CHR729 Is a CHD3 Protein That Controls Seedling Development in Rice

<div><p>CHD3 is one of the chromatin-remodeling factors that contribute to controlling the expression of genes associated with plant development. Loss-of-function mutants display morphological and growth defects. However, the molecular mechanisms underlying CHD3 regulation of plant development remain unclear. In this study, a rice CHD3 protein, CHR729, was identified. The corresponding mutant line (<i>t483</i>) exhibited late seed germination, low germination rate, dwarfism, low tiller number, root growth inhibition, adaxial albino leaves, and short and narrow leaves. <i>CHR729</i> encoded a nuclear protein and was expressed in almost all organs. RNA-sequencing analysis showed that several plant hormone-related genes were up- or down-regulated in <i>t483</i> compared to wild type. In particular, expression of the gibberellin synthetase gibberellin 20 oxidase 4 gene was elevated in the mutant. Endogenous gibberellin assays demonstrated that the content of bioactive GA₃ was reduced in <i>t483</i> compared to wild type. Moreover, the seedling dwarfism, late seed germination, and short root length phenotypes of <i>t483</i> were partially rescued by treatment with exogenous GA₃. These results suggest that the rice CHD3 protein CHR729 plays an important role in many aspects of seedling development and controls this development via the gibberellin pathway.</p></div

Expression analysis of <i>CHR729</i> and subcellular localization of the encoding protein.

<p>A, qRT-PCR analysis of <i>CHR729</i> expression in WT roots (R), culms (C), leaves (L), leaf sheaths (Ls), and young 3 cm inflorescence (In). Dissected anthers (An), pistils (Pi), lemmas (Le) and paleas (Pa) at inflorescence stage 9 were also analyzed. Values are means ±SD of three replicates. B–D, <i>CaMV35S</i>:CHR729-GFP fusion protein localization in rice protoplast. B, Subcellular localization of CHR729-GFP fusion protein. C, Subcellular localization of MADS3-mCherry fusion protein (nuclear marker). D, Merged image of (B) and (C) in bright field. Scale bars: 10 μm (B–D).</p

Seed germination of WT and <i>t483</i>.

<p>A, Germination of WT and <i>t483</i> seeds after 3 days. B–E, Seedlings of WT and <i>t483</i> at 8 days post-germination. F, Seed germination rate. Values are means ±SD of three independent experiments. Significance of differences between WT and <i>t483</i> was determined by Student’s <i>t-</i>test (**<i>P</i><0.01). Scale bars: 2 cm (A–D); 1 cm (E).</p

Phenotypic comparisons of control and homozygous T₂ transgenic plants of RNAi-3 line (Sanya).

<p>Data are presented as means ± SE. Significance of differences between control and RNAi-3 was detected using Student’s <i>t</i>-test (n = 5).</p><p>Phenotypic comparisons of control and homozygous T₂ transgenic plants of RNAi-3 line (Sanya).</p

Map-based cloning and confirmation of the <i>CHR729</i> gene.

<p>A, Map of the genomic region containing the <i>t483</i> mutant locus of interest. Numerals below the corresponding markers indicate the number of recombinants identified among F₂ plants with the mutant phenotype mutant. The mutated gene was located in a 27 kb region between markers IN27 and IN35. Three ORFs were predicted in the mapped region. Sequencing analysis revealed that an A to T substitution in the fifth exon of the ORF3 resulted in a stop codon in <i>t483</i>. B, Phenotypes of control and typical T₂ transgenic knockdown plants (RNAi-3) at the heading stage. C, Germination of control and RNAi-3 seeds at 3 day. D, Control and RNAi-3 seedlings 8 days post-germination. E, Two-week-old seedlings. F, Expression analysis of <i>CHR729</i> in leaves of control and RNAi-3 by qRT-PCR. G, Seed germination rates in control and RNAi-3 plants. H, Chlorophyll contents in control and RNAi-3 plants. Chlorophyll was extracted from above-ground parts of plants shown in (E). Values are means ±SD (n = 3, **<i>P</i><0.01). Scale bars: 25 cm (B); 2 cm (C, D); 5 cm (E).</p

Phenotypic comparisons of the <i>t483</i> mutant and wild-type Nipponbare (Beijing).

<p>Data are presented as means ± SE. Flag leaf widths were measured through the middle region of leaves at the mature stage. Significance of differences between WT and <i>t483</i> was detected using Student’s <i>t</i>-test (n = 20).</p><p>Phenotypic comparisons of the <i>t483</i> mutant and wild-type Nipponbare (Beijing).</p

Phenotypic characteristics of WT and <i>t483</i>.

<p>A, Plants at the tillering stage after removal of soil. B, Mature plant stage. C, Three uppermost internodes from the main tiller. D, Panicles of WT and <i>t483</i>. E, Adaxial side of leaf segments. F, Leaf chlorophyll contents in WT and <i>t483</i>. Values are means ±SD (n = 3) (**<i>P</i><0.01). G–H, Ultrastructure of chloroplasts in adaxial mesophyll cells of WT (G) and <i>t483</i> (H). I–J, Thylakoid lamellar structure of WT (I) and <i>t483</i> (J). <i>C</i> chloroplast, <i>Thy</i> thylakoid lamellar. Scale bars: 10 cm (A, C); 25 cm (B); 5 cm (D); 1cm (E); 0.5 μm (G, H); 0.2 μm (I, J).</p

Avr3b specifically interacts with GmCYP1 in soybean.

