A conserved clathrin-coated vesicle component, OsSCYL2, regulates plant innate immunity in rice

Abstract

Clathrin-mediated vesicle trafficking (CMVT) is a fundamental process in all eukaryotic species, and indispensable to organism's growth and development. Recently, it has been suggested that CMVT also plays important roles in the regulation of plant immunity. However, the molecular link between CMVT and plant immunity is largely unknown. SCY1-LIKE2 (SCYL2) is evolutionally conserved among the eukaryote species. Loss-of-function of SCYL2 in Arabidopsis led to severe growth defects. Here, we show that mutation of OsSCYL2 in rice gave rise to a novel phenotype-hypersensitive response-like (HR) cell death in a light-dependent manner. Although mutants of OsSCYL2 showed additional defects in the photosynthetic system, they exhibited enhanced resistance to bacterial pathogens. Subcellular localisation showed that OsSCYL2 localized at Golgi, trans-Golgi network and prevacuolar compartment. OsSCYL2 interacted with OsSPL28, subunit of a clathrin-associated adaptor protein that is known to regulate HR-like cell death in rice. We further showed that OsSCYL2-OsSPL28 interaction is mediated by OsCHC1. Collectively, we characterized a novel component of the CMVT pathway in the regulation of plant immunity. Our work also revealed unidentified new functions of the very conserved SCYL2. It thus may provide new breeding targets to achieve both high yield and enhanced resistance in crops.

Keywords: Oryza sativa; cell death; clathrin-mediated vesicle trafficking; disease resistance; lesion mimic mutant.

Figure 1

Map‐based cloning of OsSCYL2 gene. (a) The scyl2‐1 mutation was located on chromosome 1 between Indel markers ID2 and ID5. (b) The scyl2‐1 mutation was delimited to an interval about 18 kb between Indel markers ID3 and ID4 using 482 mutant F₂ individuals. (c) Only 1 open reading frame, LOC_Os01g42950 (OsSCYL2) which has 14 exons and 13 introns, was predicted in the ID3–ID4 interval. Exons and introns are indicated by black rectangles and black lines, respectively. (d) A 12 bp‐deletion in the fourth exon of LOC_Os01g42950 (OsSCYL2) was identified in the scyl2‐1 [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

(a) Schematic diagram shows the mutation position of scyl2‐1, scyl2‐2, scyl2‐3 and scyl2‐4. The dotted box indicated the region encoding the pseudokinase domain. (b) Wild type (WT) and scyl2‐1 plants at maximum tillering stage. Scale Bar = 20 cm. (c) Leaf phenotype of WT and scyl2‐1 at maximum tillering stage. Note the increasing number of necrotic spots and leaf senescence with the growth of leaf in scyl2‐1 mutant. Arrows indicate the early leaf senescence in scyl2‐1 mutant. Scale Bar = 1 cm. (d) WT and scyl2‐1 plants at maturity stage. Scale Bar = 20 cm. (e) Comparison of the flag leaf blade and leaf sheath at maturity stage. Scale Bar = 1 cm. (f) Light avoidance assay of WT and scyl2‐1 flag leaf. Scale Bar = 1 cm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

