（1）相关性找到目标微生物

（2）Exiguobacterium菌株产生的环(亮氨酸-脯氨酸)通过降解OsD53

蛋白减少水稻分蘖

Root microbiota regulates tiller number in rice 根系微生物群调节水稻分蘖数

摘要

（1）Rice tillering is an important agronomic trait regulated by plant genetic and environmental factors 水稻分蘖是受植物遗传和环境因素调控的重要农艺性状 .

（2）However, the role and mechanism of the root microbiota in modulating rice tillering have not been explored 然而，根微生物群在调节水稻分蘖中的作用和机制尚未被研究 .

（3）Here, we examined the root microbiota composition and tiller numbers of 182 genome-sequenced rice varieties grown under field conditions and uncovered a significant correlation between root microbiota composition and rice tiller number. 在这里，我们检测了在田间条件下生长的182个基因组测序的水稻品种的根微生物群组成和分蘖数，并揭示了根微生物群组成和水稻分蘖数之间的显著相关性。

（4）Using cultivated bacterial isolates, we demonstrated that various members of the root microbiota can regulate rice tillering in both laboratory and field conditions 使用培养的细菌分离物，我们证明了在实验室和田间条件下，根微生物群的各种成员可以调节水稻分蘖 .

（5）Genetic, biochemical, and structural analyses revealed that cyclo(Leu-Pro), produced by the tiller-inhibiting bacterium Exiguobacterium R2567, activates the rice strigolactone (SL) signaling pathway by binding to the SL receptor OsD14, thus regulating tillering. The present work provides insight into how the root microbiota regulates key agronomic traits and offers a promising strategy for optimizing crop growth by harnessing the root microbiota in sustainable agriculture 遗传、生化和结构分析表明，由抑制分蘖的细菌微小杆菌R2567产生的cyclo(Leu-Pro)通过与SL受体OsD14结合来激活水稻strigolactone (SL)信号通路，从而调节分蘖。目前的工作提供了根微生物群如何调节关键农艺性状的见解，并通过在可持续农业中利用根微生物群提供了优化作物生长的有前途的策略。

（6）Result结果

（7）The root microbiota is correlated with tiller number in a field-grown rice population 大田水稻群体中根系微生物区系与分蘖数相关

Figure 1 The root microbiota is correlated with tiller number variation in a field-grown rice population 图1在大田种植的水稻群体中，根系微生物区系与分蘖数变化相关

(A) Experimental design used for the field trials. The 182 rice varieties were arranged randomly and replicated in two fields (Ⅰ and Ⅱ). Root microbiota samples were harvested, and the tiller number was counted for each variety. For each variety, 3–6 replicates were performed. (A)用于田间试验的实验设计。将182个水稻品种随机排列，在两块田(ⅰ和ⅱ)中重复试验。收获根部微生物群样品，并计算每个品种的分蘖数。对于每个品种，进行3-6次重复。 

(B) The root microbiota and its interaction with plant genotype presented a considerable contribution to the variation observed in rice tiller number. The proportions of the variation explained by the root microbiota, rice genotype, and their interactions with the total tiller number variation in the field-grown rice population by linear regression models. Similar trends were observed in the other field (B)根微生物群及其与植物基因型的相互作用对观察到的水稻分蘖数的变异有相当大的贡献。通过线性回归模型解释了根系微生物区系、水稻基因型及其与大田水稻群体总分蘖数变异的交互作用所占的变异比例。在其他领域也观察到类似的趋势 (Figure S1D).（这个饼图告诉你，影响水稻分蘖数的三个因素各占多大比例：根里的细菌：占了28.2%，是最大的影响因素。水稻本身的品种（基因）：占了26.9%，和细菌的影响差不多。细菌和水稻品种的互动：占了21.5%。）

