Centro de Energia Nuclear na Agricultura, Universidade de São Paulo; Ave. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil.
Mol Plant Pathol. 2007 May;8(3):279-92. doi: 10.1111/j.1364-3703.2007.00393.x.
SUMMARY The basidiomycete Crinipellis perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cocoa). Hypertrophic growth of infected buds ('brooms') is the most dramatic symptom, but the main economic losses derive from pod infection. To identify cocoa genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from infected shoot-tips of the susceptible genotype 'ICS 39' and the resistant 'CAB 214', in both directions. A total of 187 unique sequences were obtained, with 83 from the library enriched for the susceptible 'ICS 39' sequences, and 104 for the resistant 'CAB 214'. By homology search and ontology analyses, the identified sequences were mainly putatively categorized as belonging to 'signal transduction', 'response to biotic and abiotic stress', 'metabolism', 'RNA and DNA metabolism', 'protein metabolism' and 'cellular maintenance' classes. Quantitative reverse transcription amplification (RT-qPCR) of 23 transcripts identified as differentially expressed between genotypes revealed distinct kinetics of gene up-regulation at the asymptomatic stage of the disease. Expression induction in the susceptible 'ICS 39' in response to C. perniciosa was delayed and limited, while in 'CAB 214' there was a quicker and more intense reaction, with two peaks of gene induction at 48 and 120 h after inoculation, corresponding to morphological and biochemical changes previously described during colonization. Similar differences in gene induction were validated for another resistant genotype ('CAB 208') in an independent experiment. Validation of these genes corroborated similar hypothetical mechanisms of resistance described in other pathosystems.
摘要 担子菌 Crinipellis perniciosa 是可可树（Theobroma cacao）的病原菌，可引起女巫扫帚病。受感染芽的异常生长（“扫帚状”）是最显著的症状，但主要的经济损失源于荚果感染。为了鉴定可可在感染早期差异表达的基因，我们使用抑制性消减杂交（SSH）方法构建了两个 cDNA 文库。在易感性基因型“ICS 39”和抗性基因型“CAB 214”的受感染嫩梢 cDNA 之间进行了消减杂交，两个方向都进行了杂交。共获得了 187 个独特的序列，其中 83 个来自文库，富集了易感型“ICS 39”序列，104 个来自抗性型“CAB 214”序列。通过同源搜索和本体分析，鉴定的序列主要被归为“信号转导”、“对生物和非生物胁迫的反应”、“代谢”、“RNA 和 DNA 代谢”、“蛋白质代谢”和“细胞维持”类别。对被鉴定为基因型间差异表达的 23 个转录本进行定量反转录 PCR（RT-qPCR）分析显示，在疾病无症状阶段，基因的上调具有不同的动力学。在易感型“ICS 39”中，C. perniciosa 的诱导表达延迟且有限，而在“CAB 214”中，反应更快、更强烈，在接种后 48 和 120 小时有两个基因诱导高峰，与先前描述的定植过程中的形态和生化变化相对应。在另一个独立实验中，对另一个抗性基因型（“CAB 208”）进行了基因诱导差异的验证。这些基因的验证支持了在其他病理系统中描述的类似抗性假设机制。