<p>(<b>A, C</b>) Avr3b does not interact with other GmCYP1 homologs in soybean or two CYPs of <i>P</i>. <i>sojae</i>. Selected cyclophilin genes were cloned into pGADT7. Yeast cells were co-transformed with the empty prey vector, pGADT7 or pGADT7 containing one of homologue genes and the bait vector pGBKT7-Avr3b. Yeast transformants were grown on the selective minimal SD medium lacking tryptophan, and leucine (SD-2) or the selective SD medium lacking adenine, tryptophan, histidine, and leucine (SD-4). The plates were photographed 2 days after inoculation. (<b>B, D</b>) Western blots showing protein expression of cyclophilin homologs in yeast cells. Anti-HA antibody was used to detect the expression of cyclophilin homologous proteins of soybean and <i>P</i>. <i>sojae</i>. The same protein gel was stained with Coomassie brilliant blue (CBB) to show loading. WB, western blot. (<b>E</b>) A schematic summary illustrating maturation of Avr3b by plant cyclophilin proteins during <i>Phytophthora</i> infection. Inactive Avr3b enters plant cell and interacts with plant cyclophilin to gain the Nudix hydrolase activity. Active Avr3b suppresses ETI-associated cell death triggered by Avr1b. However, in the presence of Rps3b, active Avr3b triggers Rps3b-induced HR.</p

NbCYP3 and NbCYP4 are required for the Nudix hydrolase and the virulence activity of Avr3b in <i>N</i>. <i>benthamiana</i>.

<p>(<b>A</b>) NbCYP3/4-silenced <i>N</i>. <i>benthamiana</i> can not activate the Nudix hydrolase activity of Avr3b. Avr3b and RFP (as a negative control) were transiently expressed in <i>NbCYP3/4</i>-silenced <i>N</i>. <i>benthamiana</i> leaves. Total proteins were extracted at 48 hpi, and the Nudix hydrolase activity was measured using NADH as substrate. Means and standard errors from four measurements are shown. **, <i>t</i> test P<0.01. (<b>B</b>) NbCYP3 and NbCYP4 are required for the virulence activity of Avr3b in <i>N</i>. <i>benthamiana</i>. FLAG-RFP or FLAG-Avr3b proteins were transiently expressed in <i>N</i>. <i>benthamiana</i> leaves by <i>Agro</i>-infiltration. The leaves were inoculated with <i>P</i>. <i>capsici</i> at 48 hours post <i>Agro</i>-infiltration. Infection was determined using quantitative PCR to measure the ratios of <i>P</i>. <i>capsici</i> and <i>N</i>. <i>benthamiana</i> DNA at 36 hours post inoculation. Means and standard errors from four measurements are shown. ** representing <i>t</i> test <i>P</i> < 0.01. (<b>C</b>) NbCYP3 and NbCYP4 can enhance the Nudix hydrolase activity of Avr3b in <i>N</i>. <i>benthamiana</i>. FLAG-Avr3b was co-expressed in <i>N</i>. <i>benthamiana</i> leaves with GFP-NbCYP3 or GFP-NbCYP4 by <i>Agro</i>-infiltration method, and the Nudix hydrolase activity was measured using NADH as substrate comparing with <i>GFP</i>-expressing leaves. Bars represent standard errors from three biological replicates. The same letter indicates no significant difference between values, and different letters indicate significant differences between values (P < 0.01, nonparametric Kruskal-Wallis test).</p

The enzymatic activity of Avr3b is activated by plant factors.

<p>(<b>A</b>) Avr3b only has Nudix hydrolase activity when produced in plants. Avr3b was ectopically expressed in <i>E</i>. <i>coli</i> (Ec) or <i>N</i>. <i>benthamiana</i> (Nb). Relative Nudix hydrolase activity was calculated by comparing enzymatic activity of purified GST-Avr3b with GST produced in <i>E</i>. <i>coli</i> or purified FLAG-Avr3b with FLAG-GFP in <i>N</i>. <i>benthamiana</i>. NADH was applied as substrate in Nudix hydrolase activity assays. Means and standard errors from three replicates are shown. ** representing <i>t</i> test <i>P</i> < 0.01. (<b>B</b>) <i>E</i>. <i>coli</i> produced Avr3b protein can be activated by incubation with plant protein extracts. Recombinant GST-Avr3b protein was purified from <i>E</i>. <i>coli</i>. The recombinant protein (2 μg) was incubated with 100 μg dialyzed soybean (<i>Glycine max</i>, Gm) extract, 100 μg dialyzed <i>N</i>. <i>benthamiana</i> extract, or 100 μg dialyzed <i>P</i>. <i>sojae</i> (Ps) mycelium extract at 25°C for 15 hours. The Nudix hydrolase activity was then determined using FLAG-GFP as a control. Means and standard errors from three measurements are shown. ** or *, representing <i>t</i> test <i>P</i> < 0.01 or 0.05, respectively.</p