OsGELP77, a QTL for broad-spectrum disease resistance and yield in rice, encodes a GDSL-type lipase

Abstract

Lipids and lipid metabolites have essential roles in plant-pathogen interactions. GDSL-type lipases are involved in lipid metabolism modulating lipid homeostasis. Some plant GDSLs modulate lipid metabolism altering hormone signal transduction to regulate host-defence immunity. Here, we functionally characterized a rice lipase, OsGELP77, promoting both immunity and yield. OsGELP77 expression was induced by pathogen infection and jasmonic acid (JA) treatment. Overexpression of OsGELP77 enhanced rice resistance to both bacterial and fungal pathogens, while loss-of-function of osgelp77 showed susceptibility. OsGELP77 localizes to endoplasmic reticulum and is a functional lipase hydrolysing universal lipid substrates. Lipidomics analyses demonstrate that OsGELP77 is crucial for lipid metabolism and lipid-derived JA homeostasis. Genetic analyses confirm that OsGELP77-modulated resistance depends on JA signal transduction. Moreover, population genetic analyses indicate that OsGELP77 expression level is positively correlated with rice resistance against pathogens. Three haplotypes were classified based on nucleotide polymorphisms in the OsGELP77 promoter where OsGELP77^Hap3 is an elite haplotype. Three OsGELP77 haplotypes are differentially distributed in wild and cultivated rice, while OsGELP77^Hap3 has been broadly pyramided for hybrid rice development. Furthermore, quantitative trait locus (QTL) mapping and resistance evaluation of the constructed near-isogenic line validated OsGELP77, a QTL for broad-spectrum disease resistance. In addition, OsGELP77-modulated lipid metabolism promotes JA accumulation facilitating grain yield. Notably, the hub defence regulator OsWRKY45 acts upstream of OsGELP77 by initiating the JA-dependent signalling to trigger immunity. Together, OsGELP77, a QTL contributing to immunity and yield, is a candidate for breeding broad-spectrum resistant and high-yielding rice.

Keywords: OsGELP77; OsWRKY45; QTL; disease resistance; lipase; rice.

Figure 1

OsGELP77 positively confers rice resistance to various pathogens. (a) Response of the OsGELP77‐OE plants and osgelp77 mutants to different Xoo strains. Plants were inoculated with Xoo at the booting stage. (b) Phenotype of the OsGELP77‐OE plants and osgelp77 mutants after Xoo PXO341 infection. Scale bar: 1 cm. (c) Growth of Xoo PXO341 in leaves of the OsGELP77‐OE6 plant and osgelp77‐1 mutant. (d) Response of the OsGELP77‐OE plants and osgelp77 mutants to different Xoc strains. Plants were inoculated with Xoc at the tillering stage. (e) Phenotype of the OsGELP77‐OE plants and osgelp77 mutants after Xoc RS105 infection. Scale bar: 1 cm. (f) Growth of Xoc RS105 in leaves of the OsGELP77‐OE6 plant and osgelp77‐1 mutant. (g) Response of the OsGELP77‐OE plants and osgelp77 mutants to M. oryzae isolate 99–20‐2. Plants were inoculated with M. oryzae at the tillering stage. (h) Phenotype of the OsGELP77‐OE plants and osgelp77 mutants after M. oryzae infection. Scale bar: 1 cm. (i) Relative fungal biomass of M. oryzae in leaves of the OsGELP77‐OE6 plant and osgelp77‐1 mutant. dpi, days post infection. Data represent means ± SD. n = 30 (a, d, g), n = 6 (c, f, i). Asterisks in (a, c, d, f, g, i) indicate significant differences between wild type (WT) and the OsGELP77‐OE plants or osgelp77 mutants determined by two‐tailed Student's t‐test at **P < 0.01 or *P < 0.05.