(C and D) The structure of the root microbiota is linked to variations in tiller number at the rice population level. The scatterplots illustrate the associations between tiller number and the Shannon index of the root microbiota (C, p = 5.3 × 10−10, r = 0.44) as well as the associations between tiller number and the root microbiota on the first axis of the principal coordinate analysis (PCoA) using Bray-Curtis dissimilarity across 182 rice varieties in field Ⅰ (D, p = 2.0 × 10−12, r = 0.49). (C和D)根微生物群的结构与水稻群体水平上分蘖数的变化有关。散点图显示了分蘖数和根系微生物区系的Shannon指数之间的关系(C，p = 5.3×1010，r = 0.44)，以及分蘖数和根系微生物区系之间在主坐标分析(PCoA)的第一轴上的关系(使用Bray-Curtis相异度，跨越田间ⅰ的182个水稻品种(D，p = 2.0×1012，r = 0.49)。（根部细菌种类越丰富、越均衡，水稻的分蘖数就越多，说明根部的细菌群落有某种特定的“风格”，这种风格也强有力地关联着更高的分蘖数） 

(E and F) Overlap of bacterial genera positively (E, tiller [+]) or negatively (F, tiller [−]) correlated with the tiller number of the rice varieties grown in the two fields (FDR adjusted p < 0.05, Wilcoxon rank-sum test). (E和F)细菌属的重叠与两地水稻品种的分蘖数正相关(E，分蘖[+])或负相关(F，分蘖[-])(FDR校正p < 0.05，Wilcoxon秩和检验)。 

(G) Heatmap showing the relative abundances of bacterial genera that were consistently positively (E) or negatively (F) correlated with rice tiller number across the two fields within the different rice tiller number ranges in field Ⅰ. The data obtained from field Ⅱ showed the same trend (G)热图显示了在田间ⅰ的不同水稻分蘖数范围内，与水稻分蘖数一致正相关(E)或负相关(F)的细菌属的相对丰度。从田地ⅱ获得的数据显示了同样的趋势 （这张热图直观地展示了E、F图中找出的那些关键细菌。）

（8）Root microbiota isolates regulate rice tiller number 根微生物群分离物调节水稻分蘖数

Figure 2 Cultivated root microbiota members play causal roles in regulating rice tiller number 图2栽培根系微生物群成员在调节水稻分蘖数中起因果作用

(A and B) Cultivated bacterial isolates of tiller-related genera regulate rice tiller number under laboratory conditions. Plots illustrating the distributions (A) and numbers (B) of rice tillers after inoculation with individual isolates from tiller-related genera. The results are confirmed in two independent inoculation experiments under hydroponic conditions; each treatment included 20–24 individual plants. The error bars represent the standard errors. (A和B)在实验室条件下，分蘖相关属的培养细菌分离物调节水稻分蘖数。说明接种来自分蘖相关属的单个分离物后水稻分蘖的分布(A)和数量(B)的图。该结果在溶液培养条件下的两个独立接种实验中得到证实；每个处理包括20-24株植物。误差线代表标准误差。 

(C) Gross morphologies (up) and tillers (down) of representative wild-type rice with and without inoculation by five individual tiller-related bacteria. Scale bars, 20 cm (up) and 1 cm (down). (C)接种和未接种五种单独的分蘖相关细菌的代表性野生型水稻的总体形态(上)和分蘖(下)。比例尺，20厘米(向上)和1厘米(向下)。 

(D) Isolates of five tiller-related bacterial genera regulate rice tiller number under field conditions. Bar plots showing rice tiller numbers after the inoculation of individual isolates from tiller-related genera (FDR adjusted p < 0.05, Wilcoxon rank-sum test). (D)五个分蘖相关细菌属的分离物在田间条件下调节水稻分蘖数。柱状图显示了接种来自分蘖相关属的单个分离物后的水稻分蘖数(FDR调整p < 0.05，Wilcoxon秩和检验)。 

(E and F) Bar plots showing that the spike number (E) and dry weight (F) are affected by the inoculation of tiller-related bacteria under field conditions (FDR adjusted p < 0.05, Wilcoxon rank-sum test). Each treatment included 50 individual plants. (E和F)柱状图表明，穗数(E)和干重(F)受田间条件下接种分蘖相关细菌的影响(FDR调整p < 0.05，Wilcoxon秩和检验)。每个处理包括50株植物。

（9）Exiguobacterium R2567 and Roseateles R780 regulate rice tillering via the SL pathway 微小杆菌R2567和Roseateles R780通过SL途径调节水稻分蘖

Exiguobacterium R2567 and Roseateles R780 regulate rice tillering through the SL pathway 微小杆菌R2567和Roseateles R780通过SL途径调节水稻分蘖

