Additional file 1: of Overexpression of OsbHLH107, a member of the basic helix-loop-helix transcription factor family, enhances grain size in rice (Oryza sativa L.)

Figure S1. Characterization of ‘Dongjin’ (WT) and lgs1 plants. Figure S2. Identification of transcripts after the T-DNA insertion site by 5’ RACE. Figure S3. Multiple sequence alignment of OsbHLH107 and its homologs. Figure S4. Relative expression analysis and molecular identification of OsbHLH107 overexpression and CRISPR/Cas9 transgenic plants. Figure S5. Characterization of OsbHLH107-RNAi seeds on a ‘Dongjin’ (WT) background. Figure S6. OsbHLH107 is broadly expressed in various tissues. Figure S7. Subcellular-localization of the truncated forms of OsbHLH107. Figure S8. Y2H assays showed that the truncated form of OsbHLH107 physically interacts with itself. Figure S9. Phylogenetic analysis of the reported OsbHLHs. Figure S10. Identification of OsPIL11 transgenic plants. Figure S11. Relative expression levels of OsPILs in ‘Dongjin’ and lgs1. Figure S12. Statistical analysis of grain length of OsbHLH109-CRISPR/Cas9 (107CR) and OsPIL11-CRSPR/Cas9 (PIL11CR) T1 generation plants. Table S1. Primers used in this study. Table S2. Information regarding cell cycle genes used in this study. Table S3. Information regarding bHLH genes used in this study. (DOCX 5206 kb

OsCNGC13 promotes seed-setting rate by facilitating pollen tube growth in stylar tissues

<div><p>Seed-setting rate is a critical determinant of grain yield in rice (<i>Oryza sativa</i> L.). Rapid and healthy pollen tube growth in the style is required for high seed-setting rate. The molecular mechanisms governing this process remain largely unknown. In this study, we isolate a dominant low seed-setting rate rice mutant, <i>sss1-D</i>. Cellular examination results show that pollen tube growth is blocked in about half of the mutant styles. Molecular cloning and functional assays reveals that <i>SSS1-D</i> encodes OsCNGC13, a member of the cyclic nucleotide-gated channel family. <i>OsCNGC13</i> is preferentially expressed in the pistils and its expression is dramatically reduced in the heterozygous plant, suggesting a haploinsufficiency nature for the dominant mutant phenotype. We show that OsCNGC13 is permeable to Ca²⁺. Consistent with this, accumulation of cytoplasmic calcium concentration ([Ca²⁺]_cyt) is defective in the <i>sss1-D</i> mutant style after pollination. Further, the <i>sss1-D</i> mutant has altered extracellular matrix (ECM) components and delayed cell death in the style transmission tract (STT). Based on these results, we propose that OsCNGC13 acts as a novel maternal sporophytic factor required for stylar [Ca²⁺]_cyt accumulation, ECM components modification and STT cell death, thus facilitating the penetration of pollen tube in the style for successful double fertilization and seed-setting in rice.</p></div

Assessment of a SARS-CoV-2 wastewater monitoring program in El Paso, Texas, from November 2020 to June 2022

The border city of El Paso, Texas, and its water utility, El Paso Water, initiated a SARS-CoV-2 wastewater monitoring program to assess virus trends and the appropriateness of a wastewater monitoring program for the community. Nearly weekly sample collection at four wastewater treatment facilities (WWTFs), serving distinct regions of the city, was analyzed for SARS-CoV-2 genes using the CDC 2019-Novel coronavirus Real-Time RT-PCR diagnostic panel. Virus concentrations ranged from 86.7 to 268,000 gc/L, varying across time and at each WWTF. The lag time between virus concentrations in wastewater and reported COVID-19 case rates (per 100,00 population) ranged from 4–24 days for the four WWTFs, with the strongest trend occurring from November 2021 - June 2022. This study is an assessment of the utility of a geographically refined SARS-CoV-2 wastewater monitoring program to supplement public health efforts that will manage the virus as it becomes endemic in El Paso.</p

Pollen tube growth is impaired in <i>sss1-D</i>.