scyl2‐1 exhibited alterations in several important agronomic traits. (a) Grain phenotypes of wild type (WT) and scyl2‐1. The grain size of scyl2‐1 was smaller as compared WT, and lesion mimic phenotype could be observed on the surface of grain hulls of WT and scyl2‐1. Scale Bar = 1 cm. Plant height of WT and scyl2‐1 at the maturation stage. (c) Tiller number of WT and scyl2‐1.(d) Panicle length of WT and scyl2‐1. (e) Grain number per panicle of WT and scyl2‐1. (f) Seed setting rate of WT and scyl2‐1. (g) Grain length of WT and scyl2‐1. (h) Grain width of WT and scyl2‐1. (i) Grain thickness of WT and scyl2‐1. (j) 1000‐Grain weight of WT and scyl2‐1. Error bars indicate standard deviation of twenty biological replicates, **p < 0.01 [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Mutation of OsSCYL2 led to cell death, ROS accumulation, and abnormal chloroplast morphology and activities. (a) Dead cells were detected by trypan blue staining. Scale Bar = 1 cm. (b) MDA content in the WT and scyl2‐1 flag leaves. Error bars indicate the standard deviation of three biological replicates, **p < 0.01. (c) Soluble protein in the WT and scyl2‐1 flag leaves. Error bars indicate the standard deviation of three biological replicates, **p < 0.01. (d) In situ detection of H₂O₂ in leaves by DAB staining. Scale Bar = 1 cm. (e) The enzymatic activities of SOD in the WT and scyl2‐1 flag leaves. Error bars indicate the standard deviation of three biological replicates. (f) The enzymatic activities of POD in the WT and scyl2‐1 flag leaves. Error bars indicate the standard deviation of three biological replicates, **p < 0.01. (g, h) Chloroplast ultrastructure in WT (g) and scyl2‐1. (h) Flag leaves at a flowering stage when the lesion symptom was observed in scyl2‐1. Scale Bar = 1 μm. (i) Chlorophyll content in the flag leaves of WT and scyl2‐1 plants. Error bars indicate the standard deviation of three biological replicates, **p < 0.01. (j) Net photosynthetic rate in the WT and scyl2‐1 flag leaves. Error bars indicate standard deviation of eight biological replicates, **p < 0.01. Car, carotenoid; Chl a, chlorophyll a; Chl b, chlorophyll b; CM, chloroplast membrane; DAB, 3,3ʹ‐diaminobenzidine; MDA, malondialdehyde; OG, osmiophilic globules; POD, peroxidase; SG, starch granule; SOD, superoxide dismutase; Thy, thylakoid lamellae; WT, wild type [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Defence responses are constitutively activated in the scyl2‐1 mutant. (a) The leaves of the WT and scyl2‐1 mutant 2 weeks after inoculation with Xanthomonas oryzae pv. Oryzae (Xoo) isolates P6, P7 and XOO4, respectively. (b) Lesion length in the WT and scyl2‐1 2 weeks after inoculation with Xoo isolates P6, P7 and XOO4, respectively. Error bars indicate standard deviation of 15 biological replicates, **p < 0.01. (c) Comparison of expression levels of defence response genes involved in SA‐ and JA‐signalling pathway between the WT and scyl2‐1 using the qRT‐PCR assay. The second fully‐expanded leaves at the tillering stage were collected for analysis. Error bars indicate the standard deviation of three biological replicates, **p < 0.01. qRT‐PCR, quantitative real‐time polymerase chain reaction; WT, wild type [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Expression pattern of OsSCYL2 and subcellular localisation of OsSCYL2. (a) qRT‐PCR analysis of OsSCYL2 expression in various tissues. Tissues were from plants at the heading stage. Error bars indicate the standard deviation of three biological replicates. (b) Gus staining of SPL38pro:GUS transgenic plants at heading stage. (c) Tobacco leaf epidermal cells expressing OsSCYL2:YFP (green) and 35S::CFP (red). Scale Bar = 20 μm. (d) Tobacco leaf epidermal cells expressing OsSCYL2:YFP (green). Chloroplasts in the cell were visualized by chlorophyll autofluorescence (red). Scale Bar = 20μm. (e–g) Tobacco leaf epidermal cells were cotransformed with OsSCYL2:YFP and the subcellular organelles markers for Golgi (e), TGN (f) and PVC (g). Scale Bars = 20 μm. qRT‐PCR, quantitative real‐time polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

OsSCYL2 interact with OsSPL28 via OsCHC1. (a) Pairwise interaction of OsSCYL2, OsSPL28 and OsCHC1 using the split‐luciferase assays in tobacco leaf. (b) Pairwise interaction of OsSCYL2, OsSPL28 and OsCHC1 using the BiFC assays in tobacco leaf epidermal cells. Scale Bars = 25 μm. (c) OsSCYL2 and OsSPL28 interact with OsCHC1 in a yeast two‐hybrid assay. It is noted that OsSPL28 and OsSCYL2 do not interact with each other in yeast two‐hybrid (indicated by black box). AD, activating domain; BD, DNA‐binding domain; BiFC, bimolecular fluorescence complementation; cYFP, C‐terminal yellow fluorescent protein; nYFP, N‐terminal yellow fluorescent protein; SD, synthetic defined medium [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8

Gene Ontology functional enrichment analysis. (a) Cellular component category. (b)Biological process category. (c) Molecular function category [Color figure can be viewed at wileyonlinelibrary.com]

Figure 9

Divergent biological functions of SCYL2 in mouse, Arabidopsis and rice. (a) SCYL2, by directly binding to both clathrin and the clathrin‐associated adaptor protein complex 2 (AP2), plays a critical role for the normal functioning of the nervous system in mouse (Conner & Schmid, ; Gingras et al., ; Pelletier, 2016). (b) SCYL2A and SCYL2B are functionally redundant and essential for plant growth and development, and directly binds to CHC1 in Arabidopsis (Jung et al., 2017). (c) OsSCYL2–OsSPL28 interaction is mediated by OsCHC1, and is involved in plant immunity [Color figure can be viewed at wileyonlinelibrary.com]