(A) Diagram of the biosynthesis and signaling of the rice strigolactone (SL) pathway. OsD14 is an SL receptor. OsD53 is a repressor that undergoes SL-induced degradation. (A)水稻内酯(SL)途径的生物合成和信号图。OsD14是一种S1受体。OsD53是一种经历SL诱导降解的阻遏物。 这展示了一种叫独脚金内酯（SL） 的激素在水稻体内的产生和作用路径。这种激素就像一个“少分叉”的信号：左边（合成路径）：水稻通过一系列步骤（D27, D17, D10等基因参与）合成这种激素。右边（信号路径）：激素（如Orobanchol）被合成后，会与受体OsD14结合。然后，这个复合体会去降解一个叫OsD53的“抑制蛋白”。一旦抑制蛋白被清除，水稻就会执行“少分叉”的指令。 简单说：SL激素水平高 -> 分蘖少；SL激素水平低 -> 分蘖多

(B) Exiguobacterium R2567 and Roseateles R780 affect OsD53 protein levels in rice. Immunoblotting images (up) and bar plots (down) show that OsD53 protein levels decrease 3 h after treatment with exudates from Exiguobacterium R2567 and increase after treatment with exudates from Roseateles R780 relative to levels in the mock control. The OsD53 protein levels were determined by densitometry and normalized to those of actin. The error bars represent the standard errors (n = 4, FDR adjusted p < 0.05, two-tailed Student’s t test). (B)微小杆菌R2567和Roseateles R780影响水稻中的OsD53蛋白水平。免疫印迹图像(上)和柱状图(下)显示，相对于模拟对照中的水平，用微小杆菌R2567的分泌物处理后3小时，OsD53蛋白水平降低，而用Roseateles R780的分泌物处理后，OSD 53蛋白水平升高。通过光密度测定法测定OsD53蛋白水平，并将其标准化为肌动蛋白水平。误差条代表标准误差(n = 4，FDR调整p < 0.05，双尾学生t检验)。 Exiguobacterium R2567（好细菌）：它的代谢物能降低 OsD53蛋白的水平。这相当于它模仿了SL激素的作用，强行让水稻“少分叉”。Roseateles R780（坏细菌）：它的代谢物能增加 OsD53蛋白的水平。这相当于它阻断了SL激素的作用，让“少分叉”的指令无法下达，从而使水稻“多分叉”

(C–E) Contents of the predominant rice SLs 4-deoxyorobanchol (4DO) (C), orobanchol (D), and 4-oxo-MeCLA (E) in the root exudates of wild-type rice seedlings after inoculation with Roseateles R780 or Exiguobacterium R2567. The error bars represent the standard errors. n = 4. (C–E)接种Roseateles R780或微小杆菌R2567后，野生型水稻幼苗的根分泌物中优势水稻SLs 4-脱氧列当醇(4DO) (C)、列当醇(D)和4-氧代-甲基丙烯酸(E)的含量。误差线代表标准误差。n = 4。 （这几张图显示，这两种细菌不仅影响信号接收（B图），还会影响水稻自身SL激素的分泌量。Roseateles R780（坏细菌）：让水稻分泌的几种SL激素（4DO, Orobanchol, 4-oxo-MeCLA）都显著减少。这从激素源头上导致了“多分叉”的信号。Exiguobacterium R2567（好细菌）：对水稻分泌SL激素的量没有显著影响。这说明它主要是在信号通路上做文章（如B图所示），而不是影响激素合成）

(F and G) Effects of Roseateles R780 (F) and Exiguobacterium R2567 (G) on the tillering of wild-type and mutant rice during SL biosynthesis and signaling. The results are confirmed in two experiments with independent inoculations; each genotype included 24 individual plants. The sloped arrows from mock to inoculated plants were to show the direction of tiller number changes. (F和G)Roseateles R780(F)和微小杆菌R2567 (G)在SL生物合成和信号传导期间对野生型和突变型水稻分蘖的影响。结果在两个独立接种的实验中得到证实；每个基因型包括24株个体植物。从模拟植株到接种植株的倾斜箭头表示分蘖数变化的方向。（Exiguobacterium R2567（好细菌）的效果，在正常水稻（Wild-type）中，接种它后分蘖数减少（红色箭头向下），因为它增强了SL信号。在d14和d27这两种SL信号有缺陷的突变体中，这种细菌也完全无效了。这强有力地证明，它必须通过完整的SL信号通路才能发挥“减少分蘖”的作用）