<p>(A) Comparison of mature panicle of wild type (WT) and <i>sss1-D</i>. Arrows indicate the sterile spikelets. (B) Seed-setting rate. Data are means ± SD (n > 5). **P<0.01 by the Student’s <i>t</i> test. (C) Floral structure of WT and <i>sss1-D</i>. (D and E) I₂-KI staining of pollen grains of WT and <i>sss1</i>-<i>D</i>. (F and G) Aniline blue staining of the WT and <i>sss1</i>-<i>D</i> pollen germination in vitro. (H-K) Aniline blue staining showing normal germination of pollen grains on stigmas in selfed WT (H and I) and <i>sss1</i>-<i>D</i> (J and K) at 5 minutes after pollination (MAP). (L-O) Retardation of pollen tube growth in <i>sss1-D</i>. WT pollen tubes reach the bottom part of the style in most WT pistils at 30 MAP (L and M), whereas in some <i>sss1</i>-<i>D</i> pistils, pollen tubes stay at the stigma–style boundary (N and O). (P-V) Multiple pollen tubes can be observed in the WT ovule at 120 MAP (P), whereas the <i>sss1-D</i> ovule contains fewer or no pollen tubes (Q and R). Pollen tubes stay at the stigma–style boundary (S) or at the middle part of the style in majority of <i>sss1-D</i> pistils (T). (U) Quantification of ovaries with pollen tubes observed in the ovule at 120 MAP, showing defective pollen tube growth in more than half of <i>sss1-D</i> pistils. (V) Verification of the maternal effect of <i>sss1-D</i> for defective pollen tube growth. The <i>sss1-D</i> pollen tubes are capable of growing to ovule at 120 MAP in WT pistils, whereas the WT pollen tubes fail to reach the ovule in more than 50% of <i>sss1-D</i> pistils. Arrows point to pollen tubes in I and K, and pollen tube tips in L-O and S-T. Data in U and V are means ± SD from 3 replicates with > 30 pistils observed per replicate, and different letters indicate a significant difference at <i>P</i> < 0.01 by the Student’s <i>t</i>-test. Scale bars, 5 cm in (A); 2 cm in (C); 100 μm in (D-G,M,O,S,T); 300 μm in (H,J,L,N,P-R); 50 μm in (I and K).</p

OsCNGC13 exhibits permeability to inward Ca²⁺ in HEK293 cells and is required for [Ca²⁺]_cyt accumulation in the style.

<p>(A) Patch-clamp whole-cell recordings of inward Ca²⁺ currents in HEK293 cells transfected with <i>GFP</i> (as a control), <i>OsCNGC13</i>, or <i>OsCNGC13-D</i>. The voltage protocols, as well as time and current scale bars for the recordings are shown. (B) The I-V relationship of the steady-state whole-cell inward Ca²⁺ currents. The data are derived from the recordings shown in A, and presented as means ± SD. (C) Style Ca²⁺ content measurement using SEM-EDX. Increased Ca²⁺ concentrations are detected in WT but not in <i>sss1-D</i> after pollination. (D-G) Fluo-3/AM showing Ca²⁺ accumulation in the styles. Images of styles before Fluo-3/AM incubation (D), after incubation in the buffer without Fluo-3/AM (E), and after Fluo-3/AM incubation (F) are shown. Note that the samples before incubation and the samples incubated in the buffer without Fluo-3/AM were performed as the negative controls, both of which showed no fluorescence. (G) Quantification of Fluo-3/AM fluorescence intensity. (H and I) Ca²⁺ accumulation in the styles indicated by the YC3.6 protein fluorescence. (I) Quantification of YC3.6 fluorescence intensity. Data are means ± SD (n = 4 in C; n = 9 in G; n = 6 in I). *P<0.05 and **P<0.01 by the Student’s <i>t</i> test. Scale bars, 100 μm.</p

Functional complementation and gene knockout analyses.

