ORYZA SATIVA SPOTTED-LEAF 41 (OsSPL41) Negatively Regulates Plant Immunity in Rice

doi:10.1016/j.rsci.2023.02.004

Abstract

Abstract:

Identification of immunity-associated leucine-rich repeat receptor-like protein kinases (LRR-RLK) is critical to elucidate the LRR-RLK mediated mechanism of plant immunity. Here, we reported the map-based cloning of a novel rice SPOTTED-LEAF 41 (OsSPL41) encoding a putative LRR-RLK protein (OsLRR-RLK41/OsSPL41) that regulated disease responses to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). An 8-bp insertion at position 865 bp in a mutant spotted-leaf 41 (spl41) allele led to the formation of purple-brown lesions on leaves. Functional complementation by the wild type allele (OsSPL41) can rescue the mutant phenotype, and the complementary lines showed similar performance to wild type in a number of agronomic, physiological and molecular indices. OsSPL41 was constitutively expressed in all tissues tested, and OsSPL41 contains a typical transmembrane domain critical for its localization to the cell membrane. The mutant exhibited an enhanced level of resistance to Xoo in companion of markedly up-regulated expression of pathogenesis-related genes such as OsPR10a, OsPAL1 and OsNPR1, while the level of salicylic acid was significantly increased in spl41. In contrast, the over-expression lines exhibited a reduced level of H₂O₂and were much susceptible to Xoo with down-regulated expression of pathogenesis-related genes. These results suggested that OsSPL41 might negatively regulate plant immunity through the salicylic acid signaling pathway in rice.

Key words: bacterial blight, leucine-rich repeat receptor-like protein kinase, plant immunity, reactive oxygen species, rice, spotted leaf

Tan Jingyi, Zhang Xiaobo, Shang Huihui, Li Panpan, Wang Zhonghao, Liao Xinwei, Xu Xia, Yang Shihua, Gong Junyi, Wu Jianli. ORYZA SATIVA SPOTTED-LEAF 41 (OsSPL41) Negatively Regulates Plant Immunity in Rice[J]. Rice Science, 2023, 30(5): 426-436.

Figures/Tables 8

Fig. 1. Identification and functional validation of OsSPL41. A, An 8-bp insertion (red letters) was detected in the mutant allele of Os04g0487200. ATG and TAA refer to initiation codon and termination codon, respectively. B, Structure of complementary construct p1300-OsSPL41-com. RB, Right border; LB, Left border; Pro, Promoter; CDS, Coding sequence; Hyg, Hygromycin; NOS, Terminator. C, Phenotypes of IR64, spl41 and complementary line (com-1) at the tillering stage. Scale bar, 10 cm. D, Leaf phenotypes of IR64, spl41 and complementary line (com-1) at the tillering stage. Scale bar, 1 cm. E, Contents of chlorophyll a, chlorophyll b and carotenoid in IR64, spl41 and complementary line (com-1) at the tillering stage.

Fig. 2. Physiological parameters in IR64, spl41 and complementary line (com-1). A?G, Hydrogen peroxide (H2O2) content (A), catalase (CAT) activity (B), superoxide dismutase (SOD) activity (C), ascorbate peroxidase (APX) activity (D), peroxidase (POD) activity (E), total soluble protein content (F) and malondialdehyde (MDA) content (G) in IR64 (wild type), spl41 and complementary line (com-1). Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences by the Duncan’s multiple test (P < 0.05).

Fig. 3. Characterization of over-expression lines at the heading stage. A, Phenotypes of wild type (WT) Nipponbare and over-expression lines OE3 and OE5. Scale bar, 15 cm. B, Leaf phenotypes of WT and over-expression lines OE3 and OE5. Scale bar, 1 cm. C, Relative expression of OsSPL41 in WT and over-expression lines. * and ** indicate significant differences at P < 0.05 and P < 0.01 by the Student’s t-test, respectively. The first leaves from the top were sampled at the tillering stage. Ubiquitin gene was used as a reference gene. D?G, Contents of hydrogen peroxide (H2O2) (D) and malondialdehyde (MDA) (E) as well as activities of catalase (CAT) (F) and superoxide dismutase (SOD) (G) in WT and over-expression lines (OE3 and OE5). Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences by the Duncan’s multiple test (P < 0.05).

Fig. 4. Responses of OsSPL41 over-expression to bacterial blight pathogen race PXO280. A, Lesion length in reaction to bacterial blight race PXO280. Scale bar, 1 cm. B, Relative expression of defense genes in Nipponbare (Wild type, WT) and over-expression lines (OE3 and OE5) inoculated with bacterial blight race PXO280. The first leaves from the top sampled at the tillering stage were used for the gene expression analysis. Ubiquitin gene was used as a reference gene. Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences by the Duncan’s multiple test (P < 0.05).

Fig. 5. Histochemical and gene expression analysis of cell death. A, Trypan blue staining of IR64 (left, before staining; right, after staining), spl41 (left, before staining; right, after staining) and complementary plants (left, before staining; right, after staining) at the late-tillering stage. Scale bar, 1 cm. B, TDT-mediated dUTP Nick End Labeling (TUNEL) assay of IR64 and spl41 leaves, blue fluorescence represents 4’,6-diamino-phenylindole (DAPI) staining, green fluorescence represents positive TUNEL signal. Scale bar, 50 μm. C, Relative expression levels of metascaspase (MC) genes by qRT- PCR. The first leaves from the top sampled at the tillering stage were used for the gene expression analysis. Ubiquitin gene was used as a reference gene. Data are Mean ± SD (n = 3). * and ** represent significant differences at P < 0.05 and P < 0.01 by the Student’s t-test, respectively.

Fig. 6. Subcellular localization of OsSPL41. A, Subcellular localization of OsSPL41-GFP fusion proteins. FM4-64, Cell membrane specific dye. Scale bar, 5 μm. B, Subcellular localization of extracellular domain. Scale bar, 5 μm. C, Subcellular localization of intracellular domain. Scale bar, 2 μm. D, Structural identification of OsSPL41 amino acid sequence. Red and green boxes indicate the extracellular and intracellular domains, respectively.

Fig. 7. Spatial-temporal expression pattern of OsSPL41. A, β-glucuronidase (GUS) transient expression driven by OsSPL41 promoter in root, stem, leaf, sheath and spikelet of Nipponbare. Scale bar, 0.2 cm. B, Relative expression levels of OsSPL41 in roots, stems, leaves, sheaths and spikelets of IR64 at the heading stage. Ubiquitin gene was used as a reference gene. Data are Mean ± SD (n = 3).

Fig. 8. Disease evaluation, gene defense genes in IR64 and spl41. The first leaves from the top sampled at the tillering stage were used for the gene expression analysis. Ubiquitin gene was used as a reference gene. D, Hormone levels in IR64 and spl41. Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences by the Duncan’s test (P < 0.05). * and ** represent significant differences at P < 0.05 and P < 0.01 by the Student’s t-test, respectively.

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