（10） Exiguobacterium R2567-derived compound activates the SL signaling pathway and regulates tiller development 源自微小杆菌R2567的化合物激活SL信号通路并调节分蘖发育

Figure 4 Exiguobacterium R2567-derived cyclo(Leu-Pro) activates the rice SL signaling pathway 图4来源于微小杆菌R2567的cyclo(Leu-Pro)激活水稻SL信号通路 

(A) Chemical structure of the cyclo(Leu-Pro) compound derived from Exiguobacterium R2567. (A)来源于微小杆菌R2567的环(Leu-Pro)化合物的化学结构（这就是从“好细菌”里分离提纯出来的有效成分，化学名称是环(亮氨酸-脯氨酸)）。 

(B and C) The OsD53 protein levels after treatment with the SL analog rac-GR24 and cyclo(Leu-Pro). OsD53 and actin were detected by immunoblotting (B) with anti-D53 polyclonal antibodies and anti-actin monoclonal antibodies, respectively. The bar plots (C) show the quantitative OsD53 protein levels in three independent immunoblots (n = 3, FDR adjusted p < 0.05, two-tailed Student’s t test). (B和C)用SL类似物rac-GR24和cyclo(Leu-Pro)处理后的OsD53蛋白水平。分别用抗D53多克隆抗体和抗肌动蛋白单克隆抗体通过免疫印迹(B)检测OsD53和肌动蛋白。柱状图(C)显示了三个独立免疫印迹中的定量OsD53蛋白水平(n = 3，FDR调整p < 0.05，双尾学生t检验)。 （cyclo(Leu-Pro)和SL激素类似物（rac-GR24）一样，都能显著降低OsD53蛋白的水平）

(D) Representative wild-type rice plants treated with the SL analog rac-GR24 and cyclo(Leu-Pro). Inoculation with 2 μM cyclo(Leu-Pro) or the SL analog rac-GR24 inhibited tillering in 41-day-old rice plants. Scale bars, 5 cm. (D)用SL类似物rac-GR24和cyclo(Leu-Pro)处理的代表性野生型水稻植株。用2微米环(Leu-Pro)或SL类似物rac-GR24接种抑制了41天龄水稻植株的分蘖。比例尺，5厘米。 （）

(E) Inoculation with cyclo(Leu-Pro) significantly decreased the tiller number of wild-type rice in a time-dependent manner under laboratory conditions (FDR adjusted p < 0.05, Wilcoxon rank-sum test). Each treatment included 12 individual Nipponbare wild-type plants. (E)在实验室条件下，接种cyclo(Leu-Pro)以时间依赖方式显著降低野生型水稻的分蘖数(FDR调整p < 0.05，Wilcoxon秩和检验)。每个处理包括12株单独的Nipponbare野生型植物。 

(F–I) Inoculation with cyclo(Leu-Pro) significantly inhibited the tiller number of diverse rice varieties under field conditions (FDR adjusted p < 0.05, Wilcoxon rank-sum test). Each treatment included 18–20 individual varieties, including Nipponbare (F), 9311 (G), NC1/536 (H), and Karabaschak (I). The bars represent the standard errors. Statistical significance between the chemical treatment and mock control is indicated by ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05. (F–I)接种cyclo(Leu-Pro)显著抑制田间条件下不同水稻品种的分蘖数(FDR校正p < 0.05，Wilcoxon秩和检验)。每个处理包括18-20个品种，包括日本晴(F)、9311 (G)、NC1/536 (H)和卡拉巴斯查克(I)。条形代表标准误差。化学处理和模拟对照之间的统计学显著性由∑∑p < 0.001、∑p < 0.01和∑p < 0.05表示。

（11） Cyclo(Leu-Pro) regulates tiller number through binding with the SL receptor OsD14 Cyclo(Leu-Pro)通过与SL受体OsD14结合来调节分蘖数

Cyclo(Leu-Pro) regulates rice tiller number through binding with the SL receptor OsD14 Cyclo(Leu-Pro)通过与SL受体OsD14结合调节水稻分蘖数

