Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway.

<p><b>A</b>, wild type cells or cells with the integrated mutations were treated with (+) or without (−) MMS at 30°C for 1 hour. Phosphorylation of Chk1 was monitored by the mobility shift assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092936#s2" target="_blank">Materials and Methods</a>. A section of the Ponceau S stained membrane is shown for the loading (bottom). <b>B</b>, Rad3-dependent phosphorylation of Cds1-Thr¹¹ was examined by phosphor-specific antibody (top panel). Loading of Cds1 is shown in the lower panel. <b>C</b>, sensitivity of the cells to acute HU (left) or MMS (right) treatment. Cells were treated with the drugs in YE6S medium at 30°C. At each time point, an aliquot of the culture was removed and spread on YE6S plates for the cells to recover. Colonies were counted and viability was presented as percentages of the untreated cells. Each data point represents an average of three independent experiments for each mutant. <b>D</b>, synthetic effects of the double mutants containing Δ<i>cds1</i> or Δ<i>chk1</i> with the indicated <i>rad4</i> mutations were examined by spot assay.</p

The C13Y and K56R mutations abolish the scaffolding function of Rad4 <i>in vitro</i>.

<p><b>A</b>, schematic diagram of the Rad4 fragments used in this experiment. Each fragment was fused with a GST tag for protein purification and detection in the binding assay using anti-GST antibodies. <b>B</b>, preferential binding of the N- and C-terminal pair of BRCT repeats of Rad4 to Crb2 and Rad9, respectively. Recombinant GST or GST-tagged Rad4 fragments were incubated with immunopurified Crb2 (middle panels) or Rad9 (right panels) bound to the anti-HA antibody beads. The bound proteins were released from the beads in gel loading buffer and analyzed by Western blotting. The membrane was striped and reprobed with anti-HA antibody for Crb2 and Rad9 (bottom panels). Aliquots of the binding reaction were analyzed separately by SDS PAGE as the input (left panel). <b>C</b>, equal amount of the Rad4 fragment b with or without the C13Y-K56R mutation was incubated with Crb2 bound on beads. The input and the Rad4 protein bound to Crb2 were analyzed as in B. <b>D</b>, fragment e with or without the E368K mutation was similarly tested for binding to phosphorylated Rad9. Phosphorylation of Rad9-Thr⁴¹² was detected by blotting of the same membrane with the phospho-specific antibody.</p

<i>In vitro</i> kinase assay using purified Rad3-Rad26 and Cds1(D312E) as the substrate.

<p><b>A</b>, time course of the <i>in vitro</i> kinase reaction. Rad3-Rad26 was affinity-purified from <i>S. pombe</i> using anti-Flag antibody resin, eluted with Flag peptide, and incubated with the kinase-inactive Cds1(D312E) as the substrate in the standard kinase buffer containing 200 μM ATP. At each time point, a small aliquot of the reaction was taken out and analyzed by SDS PAGE followed by Western blotting to examine the phosphorylation of Cds1-Thr¹¹. Rad3 and Rad26 in the same samples were detected by using anti-HA and anti-flag antibody, respectively. Loading of the substrate was shown by Ponceau S staining. The kinase dead Rad3(D2249E)-Rad26 was similarly prepared and used as the control (last lane on the right). <b>B</b>, phosphorylation of Cds1-Thr¹¹ was examined in the presence of increasing concentrations of Rad3-Rad26. The reaction was carried out at 30°C for 30 min. <b>C</b>, full-length Rad4 was purified from <i>S. pombe</i>. Removal of the N-terminal GST tag was analyzed by SDS PAGE (left) and confirmed by Western blotting (right) using anti-Rad4 antibodies. Asterisks indicate the degradation products of Rad4. <b>D</b>, the Rad3 kinase reactions were carried out at 30°C for 20 min in the absence or presence of increasing amount of purified Rad4 and analyzed as in A.</p

Genetic screen of novel <i>rad4</i> mutants with enhanced sensitivities to HU and MMS in the <i>rad4⁺</i> shut-off strain.

<p><b>A</b>, thiamine-controlled cell growth of the shut-off strain (nmt-rad4) in which the endogenous promoter of <i>rad4⁺</i> was replaced with a thiamine repressive <i>nmt81</i> promoter. Logarithmically growing cells were diluted in fivefold steps and spotted on EMM6S plates with (+) or without (-) thiamine. The plates were incubated at 30°C for 3 days before being photographed. <b>B</b>, thiamine-regulated expression of Rad4 was examined by Western blotting. Untagged Rad4, Rad4 with the deletion of whole C-terminus (ΔC) and HA-tagged Rad4 were expressed on a vector in the shut-off strain. Expression of Rad4 in the presence (+) or absence (-) of thiamine was examined by using anti-Rad4 antibodies (Top). The same membrane was stripped and blotted with anti-HA antibody (bottom). Asterisks indicate the cross-reacting materials. <b>C</b>, molecular architecture of Rad4 with relative locations of the newly isolated (solid circles) and previously reported (open circles) mutations and the AAD domain. The four BRCT repeats are marked by roman numerals. The regions covered by mutational PCRs in the genetic screening are also shown. <b>D</b>, sensitivities of the <i>rad4</i> mutants to HU and MMS were assessed by spot assay in the shut-off strain. The newly screened (top part) and the previously reported (lower part) mutations are marked on the right. Wild type cells, Δ<i>rad3</i> mutant, and the shut-off strain carrying an empty vector or the vector expressing wild-type Rad4 were used as controls.</p

The C13Y and K56R mutations abolish the scaffolding function of Rad4 <i>in vivo</i>.