Figure 2

Expression patterns of OsGELP77 and lipase activity of OsGELP77. (a) OsGELP77 expression in different tissues. Data represent means ± SD (n = 3). Gene expression analysis was performed by RT‐qPCR and normalized to Actin. (b) Histochemical staining of different tissues of OsGELP77pro:Gus transgenic lines. Scale bars: 1 cm. (c) Subcellular localization of OsGLEP77 in rice protoplasts. HDEL protein fused to RFP was used as an endoplasmic reticulum marker. Auto, chlorophyll autofluorescence. Scale bars: 10 μm. (d) Expression and purification of recombinant OsGELP77ΔSP‐GST and OsGELP77^4AΔSP‐GST proteins in E. coli. (e) Recombinant OsGELP77ΔSP‐GST and OsGELP77^4AΔSP‐GST proteins were incubated with p‐nitrophenyl butyrate, p‐nitrophenyl acetate, p‐nitrophenyl octanoate, or p‐nitrophenyl palmitate. The absorbance readings were collected every 5 min in a time course of 90 min. (f) Total proteins from the leaves of transgenic plants and wild type (WT) were incubated with p‐nitrophenyl butyrate and p‐nitrophenyl acetate. The absorbance readings were collected every 5 min in a time course of 60 min. Data represent means ± SD. n = 6 (e, f).

Figure 3

Lipidomic profiling of the OsGELP77‐OE plants and osgelp77 mutants. (a) Total lipid composition in the leaves of transgenic plants and wild type (WT). (b) Contents of eight PA species. (c) Contents of seven PS species. (d) Contents of 31 PG species. (e) Contents of 12 PI species. The individual lipid species is presented as the XX:Y nomenclature where XX is the number of carbon atoms and Y is the number of double bonds in the fatty acyl groups. Data represent means ± SD (n = 3). Asterisks in (a–e) indicate significant differences between WT and the OsGELP77‐OE plants or osgelp77 mutants determined by two‐tailed Student's t‐test at **P < 0.01 or *P < 0.05.

Figure 4

OsGELP77 Alters JA homeostasis. (a) JA contents in the leaves of transgenic plants and wild type (WT). (b) Relative transcript levels of OsLOX2 and OsAOS2 in the leaves of transgenic plants and WT. (c) Relative transcript levels of JA‐responsive PR genes in the leaves of transgenic plants and WT. (d) SA contents in the leaves of transgenic plants and WT. (e) Relative transcript levels of OsWRKY45 and OsNPR1 in the leaves of transgenic plants and WT. Data represent means ± SD (n = 3). Gene expression analysis was performed by RT‐qPCR and normalized to Actin. Asterisks in (a–c) indicate significant differences between WT and the OsGELP77‐OE plants or osgelp77 mutants determined by two‐tailed Student's t‐test at **P < 0.01 or *P < 0.05.

Figure 5

OsGELP77‐mediated resistance is JA signalling dependent. (a) JA contents in the leaves of transgenic plants and the corresponding background. (b) Relative transcript level of OsVSP in the leaves of transgenic plants and the corresponding background. (c) Responses of the transgenic plants and the corresponding background to Xoo strain PXO99. (d) JA contents in the leaves of transgenic plants. (e) Relative transcript level of OsVSP in the leaves of transgenic plants. (f) Responses of the transgenic plants to Xoo strain PXO99. (g) Response of the transgenic plants and wild type (WT) to Xoo strain PXO99. (h) Response of the transgenic plants and WT to Xoc strain GX01. Plants were pretreated with 250 mM JA and two days later were inoculated with Xoo or Xoc. Water inoculation was used as mock control. Data represent means ± SD. n = 3 (a, b, d, e). n = 30 (c, f, g, h). The different letters above each bar in (a, b, c, d, e, f, g, h) indicate statistically significant differences, as determined by one‐way ANOVA analysis followed by Tukey's multiple test (P < 0.05).