<p>(A) Genetic scheme of W109 selection. (B-D) The seed-setting rate (B) and qRT-PCR analysis of <i>ORF1</i> (primer pair 6F/6R shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006906#pgen.1006906.g002" target="_blank">Fig 2D</a>) (C) and <i>ORF3</i> transcript levels (D) in W109 and the transgenic plants. Three independent transgenic lines individually transformed with <i>pORF1</i>::<i>ORF1</i>, <i>35S</i>::<i>ORF1-GFP</i>, <i>gORF3</i>, and <i>35S</i>::<i>ORF3-GFP</i> are shown. (E) Observation of pollen tube growth in W109, <i>35S</i>::<i>ORF1-GFP-1</i> transgenic plant, and <i>gORF3-1</i> transgenic plant. Arrow indicates the pollen tube tip. Scale bars, 300 μm. (F) Frequency of the pistils with at least one pollen tube reaching the ovaries at 120 MAP. (G and H) Sketch map of the mutations of <i>ORF1</i> (G) and <i>ORF3</i> (H) in knockout lines. The mutation site, the corresponding seed-setting rate, and the percentage of ovules with pollen tube of each line are shown. Minus (-) and plus (+) signs indicate the number of nucleotides deleted and inserted, respectively. Data are means ± SD (n > 5 in B; n = 3 in G and H); data in F are means ± SD from 3 replicates with > 30 pistils observed per replicate. ns, no significance; **P<0.01 by the Student’s <i>t</i> test.</p

<i>OsCNGC13</i> mRNA expression and subcellular localization of its protein.

<p>(A and B) GUS staining of the spikelets after removing the lemma and palea. Mature pistil before flowering (A) and pistil at 30 MAP (B) are shown. (C-L) <i>In situ</i> hybridization analysis. Longitudinal sections of the floral primodia (C and D), stigma (E) and style (F) before flowering, and the style of the pistil at 30 MAP (I) are shown. (D) The magnified image of the selected area in C. Transverse sections of the stigmas (G and J) and styles (H, K and L) of the pistils before flowering (G and H) and the pistils at 30 MAP (J-L) are shown. (L) Negative control with sense probe. (M-P) Localization of GFP protein (M), OsCNGC13-GFP fusion protein (N), and PIP2;1-mCherry fusion protein (O) in rice protoplasts. (P) The merged image of N and O. (Q-T) Localization of GFP protein in the root tip cells of transgenic rice plants expressing 35S promoter-driven <i>GFP</i> (Q) and localization of OsCNGC13-GFP fusion protein in the root cells of the transgenic rice plants expressing <i>35S</i>::<i>OsCNGC13-GFP</i> (R). (S) Cellular outlines of the root cells were stained with FM4-64 for 5 min on ice. (T) The merged image of R and S. Scale bars, 1 mm in (A and B); 200 μm in (C and D); 50 μm in (E-L); 10 μm in (M-P); 20 μm in (Q-T).</p

Molecular cloning.

<p>(A) The <i>sss1-D</i> locus was mapped to a ~52 kb region on the short arm of chromosome 6 between the markers Q-20 and Y-80. (B) Annotated open reading frames (ORF) in the ~52 kb region. Note that the inversion of a 44 kb genomic fragment in <i>sss1-D</i> disrupts both ORF1 and ORF3. (C) Verification of the inversion in <i>sss1-D</i>. Primers used for PCR analysis are shown in B. (D) A diagram of ORF1 and ORF3 in wild type and their mutant version in <i>sss1-D</i>. The inversion results in a truncated ORF1 with a premature stop (mORF1) and a stop codon-less ORF3 (mORF3). Filled box, exon; bold line, intron. (E) qRT-PCR analysis of the transcript levels of <i>ORF1</i>/<i>mORF1</i> (primer pair 5F/5R in D), <i>ORF2</i>, and <i>ORF3</i>. Data are means ± SD (n = 3).</p

The expression of <i>OsCNGC13</i> (<i>ORF1</i>) positively affects the plant seed-setting rate.

