Gene Expression profiling of rice leaves undergoing an innate immune response induced by XOO secreted cell wall degrading enzyme LipA (Lipase/Esterase A)

The plant cell wall degrading enzyme LipA (Lipase/Esterase A) is a Type II secretion system secreted protein of Xanthomonas oryzae pv. oryzae (Xoo; the casual of bacterial leaf blight of rice). LipA is an Xoo virulence factor. However, LipA is a double edged sword for Xoo as it induces rice defense responses such as programmed cell death/hypersensitive response like reaction (HR) and callose deposition. Prior treatment with LipA enhances resistance against subseqent Xoo infection. In order to understand the molecular events associated with Esterase (LipA) induced innate immune responsein rice , whole genome transcriptional profiling was performed using Affymetrix Rice GeneChips Leaves of two weeks old green house grown susceptible rice cultivar (Taichung Native-1; TN-1) were infiltrated with either 30-40μl of purified Xoo Lipase/Esterase A (LipA) (500μg/ml) or with buffer (10mM potassium phosphate buffer pH 6.6) alone (as described in Jha et al. 2007; MPMI vol 20, pp 31-40). The plants were shifted to a growth chamber (28oC; 80% relative humidity; 12/12h light/dark cycle) 48 hours before the treatment. 20-30 leaf pieces covering the infiltrated zone from each of the treatments were harvested 12 h after infiltration. Total RNA isolated from the pooled samples was subjected for expression analysis using Affymetrix GeneChip System. The experiment was repeated with three different biological replicates using RNA isolated from three batches of rice leaves treated with the freshly purified Xoo LipA and the buffer.

植物细胞壁降解酶LipA（脂肪酶/酯酶A）是稻黄单胞菌稻致病变种（Xanthomonas oryzae pv. oryzae，简称Xoo，水稻细菌性叶枯病的病原菌）的II型分泌系统（Type II secretion system）分泌蛋白。LipA是Xoo的毒力因子，但对Xoo而言却是一把双刃剑：它既能诱导水稻产生防卫反应，包括程序性细胞死亡/类似超敏反应（hypersensitive response，HR）以及胼胝质沉积；经LipA预处理的水稻可增强对后续Xoo侵染的抗性。为阐明酯酶LipA诱导水稻先天免疫反应的分子机制，研究人员采用Affymetrix水稻基因芯片（Rice GeneChips）开展全基因组转录组分析。实验材料为温室培养两周的感病水稻品种台中本地1号（Taichung Native-1，简称TN-1）的叶片，分别以30~40 μL浓度为500 μg/mL的纯化Xoo脂肪酶/酯酶A（LipA）溶液，或仅用10 mM磷酸钾缓冲液（pH 6.6）进行叶片浸润处理（参照Jha等2007年发表于《Molecular Plant-Microbe Interactions》（MPMI）第20卷第31-40页的实验方法）。处理前48小时，将植株转移至生长箱中培养，培养条件设置为28℃、相对湿度80%、光暗周期12/12 h。浸润处理12小时后，采集各处理组中覆盖浸润区域的20~30片叶组织样本。从混合样本中提取总RNA，借助Affymetrix基因芯片系统完成表达谱分析。本实验设置3次生物学重复，分别采用三批经新鲜纯化的Xoo LipA及缓冲液处理的水稻叶片提取的RNA完成重复实验。