CBQF - Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Centro Regional do Porto da Universidade Católica Portuguesa, Rua Dr, António Bernardino Almeida, Porto, 4200-072, Portugal.
BMC Genomics. 2012 Nov 7;13:599. doi: 10.1186/1471-2164-13-599.
Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant's molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN).
Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species.
Defense-related genes triggered by nematode infestation were detected in both P. pinaster and P. pinea transcriptomes utilizing 454 pyrosequencing technology. P. pinaster showed higher abundance of genes related to transcriptional regulation, terpenoid secondary metabolism (including some with nematicidal activity) and pathogen attack. P. pinea showed higher abundance of genes related to oxidative stress and higher levels of expression in general of stress responsive genes. This study provides essential information about the molecular defense mechanisms utilized by P. pinaster and P. pinea against PWN infestation and contributes to a better understanding of PWD.
松材线虫（Bursaphelenchus xylophilus）引起的松萎蔫病（PWD）会损害和杀死松树，给全世界造成严重的经济损失。尽管已经很好地描述了侵染的生态机制，但植物对病原体的分子反应还不是很清楚。这主要是由于缺乏基因组信息和疾病的复杂性。高通量测序现在是一种检测非模式生物中基因表达的有效方法，因此即使缺乏基因组序列，也能提供有价值的信息。为了揭示潜在参与松树防御病原体的基因，我们在此报告对易受松材线虫（PWN）感染的栓皮栎（Pinus pinaster）和较不易受 PWN 感染的欧洲赤松（Pinus pinea）的感染和未感染茎进行高通量比较序列分析。
从易受 PWN 感染和未感染的栓皮栎和欧洲赤松茎中分别构建了 4 个 cDNA 文库，并在全 454 GS FLX 运行中进行了测序，共产生了 2083698 条reads。组装的转录本所编码的假定氨基酸序列根据基因本体论进行了注释，将松属 contigs 分配到生物过程、细胞成分和分子功能类别中。大多数注释的转录本与云杉基因相对应-25.4-39.7%，而较小比例的转录本与松属基因相对应，为 1.8-12.8%，这可能是因为云杉的公共基因组信息比松属多。比较转录组分析表明，当栓皮栎被松材线虫感染时，苹果酸脱氢酶、ABA、水分胁迫相关基因和 PAR1 高度表达，而在感染松材线虫的欧洲赤松中，高度表达的基因是蓖麻 B 相关凝集素和 SNARE 和高迁移率族家族的基因。定量 PCR 实验证实了两种松树之间差异表达的基因。
利用 454 焦磷酸测序技术，在栓皮栎和欧洲赤松转录组中检测到了线虫侵染触发的防御相关基因。栓皮栎表现出与转录调控、萜类次生代谢（包括一些具有杀线虫活性）和病原体攻击相关的基因丰度更高。欧洲赤松表现出与氧化应激相关的基因丰度更高，一般应激响应基因的表达水平更高。本研究提供了关于栓皮栎和欧洲赤松对抗松材线虫侵染的分子防御机制的重要信息，有助于更好地理解 PWD。