<p>(A) qRT-PCR analysis of <i>OsCNGC13</i> and <i>OsCNGC13-D</i> in WT, <i>sss1-D</i>, and heterozygous plants with the 5F/5R primer pair shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006906#pgen.1006906.g002" target="_blank">Fig 2D</a>. Note that the expression of <i>OsCNGC13</i> and <i>OsCNGC13-D</i> in the heterozygous plants was significantly reduced. (B and C) qRT-PCR analysis of <i>OsCNGC13</i> and <i>OsCNGC13-D</i> in WT, <i>sss1-D</i> and heterozygous plants with allele-specific primers. Unequal reduction of expression of <i>OsCNGC13</i> and <i>OsCNGC13-D</i> is observed in the heterozygous plant as normalized to WT (B) and <i>sss1-D</i> (C). (D) Comparison of mature panicles of wild type Kitaake and two independent lines (-1 and -2) of <i>pOsCNGC13</i>::<i>OsCNGC13-D</i>. Scale bars, 5 cm. (E and F) The seed-setting rate (E) and the percentages of ovules with pollen tube (F) in Kitaake and the transgenic plants. Data in E are means ± SD (n > 5). Data in F are means ± SD from 3 replicates with > 30 pistils observed per replicate. Different letters indicate a significant difference at <i>P</i> < 0.01 by the Student’s <i>t</i>-test. (G) qRT-PCR analysis of <i>OsCNGC13</i> and <i>OsCNGC13-D</i> in Kitaake and the transgenic plants. The primer pairs used in A (5F/5R for both <i>OsCNGC13</i> and <i>OsCNGC13-D</i>), B (6F/6R specific for <i>OsCNGC13</i>), C (7F/7R specific for <i>OsCNGC13-D</i>), G (6F/6R and 7F/7R) are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006906#pgen.1006906.g002" target="_blank">Fig 2D</a>. Data are means ± SD (n = 3). (H) Positive correlation between the seed-setting rates and expression levels of <i>OsCNGC13</i> in several RNAi transgenic lines. (I) Positive correlation between the seed-setting rates and the percentages of ovules with pollen tube in several RNAi transgenic lines. <i>r</i> value is based on two-tailed Pearson correlation analyses.</p

Defective PCD in styles of <i>sss1-D</i> during pollen tube growth and a proposed model of OsCNGC13 function.

<p>(A-H) Transverse paraffin (A-D) and plastic (E-H) sections at the middle of the style. The boxed regions in A, B, E, and F are magnified in C, D, G, and H, respectively. Intercellular space (red arrows), as a result of PCD, is observed in WT but not in <i>sss1-D</i>. (I and J) TEM images of cells at the bottom of the style, showing a collapsed cell in WT (I) but not in <i>sss1-D</i> (J). Nu, nucleus; Mt, mitochondria. (K-P) TUNEL assay shows that DNA fragmentation signal (white arrow) is visible in WT but not in <i>sss1-D</i>. Longitudinal sections of the bottom (K and L), transverse sections at the middle (M and N) and bottom parts (O and P) of the style are shown. (Q) qRT-PCR analysis of the transcript levels of the genes related to PCD. Pollination-triggered expression of these genes is significantly reduced in <i>sss1-D</i>. All samples used in A-P were collected at 30 MAP. Data in Q are the mean ± SD (n = 3). **P < 0.01 by the Student’s <i>t</i>-test. Scale bars, 50 μm in (A, B, E, F and K-P); 1 μm in (I and J). (R) A sketch illustrating the patterns of pollen tube growth in the pistils of the wild type and the <i>sss1-D</i> mutant. The growth of pollen tube is normal in almost all the wild type pistils, while the pollen tube is blocked in about half of the <i>sss1-D</i> pistils. (S) A proposed model of OsCNGC13 function. OsCNGC13 is localized on the plasma membrane of the STT cells and plays an important role in linking [Ca²⁺]_cyt accumulation in the style after pollination, ECM components modification and PCD in the style to facilitate the penetration of the pollen tube, successful double fertilization, and consequently high seed-setting rate.</p