Figure 6

Natural variation in OsGELP77. (a) Natural variation and haplotype analysis of OsGELP77 in 2178 rice accessions. The three haplotypes were based on the six SNPs distributed in the promoter. (b) Geographic distributions of 2178 rice accessions. (c) Disease responses of 109 mini‐core rice accessions harbouring three classes of OsGELP77 haplotype after Xoo PXO99 infection. Plants were inoculated with Xoo at the booting stage. Data represent means ± SD (n = 30). (d) Relative OsGELP77 transcript level of the three classes of haplotypes in leaves. Data represent means ± SD (n = 3). (e) Distributions of three OsGELP77 haplotypes in wild rice. (f) Nucleotide diversity of OsGELP77 and its surrounding 120‐kb region in different rice subgroups. (g) Tajima's D values of OsGELP77 genomic sequences in the subgroups of cultivated rice. (h) Distributions of three OsGELP77 haplotypes in hybrid rice varieties. The different letters above each bar in (c, d) indicate statistically significant differences, as determined by one‐way ANOVA analysis followed by Tukey's multiple test (P < 0.05).

Figure 7

OsGELP77 is a disease‐resistance QTL. (a) Colocalization of OsGELP77 and disease resistance QTL. LOD, Logarithm of odds. (b) Relative transcript level of OsGELP77. (c) Contents of lipid species in the leaves of NIL‐OsGELP77 ^Hap3 and Zhenshan 97. (d) JA content in the leaves of NIL‐OsGELP77 ^Hap3 and Zhenshan 97. (e) Response of NIL‐OsGELP77 ^Hap3 and Zhenshan 97 to different Xoo strains. Plants were inoculated with Xoo at the booting stage. (f) Response of NIL‐OsGELP77 ^Hap3 and Zhenshan 97 to different Xoc strains. Plants were inoculated with Xoc at the tillering stage. (g) Response of NIL‐OsGELP77 ^Hap3 and Zhenshan 97 to M. oryzae isolate 99–20‐2. Plants were inoculated with M. oryzae at the tillering stage. Data represent means ± SD. n = 3 (b, c, d), n = 30 (e, f, g). Asterisks in (b, c, d, e, f, g) indicate significant differences between Zhenshan 97 and NIL‐OsGELP77 ^Hap3 determined by two‐tailed Student's t‐test at **P < 0.01 or *P < 0.05.

Figure 8

OsWRKY45 genetically acts upstream of OsGELP77. (a) OsGELP77 expression in OsWRKY45‐OE and oswrky45 mutant. (b) DNA binding activity assay of OsWRKY45 by EMSA assay. (c) Binding assay of OsWRKY45 to the promoter of OsGELP77 by ChIP‐qPCR in OsWRKY45:GFP plants using the anti‐GFP antibody. Anti‐GFP antibody was used for immunoprecipitation and IgG acted as a control. The blue capital letters indicate the intact W‐box, the red capital letters represent the mutated W‐box. (d) Activity assay of OsWRKY45 in regulating OsGELP77 expression. (e) JA contents in the leaves of transgenic plants and the corresponding background. (f) Responses of the transgenic plants and the corresponding background to Xoo strain PXO99. Plants were inoculated with Xoo at the booting stage. (g) FPKM values of OsWRKY45‐1 and OsWRKY45‐2 in rice leaf. (h) Responses of rice varieties harbouring OsGELP77 ^Hap3 and OsWRKY45‐1 or OsWRKY45‐2 to Xoo strain PXO99. Plants were inoculated with Xoo at the booting stage. Data represent means ± SD. n = 3 (a, c, d, e), n = 30 (f). Asterisks in (a, c, d) indicate significant differences between transgenic plants and WT or control determined by two‐tailed Student's t‐test at **P < 0.01 or *P < 0.05. The different letters above each bar in (e, f, g, h) indicate statistically significant differences, as determined by one‐way ANOVA analysis followed by Tukey's multiple test (P < 0.05).