Data for Figures 5 A, C, D, E

Ras activity dependent gene expression is coupled to changes in cell cycle dynamics A. RNA-seq reveals reciprocal genes expression changes in response to GefE and nutrition. RNA-seq was performed on AX3 G+, AX3 G-, and gefE- G+ cells. Ras dependent genes were identified by comparing gene expression of gefE- G+ and AX3 G+ samples. A highly significant proportion of these 45 DEGs (80%) also show a >1.5x FC in expression between AX3 G+ and AX3 G- (hypergeometric test, p = 4 x 10-15). Comparison of log2 FCs of the 36 overlapping genes shows that stalky nutritional bias results in sporey low Ras gene activity. Candidate genes are highlighted in red. C. Quantification of rrgA and rigA RNA-FISH. The relative expression of each gene in each cell was calculated as an index of expression rrgA/(rrgA+rigA). Values of 1 or 0 represent only rrgA or rigA expression, respectively. D. Ras dependent genes are maximally expressed during multicellular development. Expression profiles of RasD dependent genes were determined from RNA-seq data at different time points during development. E. rrgA and rigA mutant behaviour in stalk cell induction monolayer assays. rrgA knockout in AX3 does not significantly stalk cell differentiation, whereas the rrgA-/gefE- double mutant rescues gefE- mutant prespore bias (see Supplementary File 4 for p-values). rigA knockout in AX3 does not significantly affect stalk cell differentiation, whereas the rigA/AX3rasD(G12T) strain shows significantly reduced stalk cell differentiation compared to AX3rasD(G12T) (see Supplementary File 4 for p-values). Plotted values are the mean of three replicates and error bars depict the standard error of the mean.

Ras活性依赖性基因表达与细胞周期动态变化相偶联。
A. RNA测序（RNA-seq）揭示了响应GefE与营养条件的双向基因表达变化。本研究以AX3 G+、AX3 G-及gefE- G+细胞为材料开展RNA-seq实验。通过对比gefE- G+与AX3 G+样本的基因表达谱，成功鉴定出Ras依赖性基因。上述45个差异表达基因（DEGs, Differentially Expressed Genes）中，80%在AX3 G+与AX3 G-样本间的表达量变化倍数（FC, Fold Change）大于1.5倍，该富集结果具有极显著统计学意义（超几何检验，p=4×10^-15）。对36个重叠基因的log2转化表达倍数进行差异分析后发现，营养偏向柄细胞分化时，会导致孢子细胞相关的Ras基因活性降低。候选基因以红色标注。
C. rrgA与rigA的RNA荧光原位杂交（RNA-FISH）定量分析。以rrgA/(rrgA+rigA)作为指标计算每个细胞中各基因的相对表达量，取值为1或0时分别代表仅表达rrgA或仅表达rigA。
D. Ras依赖性基因在多细胞发育阶段的表达量达到峰值。基于发育过程中不同时间点的RNA-seq数据，我们获取了RasD依赖性基因的表达谱。
E. rrgA与rigA突变体在柄细胞诱导单层培养实验中的表型。rrgA基因敲除对AX3菌株的柄细胞分化无显著影响，而rrgA-/gefE-双突变体可挽救gefE-突变体的前孢子偏向表型（p值详见补充文件4）。rigA基因敲除对AX3菌株的柄细胞分化亦无显著影响，但与AX3rasD(G12T)相比，rigA/AX3rasD(G12T)菌株的柄细胞分化水平显著降低（p值详见补充文件4）。图中数值为三次生物学重复的平均值，误差棒代表平均值的标准误。