bottoms of lesions. Data are shown as means ± SD (<i>n</i> > 10). Asterisks indicate significant difference in comparison with the wild-type control (Student’s <i>t</i>-test, *<i>P</i> < 0.05; ** <i>P</i> < 0.01). (G) Disease development during 12 days of inoculation in the representative lines of <i>OsGLIP1-OE</i>, <i>OsGLIP2-OE</i> and <i>OsGLIP1/2</i>-<i>RNAi</i>, compared with the wild type. Data are shown as means ± SD (<i>n</i> > 10). Asterisks indicate significant difference in comparison with the wild-type control (Student’s <i>t</i>-test, ** <i>P</i> < 0.01). (H) Bacterial growth during 12 days of inoculation in the representative lines of <i>OsGLIP1-OE</i>, <i>OsGLIP2-OE</i> and <i>OsGLIP1/2</i>-<i>RNAi</i>, compared with the wild type. Data are shown as means ± SD (<i>n</i> = 3). Asterisks indicate significant difference in comparison with the wild-type control (Student’s <i>t</i>-test, *<i>P</i> < 0.05; ** <i>P</i> < 0.01).</p