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, 68 (5), 899-913

SPL33, Encoding an eEF1A-like Protein, Negatively Regulates Cell Death and Defense Responses in Rice

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SPL33, Encoding an eEF1A-like Protein, Negatively Regulates Cell Death and Defense Responses in Rice

Shuai Wang et al. J Exp Bot.

Abstract

Lesion-mimic mutants are useful to dissect programmed cell death and defense-related pathways in plants. Here we identified a new rice lesion-mimic mutant, spotted leaf 33 (spl33) and cloned the causal gene by a map-based cloning strategy. SPL33 encodes a eukaryotic translation elongation factor 1 alpha (eEF1A)-like protein consisting of a non-functional zinc finger domain and three functional EF-Tu domains. spl33 exhibited programmed cell death-mediated cell death and early leaf senescence, as evidenced by analyses of four histochemical markers, namely H2O2 accumulation, cell death, callose accumulation and TUNEL-positive nuclei, and by four indicators, namely loss of chlorophyll, breakdown of chloroplasts, down-regulation of photosynthesis-related genes, and up-regulation of senescence-associated genes. Defense responses were induced in the spl33 mutant, as shown by enhanced resistance to both the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae and by up-regulation of defense response genes. Transcriptome analysis of the spl33 mutant and its wild type provided further evidence for the biological effects of loss of SPL33 function in cell death, leaf senescence and defense responses in rice. Detailed analyses showed that reactive oxygen species accumulation may be the cause of cell death in the spl33 mutant, whereas uncontrolled activation of multiple innate immunity-related receptor genes and signaling molecules may be responsible for the enhanced disease resistance observed in spl33. Thus, we have demonstrated involvement of an eEF1A-like protein in programmed cell death and provided a link to defense responses in rice.

Keywords: Defense responses; Oryza sativa; SPL33.; eukaryotic translation elongation factor 1 alpha (eEF1A); lesion-mimic mutant; programed cell death.