<p><b>A</b>, Rad9 was IPed from the lysates of MMS-treated cells with the indicated mutation (bottom panel). Wild type or mutant Rad4 (top two panels) Co-IPed with Rad9 was detected by Western blotting using anti-Rad4 antibodies. Phosphorylated Rad9-Thr⁴¹² was shown in the third panel from the top. <b>B</b>, Co-IP of Rad4 containing the indicated mutations with phosphorylated Rad9 from HU-treated cells. <b>C</b>, Co-IP of Rad4 and Rad9 with Crb2 from MMS-treated cells.</p

Drug sensitivities of <i>S. pombe</i> with the integrated <i>rad4</i> mutations.

<p><b>A</b>, the two-step marker switching method used for integration of the mutations. The genomic locus of <i>rad4⁺</i> is shown on top. <b>B</b>, sensitivities of the cells with integrated <i>rad4</i> mutations to HU or MMS were examined by spot assay. The newly identified (upper part) and the previously reported (lower part) mutants are marked on the right. <b>C</b>, expression of Rad4 in the cells with the integrated <i>rad4</i> mutations was detected in whole cell lysates by Western blotting using anti-Rad4 antibodies. The asterisk denotes the cross-reactive material. Note: like the T45M mutant, A60P is a <i>ts</i> mutant.</p

DataSheet_1_Differential responding patterns of the nirK-type and nirS-type denitrifying bacterial communities to an Ulva prolifera green tide in coastal Qingdao areas.docx

Coastal eutrophication may be a vital inducement of green tide. Denitrification is an important nitrogen removal pathway that involves a series of enzymatic reactions. The rate-limiting step in the conversion of nitrite to nitric oxide is encoded by two functionally equivalent but structurally distinct genes, copper-containing nitrite reductase gene (nirK) and cytochrome cd1-containing nitrite reductase gene (nirS). Here, we used Illumina Miseq sequencing approach to examine the variations in denitrifying bacterial community characteristics and interactions during an Ulva prolifera green tide in coastal Qingdao areas. Our findings suggested that the variations in the denitrifying bacterial community structure during the green tide were closely related to the changes of chlorophyll a content, salinity and dissolved oxygen content. The nirK-type denitrifying bacteria were more sensitive to green tide than the nirS-type denitrifying bacteria. Additionally, the nirK-type denitrifying bacterial interactions were more stable and complex during the outbreak phase, while the nirS-type denitrifying bacterial interactions were more stable and complex during the decline phase. All of these characters demonstrated that the nirK-type and nirS-type denitrifying bacteria respond differently to the green tide, implying that they may occupy different niches during the green tide in coastal Qingdao areas.</p

DataSheet_1_Deep mowing rather than fire restrains grassland Miscanthus growth via affecting soil nutrient loss and microbial community redistribution.docx

Fire and mowing are crucial drivers of grass growth. However, their effects on soil properties, microbial communities, and plant productivity in dry-alkaline grasslands have not been well investigated. This study evaluated the effects of mowing (slightly and deeply) and fire on vegetation traits (Tiller number per cluster and plant height) and biomass (plant dry weight), and soil availability of N, P, and K, as well as soil microorganism abundance in a Miscanthus system. We designed one control and three experimental grass plots (slightly and deeply mowed, and burned) in 2020–2021 in the Xi’an Botanical Garden of Shaanxi Province, Xi’an, China. Tiller number, plant height per cluster, and soil N, P, and K availability during Miscanthus growth decreased significantly (p < 0.05) in all treatments compared to the control. However, this effect was much greater in the deep-mowing plot than in the other plots. After harvest, deep mowing induced the greatest effect on biomass among all treatments, as it induced a 5.2-fold decrease in dry biomass relative to the control. In addition, both fire and mowing slightly redistributed the community and diversity of the soil bacteria and fungi. This redistribution was significantly greater in the deep-mowing plot than in other plots. In particular, relative to the control, deep mowing increased the abundance of Firmicutes and especially Proteobacteria among soil bacterial communities, but significantly (p < 0.05) decreased Basidiomycota and increased Ascomycota abundance among soil fungal communities. We conclude that nutrient limitation (N, P, and K) is crucial for Miscanthus growth in both mowing and fire grasslands, whereas deep mowing can induce soil nutrient loss and microorganism redistribution, further restraining grass sustainability in dry-alkaline grasslands.</p

Heatmap representing exosomal proteins identified in H1975 in order of decreasing levels in TRs (R1-R2-R3, R4-R5-R6, R7-R8-R9) and in BRs (R1-R4-R7, R2-R5-R6, R3-R6-R9).

Boxes highlighted in dark red represent the most abundant, while boxes in dark blue represent the least abundant proteins.</p

Heatmap representing exosomal proteins identified in H1993 in order of decreasing levels in TRs (R1-R2-R3, R4-R5-R6, R7-R8-R9) and in BRs (R1-R4-R7, R2-R5-R6, R3-R6-R9).

Boxes highlighted in dark red represent the most abundant, while boxes in dark blue represent the least abundant proteins.</p