A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (Botrytis cinerea infection).

A network model has been generated that describes the immediate gene expression cascade surrounding three similar but distinct NAC transcription factors that have roles to play in leaf senescence and in many stress responses in Arabidopsis. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1 hybrid analysis and modeling using gene expression time course data has been used to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modeling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions.. Gene expression measurements in mutants of ANAC019 and ANAC055 at different times during leaf senescence have shown a distinctly different role for each of these genes. To assess mutant response to Botrytis cinerea infection, Col-0, myb2 and myb108 leaves were inoculated with 4-6 (depending on leaf size) evenly spaced 10μl droplets of B. cinerea spores. Infected leaves were harvested to provide 4 biological replicates. Samples for the comparison between myb108 and Col-0 were harvested at 26 and 30 hpi. Samples for the comparison between myb2 knockout line and Col-0 or myb2 knockout line and Col-0 were harvested at 26 and 30 hpi or 24 and 30 hpi respectively. Analysis of expression differences between Col-0 and myb2 and Col-0 and myb108 under each condition, using dye swaps, was performed using the R Bioconductor package limmaGUI (Wettenhall and Smyth, 2004).

本研究构建了一套基因调控网络模型，用以描述拟南芥（Arabidopsis）中3个结构相似但功能各异的NAC转录因子（NAC transcription factors）所介导的即时基因表达级联反应；这3个转录因子均参与叶片衰老与多种胁迫应答过程。ANAC019、ANAC055与ANAC072同属于NAC结构域基因的同一进化枝，且三者的表达模式存在重叠。本研究结合酵母单杂交（yeast one-hybrid）实验鉴定得到的启动子DNA-蛋白质互作数据，与基于基因表达时序数据构建的模型，对这3个基因的上游调控网络进行了预测。通过上游转录因子结合的不同组合以及模型预测，可揭示这些基因在多种胁迫应答过程中调控模式的异同。通过对潜在上游基因开展突变体分析，验证并确认了部分预测得到的调控互作关系。对ANAC019与ANAC055突变体在叶片衰老过程中不同时间点的基因表达检测结果显示，这两个基因各自发挥着截然不同的功能。为评估突变体对灰葡萄孢（Botrytis cinerea）侵染的应答反应，本研究将4~6滴（根据叶片大小调整）间距均匀的10μl灰葡萄孢孢子悬浮液接种于Col-0、myb2与myb108的叶片上。收集受侵染叶片以获得4个生物学重复样本：myb108与Col-0的比较样本于接菌后26小时（26 hpi）和30小时（30 hpi）采集；用于比较myb2敲除株系与Col-0、以及myb2敲除株系与Col-0的样本，分别于接菌后26、30小时和24、30小时采集。针对各处理条件下Col-0与myb2、Col-0与myb108之间的表达差异分析，采用染料互换（dye swap）实验设计，并通过R语言Bioconductor套件中的limmaGUI工具（Wettenhall与Smyth，2004）完成。