Supporting Information S1 - Systematic Deletion of Homeobox Genes in <em>Podospora anserina</em> Uncovers Their Roles in Shaping the Fruiting Body

Figure S1. Expression of homeobox genes during fruiting body maturation. Differential expression in space and time of the seven homeobox genes was assessed by microarray analysis in a time course of mycelium growth (Bidard and Silar, unpublished data) and perithecium maturation (Bidard and Berteaux-Lecellier, unpublished data). No difference in expression was detected for the seven genes in one-day, two-day or three-day old mycelia. In contrast, statistically significant differential expression of the homeobox genes was detected during perithecium maturation. Each gene had its own pattern of expression. However, the expression of each homeobox gene except pah7 increased after fertilization, which corresponded to the timing at which some of these genes are required for proper development of the fruiting bodies, i.e., for neck differentiation. The expression of most of the genes was decreased at the end of the development period (96 h). The expression level was measured as described by Bidard et al. [51] on three independent samples and was normalized by the median intensity for each gene. Figure S2. Southern blot analyses of the Δpah mutants. DNA was isolated from wild-type P. anserina and one or several purified transformants (T1, T2, T3…) and digested with appropriate restriction enzymes. The blots were probed either with a sequence containing the relevant CDS and its flanking region (Δpah2, Δpah3, Δpah4, Δpah5) or with the ble sequence (Δpah6, Δpah7). For each pah gene, a restriction map of the wild-type and mutant locus is presented. The sizes of the expected fragments are indicated on the maps and are reported close to the corresponding fragment on the Southern blot. The DNA sequence around pah7 is not well defined because of sequencing errors and the restriction pattern cannot be confidently predicted for the pah7 deletion. To confirm the deletion of pah7, the blot was stripped and further probed with the pah7 CDS to confirm that it was absent in the Δpah7 candidates. Figure S3. Fertility of Δpahx mutants on medium containing wood shavings. Strains of the indicated genotypes were inoculated on medium containing wood shavings as the sole carbon source. The images were taken 10 days later. Even after prolonged incubation for up to three weeks, no perithecia were observed in the Δpah1Δpah2 and Δpah2Δpah5 cultures. The small dots visible on the Δpah2Δpah5 cultures correspond to dark, insoluble product(s) excreted by the hyphae. These products are also excreted by wild-type and all the other pahx mutants. Figure S4. Fertility rescue by Δmat hyphae. Heterokaryons with the following components: pah1Δpah2Δpah3Δpah4Δpah5Δpah6 mat+, pah1Δpah2Δpah3Δpah4Δpah5Δpah6 mat− andΔmat are able to differentiate fruiting bodies of wild-type appearance, which produce abundant ascospores. (DOC)补充图S1 子实体成熟过程中同源框基因（homeobox genes）的表达谱 对7种同源框基因在菌丝体（mycelium）生长时序（Bidard与Silar，未发表数据）及子囊壳（perithecium）成熟过程（Bidard与Berteaux-Lecellier，未发表数据）中的时空差异表达模式，通过微阵列分析（microarray analysis）进行了评估。在培养1、2、3天的菌丝体中，未检测到这7种基因的表达差异。与之相反，在子囊壳成熟阶段，可检测到各同源框基因存在统计学意义显著的差异表达。各基因均有其独特的表达模式。除pah7外，其余同源框基因的表达量在受精（fertilization）后均出现上调，这一时间节点与部分基因参与子实体正常发育（即颈分化（neck differentiation））所需的时间相吻合。在发育周期末期（96小时），多数基因的表达量出现下调。基因表达量的检测参照Bidard等人[51]的方法完成，共设置3次独立生物学重复，并以每个基因的信号中值进行归一化处理。 补充图S2 Δpah突变体的Southern印迹（Southern blot）分析 从野生型鹅掌柄孢壳菌（P. anserina）及1个或多个纯化后的转化子（transformants，T1、T2、T3……）中提取基因组DNA，使用适配的限制性内切酶进行酶切。杂交膜分别使用包含目标编码序列（Coding Sequence，CDS）及其侧翼区域的探针（针对Δpah2、Δpah3、Δpah4、Δpah5突变体），或博来霉素抗性序列（ble sequence，针对Δpah6、Δpah7突变体）进行杂交。针对每个pah基因，均给出了野生型及突变体基因座的限制性酶切图谱。预期酶切片段的大小标注于图谱上，并在Southern印迹图中对应片段旁予以标注。由于测序误差，pah7基因周边的DNA序列尚未明确，因此无法准确预测Δpah7突变体的酶切图谱。为验证pah7基因的敲除成功，将杂交膜洗脱探针后，使用pah7的CDS序列重新进行杂交，以确认Δpah7候选株中不存在该基因序列。 补充图S3 Δpahx突变体在含木屑培养基上的育性 将指定基因型的菌株接种于以木屑为唯一碳源的培养基上，于接种10天后拍摄图像。即便延长培养至3周，Δpah1Δpah2与Δpah2Δpah5双突变株的培养板中仍未观察到子囊壳结构。Δpah2Δpah5培养板上可见的细小黑点为菌丝体（hyphae）分泌的深色不溶性代谢产物，该类产物在野生型菌株及其余所有pahx突变株中均有分泌。 补充图S4 Δmat菌丝体对育性的挽救作用 由以下组分构成的异核体（heterokaryons）：pah1Δpah2Δpah3Δpah4Δpah5Δpah6 mat+、pah1Δpah2Δpah3Δpah4Δpah5Δpah6 mat− 以及Δmat，均可分化出外观与野生型一致的子实体，并产生大量子囊孢子（ascospores）。 (DOC)