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, 16 (10), 2795-808

Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-box/armadillo Repeat Protein Endowed With E3 Ubiquitin Ligase Activity

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Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-box/armadillo Repeat Protein Endowed With E3 Ubiquitin Ligase Activity

Li-Rong Zeng et al. Plant Cell.

Abstract

The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice-blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.

Figures

Figure 1.
Figure 1.
Physical Delineation of the spl11 Mutation. (A) Schematic representation of the BAC contig spanning the Spl11 locus. The overlaps between BAC inserts are displayed to scale as open bars. The dotted vertical lines mark the positions of DNA markers. The BAC insert containing the Spl11 locus is highlighted in light gray. Orientation of rice chromosome 12 is indicated in the top right corner. (B) Fine physical mapping of Spl11 in BAC78. The two cross-hatched gray bars denote the sequenced regions in BAC78. The vertical dotted lines denote the positions of the respective cleaved amplified polymorphism sequence markers. The number of recombinants/number of segregants tested is indicated for each marker. Arrows above the bars mark the NotI cutting sites of the BAC78 insert. T7 and Sp6 indicate the orientation of the insert cloned into the BAC vector pBeloBAC11. The position of the subclone TAC20 insert that contains the Spl11 gene is displayed. (C) Prediction of potential coding sequences in the 27-kb region of TAC20 where the Spl11 gene was physically delimited. The gray bar depicts the sequenced area. The three solid gray lines designated as G1, G2, and G3 indicate the regions with high coding probability. The vertical lines mark the BslI cutting sites. The asterisk denotes the putative mutation site in spl11. Exons predicted in G3 by the programs GENSCAN and Fgenesh using different matrixes are displayed in dark gray. (D) RFLP fingerprinting of IR68, spl11, and Nipponbare genomic DNA at the Spl11 locus and detection of a point mutation detected in the spl11 gene. Nineteen restriction enzymes were analyzed but only the results of BslI are shown. (1) Genomic DNA was digested with BslI and then separated on a 1.0% agarose gel. A TAC20 insert digested with HindIII was used as the probe for hybridization. (2) The same blot hybridized with a TAC20 insert in (1) was used. DNA spanning the putative mutated BslI site was amplified from TAC20 and used as the probe. Gray arrows denote the polymorphic bands. M, λ/HindIII DNA marker (New England Biolabs, Beverly, MA). (3) DNA sequence in the vicinity of the spl11 mutation. The C-to-T point mutation in spl11 is denoted by an arrow. This point mutation causes a premature stop codon as marked by the underline. The asterisk marks the start codon for the SPL11 protein.
Figure 2.
Figure 2.
Analysis of IR64 Lesion Mimic Mutants Allelic to spl11. (A) Lesion phenotype of spl11 and IR64 background lesion mimic mutants. Picture was taken of leaves from 2-month-old plants. (B) DNA gel blot analysis of the Spl11 locus in wild-type plants and different mutant lines. (1) Genomic DNA was restricted by BslI and then separated on a 1.0% agarose gel. A 2.5-kb genomic DNA fragment at the 5′ end of the Spl11 gene was used as the probe. (2) Genomic DNA was digested by EcoRI and PstI, respectively. The same probe in (1) was used for the hybridization. Changes detected at the Spl11 locus in the IR64 mutants are described in the text. (C) Transcript analysis of Spl11 in IR64 lesion mimic mutants by RT-PCR. Spl11-specific primers were used to amplify a 0.84-kb Spl11 cDNA fragment from total RNA. The rice Actin1-specific primers were used in the RT-PCR to quantify the cDNA template. The experiment was repeated three times.
Figure 3.
Figure 3.
Functional Complementation Test of the Spl11 Candidate Gene. The leaves of 2-month-old plants are shown. Wild-type TP309 and mutant TP2-3 (TP309spl11/spl11) are used as nontransgenic controls. Lines pGW78-34 and pGW78-161 are Spl11 transgenic plants: spl11 mutant TP2-3 (TP309spl11/spl11) with the 8.06-kb XbaI-PacI fragment of the wild-type gene. Line pGW78-28 indicated as an example of failure in transformation.
Figure 4.
Figure 4.
