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, 99 (11), 7530-5

A Rice Spotted Leaf Gene, Spl7, Encodes a Heat Stress Transcription Factor Protein

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A Rice Spotted Leaf Gene, Spl7, Encodes a Heat Stress Transcription Factor Protein

Utako Yamanouchi et al. Proc Natl Acad Sci U S A.

Abstract

A rice spotted leaf (lesion-mimic) gene, Spl7, was identified by map-based cloning. High-resolution mapping with cleaved amplified polymorphic sequence markers enabled us to define a genomic region of 3 kb as a candidate for Spl7. We found one ORF that showed high similarity to a heat stress transcription factor (HSF). Transgenic analysis verified the function of the candidate gene for Spl7: leaf spot development was suppressed in spl7 mutants with a wild-type Spl7 transgene. Thus, we conclude that Spl7 encodes the HSF protein. The transcript of spl7 was observed in mutant plants. The levels of mRNAs (Spl7 in wild type and spl7 in mutant) increased under heat stress. Sequence analysis revealed only one base substitution in the HSF DNA-binding domain of the mutant allele, causing a change from tryptophan to cysteine.

Figures

Figure 1
Figure 1
Lesion-mimic phenotype of the spl7 mutant. Leaves of 2-month-old plants grown under various conditions. (a) Natural summer field. (b) Green-house (26°C, solar radiation). (c) Green-house (26°C, UV-filtered solar radiation). (d) Growth chamber (26°C, artificial light). (e) Growth chamber (35°C, artificial light).
Figure 2
Figure 2
Delimitation of candidate genomic region of Spl7. (A) Genetic linkage map of 298 F2 plants showing the relative position of Spl7 with the RFLP markers on chromosome 5. The numbers under the linkage map indicate the number of recombinants in the adjacent marker intervals. (B) YAC (Y) and PAC (P) clone contigs spanning the Spl7 region. Squares indicate YAC/PAC-end clones. Circles indicate STSs generated from the YAC/PAC-end sequences. (C) Fine-scale, high-resolution genetic linkage map of the Spl7 region developed from the analysis of 2,944 F2 plants. The numbers under the linkage map indicate the number of recombinants in the adjacent marker intervals. (D) Fine-scale genetic and physical map of the 16-kb Spl7 region. a to j indicate the CAPS markers. The estimated positions of recombination are shown by arrows. (E) The ORFs in the Spl7 candidate region predicted by GENSCAN. A 5.6-kb NspV-BglII wild-type genomic fragment containing the entire Spl7 candidate ORF was used in the complementation analysis.
Figure 3
Figure 3
Functional complementation test of candidate gene. (A) The leaves of 2-month-old plants. Nontransgenic control: wild type and mutant. Transgenic plants: spl7 mutant with the 5.6-kb NspV–BglII fragment of the wild-type gene (NB5.6k) and spl7 mutant with the vector but no insert (Vector). (B) Three-month-old transgenic plants. The copy number of transgenes is shown on the right. (C) RT-PCR analysis of transgenic plants. PCR products were digested with the restriction enzyme ApaI, whose recognition site was just on the mutated point of the wild-type gene. The 0.43-kb band is derived from intrinsic spl7, and the 0.38-kb band is derived from the transgene (Spl7). The copy number of transgenes is shown below the gel.
Figure 4
Figure 4
The structure of Spl7. (A) The block diagram of the Spl7 structure indicates the position of the typical functional elements. The positions of the primers used for 5′ RACE-PCR are shown by white arrowheads, and those of the primers used for RT-PCR are shown by black arrowheads. (B) Gel blot of the PCR products. W shows wild-type template and M shows mutant template. L2>L5 means that 5′ RACE used the gene-specific primer L2 for the primary PCR and L5 for the nested PCR. U9-L8 shows that the PCR products used the primer combination U9 and L8. C and G show cDNA template and genomic DNA template, respectively. (C) The predicted amino acid sequence. The sequence is shown in single-letter code. The DBD is boxed and the position of intron 2 is shown by a black triangle. The nuclear localization sequence (NLS) is written in bold. The hydrophobic heptad repeat regions are single-underlined (HR-A/B amino acids 134–191, HR-C amino acids 438–458), and an insertion of 21 aa residues between HR-A and HR-B is double-underlined. The AHA is dot-underlined. (D) Alignment of deduced amino acid sequence of Spl7 and known HSF DBD regions. The numbers on both sides indicate the amino acid positions of each protein. Because ZmHSFa and ZmHSFc are partial cDNA clones of DBDs, the temporary amino acid positions are displayed in parentheses. Conserved amino acids among all of the plant HSFs listed here are shaded. The position of the amino acid substitution is shown by an arrowhead. The secondary structure elements based on the LpHSF24 crystal structure (51) are shown below the sequence alignment: α1 to α3, α-helix; β1 to β4, β-sheet. The putative amino acids constituting the central hydrophobic core of the tertiary structure are shown by asterisks.
Figure 5
Figure 5
HSF phylogenetic tree from parsimony analysis of DBDs. Plant HSFs plus a yeast HSF were analyzed by using CLUSTALX V.1.8 software (52) with 1,000 repetitions by the bootstrap method. A consensus tree was generated with TREEVIEW software (53). At, Arabidopsis thaliana; Lp, Lycopersicon esculentum; Gm, Glycine max; Zm, Zea mays; Sc, Saccharomyces cerevisiae. Sequence data were taken from the GenBank database for the following accession numbers: AtHSF1, X76167; AtHSF3, Y14068; AtHSF21, U68561; AtHSF4, U68017; LpHSF8, X67600; LpHSF30, X67601; LpHSFA3, AF208544; LpHSF24, X55347; GmHSF21, Z46952; GmHSF34, Z46953; GmHSF5, Z46956; ZmHSFa, S61458; ZmHSFb, S61448; ZmHSFc, S61459; ScHSF, J03139.
Figure 6
Figure 6
Expression analysis of Spl7 by RT-PCR assay. (A) Change in expression with growth stage. Plants were grown in the field, and leaves were collected every 14 days after sowing (DAS) for RNA extraction. Actin primers were used in the control amplification. (B) Change in expression with heat stress. Plants were grown in a growth chamber for 6 weeks (26°C, control) and then were incubated at 35°C for 1 day and at 42°C for 1 day more.

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