Figures

Fig. 1.
Fig. 1.
Comparison of wild type (Nip) and spl33 plants. (A) Lesion mimic phenotype in spl33. a, clean leaf blade in the Nipponbare wild type (WT); b and c, lesion mimics at seedling and tillering stages of greenhouse-grown spl33 in winter; d and e, lesion mimics at seedling and tillering stages of field-grown spl33 in summer. (B) spl33 plant at tillering stage showing early senescence in spl33. (C) WT and spl33 plants at heading stage. Note that spl33 was full of lesion mimics. Nip, Nipponbare. Scale bar: 1cm in (A), 10 cm in (B, C).
Fig. 2.
Fig. 2.
Genetic and physical maps of the SPL33 gene. (A) The SPL33 gene was located on chromosome 1 between InDel markers I1-1 and S1-1-5. (B) The SPL33 gene was delimited to the V54-V19 interval using 148 F2 mutant individuals; marker names and number of recombinants are shown. (C) Fine genetic mapping of the SPL33 gene based on 476 mutant F2 individuals. (D) Eleven putative ORFs were located in an ~70-kb region. (E) Gene structure LOC_Os01g02720. Eleven exons and ten introns are indicated by green rectangles and black lines, respectively; a G to T point mutation was identified in the seventh exon (red arrow) generating a premature termination codon. (F) Sequence analysis of the G-to-T mutation site in plants of wild type and spl33.
Fig. 3.
Fig. 3.
Genetic complementation of spl33. (A) spl33 plant transformed with the genomic sequence of SPL33 (pGSPL33) was completely recovered to the wild type Nipponbare (Nip) phenotype. The insert indicates enlargement of leaf section with lesion spots. (B) Transgenic plants were verified by the presence of the hygromycin selectable marker gene. M, molecular markers; pEmV, the empty vector. Scale bar: 10 cm in (A).
Fig. 4.
Fig. 4.
SPL33 encodes a eukaryotic translation elongation factor1A (eEF1A)-like protein. (A) Predicted domains of SPL33 by Simple Modular Architecture Research Tools (SMART). SPL33 has a zinc finger domain (blue rectangle) at the N-terminus. (B) Alignment of the conserved motifs of eukaryotic translation elongation factor 1 alpha (eEF1A) and prokaryotic elongation factor (EF-Tu) from multiple organisms. The amino acid sequence alignment indicated that eEF1A is a highly conserved domain. The region corresponding to the GTP/GDP binding domain of GTP-binding proteins is indicated by white boxes, the conserved amino acid sequences are shown at the top, and the consensus amino acid residues are shown at the bottom.
Fig. 5.
Fig. 5.
Dissection of SPL33 by transformation. (A) Schematic diagram of the overexpression vectors. pUbi::SPL331–223: the 1–669 bp sequence of SPL33 (SPL331–223, encoding amino acids 1–223 of SPL33) that contains the zinc finger domain under the control of the ubiquitin promoter; pUbi::SPL33224–655: the 670–1968 bp sequence of SPL33 (SPL33224–655, encoding amino acids 224–655 of SPL33) that contains three EF-Tu structural domains under the control of the ubiquitin promoter. (B) Phenotypes of wild-type (Nip), spl33 mutant, and two transgenic T0 plants carrying pUbi::SPL331–223 and T0 pUbi::SPL33224–655, respectively. The inset indicates enlargement of leaf section with lesion spots.
Fig. 6.
Fig. 6.
Expression analysis of SPL33. (A) Expression of SPL33 in roots of 7-day-old seedling, stem, flag leaf blade and sheath, and young panicle at booting stage of wild type Nipponbare (Nip) and mutant spl33 analysed by quantitative RT-PCR. (B) Developmental expression pattern of SPL33 in different leaves along plant growth. 1L–8L represent the first to eighth leaf, respectively. (C–H) Histochemical signals in plants carrying the SPL33 promoter–GUS reporter gene. GUS signals were detected in the root (C), stem (D), leaf (E), leaf sheath (F), panicle (G), and spikelet (H). Scale bar: 2 mm in (C–H). Error bars in (A, B) indicate standard deviations of three independent samples. Data are means±SD of three biological replicates (Student’s t-test: *P<0.05; **P<0.01).
Fig. 7.
Fig. 7.
Subcellular localization of SPL33 protein. (A–C) Rice protoplast transient assay. (D–F) N. benthamiana leaf assay. SPL33–GFP, SPL33 fused to GFP; mCherry–HDEL, endoplasmic reticulum (ER) marker. Scale bar: 20 μm.
Fig. 8.
Fig. 8.
H2O2 accumulation and PCD detection in spl33. (A) DAB staining for H2O2 accumulation. (B) Trypan blue staining for cell death. (C, D) Aniline blue staining for callose accumulation under UV light; fluorescent regions indicate callose accumulation. (C) WT; (D) spl33. Scale bar: 100 μm. (E) DNA fragmentation detection in mesophyll cells by TUNEL assay. Red signal represents staining with propidium iodide, and yellow and green signals indicate TUNEL-positive nuclei of dead cells resulting from PCD. Scale bar: 100 μm. WT and spl33 leaf samples in (A–E) were analysed at 28 DAS.
Fig. 9.
Fig. 9.
Identification of early leaf senescence in spl33 at the molecular level. Relative expression of photosynthesis-related genes (A), STAY GREEN (SGR) gene (B), and senescence-associated genes (C). Wild-type (WT) and spl33 leaves were collected from 28-day-old seedling; the expression level of each gene in WT was normalized to 1. Data are means±SD of three biological replicates (Student’s t-test: *P<0.05; **P<0.01).
Fig. 10.
Fig. 10.
Enhanced resistance in the spl33 mutant to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae isolates. (A) Reactions to 12 M. oryzae isolates. The variety LTH was used as a susceptible control. (B) Reactions to 11 Xoo isolates. (C) Disease indices of WT and spl33 to M. oryzae isolates. (D) Lesion lengths of wild-type Nipponbare (Nip) and spl33 to 11 Xoo isolates. Data are means±SD of 15 plants.

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