(A and B) Quantification of the binding between the SL analog rac-GR24 or cyclo(Leu-Pro) and OsD14 by microscale thermophoresis (MST). Measurements were performed at a light-emitting diode (LED) power of 60% and medium MST power, and the results of three independent measurements are presented as the mean values of the binding affinity. The error bars represent the standard deviations. (A和B)通过微量热泳(MST)对SL类似物rac-GR24或cyclo(Leu-Pro)和OsD14之间的结合进行定量。在60%的发光二极管(LED)功率和中等MST功率下进行测量，三次独立测量的结果表示为结合亲和力的平均值。误差线代表标准偏差。 （不仅天然的SL激素类似物（rac-GR24）能结合OsD14蛋白，我们找到的细菌小分子 cyclo(Leu-Pro) 也能直接结合到OsD14上）

(C) Crystal structure of the OsD14-cyclo(Leu-Pro) complex. Cyclo(Leu-Pro) is shown in a ball-and-stick model in green. The electron density for cyclo(Leu-Pro) is shown as blue meshes at a contour level of 1.2σ. (C)OSD 14-环(亮氨酸-脯氨酸)复合物的晶体结构。Cyclo(Leu-Pro)以绿色显示在球杆模型中。cyclo(Leu-Pro)的电子密度显示为1.2σ等高线级别的蓝色网格。 （展示了小分子cyclo(Leu-Pro)（绿色）正好嵌在OsD14蛋白的“口袋”里）

(D) Local interactions between OsD14 and cyclo(Leu-Pro) (left) in comparison with those between OsD14 and rac-GR24 (right, Protein Data Bank code, PDB: 5DJ5).36 Involved residues and ligands are shown in ball-and-stick models. Only residues responsible for ABC-ring coordination are shown in the OsD14-GR24 complex structure. (D)OsD14和cyclo(Leu-Pro)(左)之间的局部相互作用与OSD 14和rac-GR24之间的相互作用的比较(右，蛋白质数据库代码，PDB:5dj 5)。36涉及的残基和配体显示在球杆模型中。OsD14-GR24复合物结构中仅显示了负责ABC环配位的残基。 

(E) MST data revealed that mutations of OsD14 reduced its binding with cyclo(Leu-Pro). The results of three independent measurements are presented as the mean values of the binding affinity. The error bars represent the standard deviations (E) MST数据显示OsD14的突变降低了其与cyclo(Leu-Pro)的结合。三次独立测量的结果表示为结合亲和力的平均值。误差线代表标准偏差 .（当OsD14发生突变后，它和细菌小分子cyclo(Leu-Pro)的结合能力就大大下降了。）

(F) Mutations in OsD14 eliminate the effect of cyclo(Leu-Pro) on OsD53 degradation. The bar plots (down) show the quantitative OsD53 protein levels in three independent immunoblots (FDR adjusted p < 0.05, two-tailed Student’s t test). (OsD14的突变消除了cyclo(Leu-Pro)对OsD53降解的影响。柱状图(下方)显示了三个独立免疫印迹中定量的OsD53蛋白水平(FDR调整p < 0.05，双尾学生t检验)。 （在突变的植物里，细菌小分子无法再降低OsD53蛋白的水平了。因为“锁”坏了，“假钥匙”转不动，信号发不出去。）

(G and H) Mutations in OsD14 eliminate the tiller-inhibiting effect of cyclo(Leu-Pro) under the hydroponic (G) and field (H) conditions (FDR adjusted p < 0.05, Wilcoxon rank-sum test). Each treatment included 11–12 individual rice plants in (G) and 18–19 in (H). The bars represent the standard errors. Statistical significances between the chemical treatment and mock control are indicated by ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05. 在水培(G)和田间(H)条件下，OsD14中的(G和H)突变消除了cyclo(Leu-Pro)的分蘖抑制效应(FDR调整p < 0.05，Wilcoxon秩和检验)。每个处理包括11-12株水稻植株(G)和18-19株(H)。条形代表标准误差。化学处理和模拟对照之间的统计学显著性由∑∑p < 0.001、∑p < 0.01和∑p < 0.05表示（在水培和大田两种条件下，在OsD14突变的植物上，细菌小分子完全失去了抑制分蘖的效果！分蘖数和没处理的一样多。）

tiller 分蘖