Transcript Abundance of Spl11 in Different Tissues, Gene Structure, and Deduced Amino Acid Sequence of Spl11. (A) RNA gel blot analysis of the Spl11 transcript accumulation in different tissues. Total RNA from 3-week-old leaves (L), stems (S), and roots (R) of IR68 were probed with a 0.84-kb cDNA fragment at the 3′ portion of Spl11. Ethidium bromide staining of rRNA was used as a loading control. (B) Spl11 gene structure. Exons are denoted as black boxes. The number below each exon indicates the length of the exon in base pairs. (C) Deduced amino acid sequence of Spl11. The N-terminal Ala-rich region is in bold. Amino acids of the coiled-coil domain (145 to 165) are highlighted. The U-box domain (272 to 346) is boxed. Amino acids of the ARM repeat motifs are displayed in italics and boldface. (D) Sequence alignments of the ARM repeat of β-catenin and SPL11. Numbers of the ranges of amino acids composing each repeat are shown on the left. The repeats are structurally similar, with each repeat containing three helices, H1, H2, and H3, as indicated. The chemically conserved hydrophobic and polar residues are highlighted in dark and light gray, respectively.
Figure 5.
Figure 5.
Amino Acid Sequence Alignments between SPL11 and U-Box-ARM Proteins from Other Plant Species. (A) Sequence alignments in the highly conserved U-box domain of SPL11 and those of other U-box-ARM proteins. The numbers on the left or right indicate the amino acid residues. Gaps, which were introduced to maximize alignment, are indicated by dashes. The residues conserved among the compared sequences are boxed in black or light gray based on the degree of conservation. AK121978 from O. sativa (GI:37991601), ARC1 from B. napus (GI:2558938), ACRE276 from N. tabacum (GI:30013679), AAM91213 from A. thaliana (GI:22136270), bg55 from B. gymnorrhiza (GI:14149112), PHOR1 from Solanum tuberosum (GI:13539578), CMPG1 from P. crispum (GI:14582202), and NtPUB4 from N. tabacum (GI:28974687) are shown. Only the one most highly related to SPL11 from rice and Arabidopsis is included in the alignment because of the large number of U-box-ARM proteins present in the rice and Arabidopsis genomes. The asterisk and arrows marked the amino acids that were mutated in the SPL11 E3 ligase activity assay. (B) Schematic representation of SPL11 and other U-box-ARM proteins of plants. The black box indicates the U-box domain, and the individual ARM repeat of the ARM domain is indicated by a numbered, shaded box. The percentage of sequence identity of the ARM repeats from plant U-box-ARM proteins to their most homologous ARM repeats in SPL11 is indicated. Detail sequence alignment in the ARM domain of SPL11 with those of other U-box-ARM proteins is indicated in Supplemental Figure 1 online. (C) Phylogenetic relationship between SPL11 and Arabidopsis U-box/ARM repeat proteins. The phylogenies were generated with neighbor joining with 400 bootstrap replicates and were rooted at midpoint. The bootstrap values are shown as percentages. AtPUB8 (locus At4g21350) was not included in the tree because no EST, SAGE tag, or cDNA was identified for the corresponding predicted gene.
Figure 6.
Figure 6.
E3 Ubiquitin Ligase Activity of SPL11. (A) MBP-SPL11 and MBP-CBL fusion proteins were assayed for E3 activity in the presence of E1 (from wheat, GI:136632), E2 (AtUBC9, GI:20136191), and 32P-labeled ubiquitins. The numbers on the left denote the molecular mass of marker proteins in kilodaltons. Mouse E3 ubiquitin ligase CBL (GI:38605691) was used as a positive control. 32P-ubiquitin is indicated by an arrow. MBP itself was used as a negative control. (B) E3 ligase activity of SPL11 and its mutants. CK, MBP; lane 1, wild-type SPL11; lane 2, SPL11 (V290R); lane 3, SPL11ΔC314P315T316. 32P-ubiquitin is indicated by arrow. The bottom panel shows two times of the corresponding amount of MBP and MBP fusion proteins used in the E3 activity assay.
Figure 7.
Figure 7.
Expression Patterns of Spl11 in Rice–Blast Interaction. Total RNA was isolated from infected leaves at the indicated hours after rice blast inoculation. Approximately 10 μg of total RNA was loaded in each lane in RNA gel blotting. 32P-labeled Spl11 cDNA fragment (0.84 kb) was used as the probe in the RNA hybridization. A pair of Spl11-specific primers was used in the RT-PCR analysis. The rRNA gel shows the loading quantification of the RNAs in the RNA gel blot analysis. The amplification of the rice Actin1 gene was used as a control for equal amount of total RNAs in the RT-PCR analysis. The numbers denote the hours after rice blast inoculation.

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