云杉腐烂病菌Cytospora piceae全基因组测序及比较基因组分析

Genomic sequencing analysis of Cytospora piceae associated with spruce canker disease and comparative genomic analysis of Cytospora species

摘要:

[目的] 壳囊孢属（Cytospora）真菌引起的林木腐烂病和枝枯病，是一类重要的、分布广泛的枝干病害，可引起重大经济损失和生态破坏。通过全基因组测序和比较基因组学分析，探究不同腐烂病菌的全基因组特征，分析其与寄主选择和致病性相关的基因或基因家族的差异性，将有助于进一步揭示腐烂病菌与寄主互作的分子机制，为腐烂病的有效防治提供基础资料。[方法] 采用PacBio测序技术对云杉腐烂病菌Cytospora piceae进行了全基因组测序和组装，并通过比较基因组学方法，从基因组水平探究引起腐烂病的4种腐烂病菌的基因组的差异，分析其共有的和特有的与致病相关的基因家族。[结果] C.piceae基因组大小为39.25 Mb，GC含量为51.79%。基于单拷贝直系同源基因构建的系统发育树显示，C.piceae与Cytospora chrysosperma的进化关系相近，Valsa mali和Valsa pyri则更相近。比较基因组学分析表明，4种腐烂病菌均具有重复诱导的点突变（RIP）活性，其中，C.piceae的RIP活性最强。与其他3种腐烂病菌相似，与木质素降解相关的AA3和AA7家族在C.piceae中显著扩张，但木质素降解关键酶AA5家族均缺失；C.piceae和C.chrysosperma基因组中果胶降解关键酶GH28和CE8家族基因的数量与V.mali和V.pyri相近。4种腐烂病菌都含有较多数量的MFS （major facilitator superfamily）超家族转运蛋白和较少的ABC （ATP-binding cassette transporter）超家族转运蛋白，但C.piceae含有更多DHA2、PDR和MDR类转运蛋白。4种腐烂病菌的分泌蛋白的GO分类分子功能主要集中在水解酶活性，其中V.mali含有最多数量的该类别基因；而生物学过程则集中在碳水化合物代谢过程、果胶分解过程和氧化还原过程。在次生代谢核心基因中，C.piceae的PKS基因明显少于V.mali和V.pyri；在C.piceae含有的4个特异性次生代谢基因中，3个为NRPS基因。[结论] 4种腐烂病菌含有的碳水化合物活性酶的种类和数量相似，且都具有较强的果胶降解能力。不同腐烂病菌的膜转运蛋白中多药转运体的选择性扩增，以及次生代谢核心基因中NRPS类基因的特异性存在和缺失，表明它们作为重要的致病因子很可能在腐烂病菌寄主选择中发挥了重要的作用。

Abstract:

[Objective] Cytospora canker diseases are among the most important forest diseases, causing devastating economic losses and ecological damage. To understand the genome structure and genetic variation of Cytospora species with different host range, we performed a high-quality genome sequencing of Cytospora piceae and the corresponding comparative genomic analysis. Our study will provide a stepping stone to indicating the molecular mechanism of the interaction between Cytospora spp. and their hosts, and controlling the Cytospora canker disease.[Methods] The draft genome of Cytospora piceae was sequenced by PacBio sequencing technology, and was annotated. The genomic variation, host range determinants and the unique virulence-related gene families of the four Cytospora species were analyzed through comparative genomics tools.[Results] The assembled genome size of C. piceae was 39.25 Mb, with a GC content of 51.79%. The phylogenetic tree based on single-copy orthologue genes showed that C. piceae was closely related to Cytospora chrysosperma, and Valsa mali and Valsa pyri was in the same clade. GC content distribution analysis indicated repeat-induced point mutation activity in all Cytospora species, and C. piceae had the strongest repeat-induced point mutation activity. The carbohydrate active enzymes of all four Cytospora spp. was similar in number. Among plant cell wall degrading enzymes, the auxiliary activity family 3 and 7 related to lignin degradation expanded significantly, while the auxiliary activity family 5, the key enzymes for lignin degradation, was absent in Cytospora spp.. The number of genes in the glycoside hydrolase family 28 and 8 of key enzymes for pectin degradation in the C. piceae and C. chrysosperma genome was similar to that of V. mali and V. pyri. C. piceae and other three Cytospora species all had more major facilitator superfamily transporters and fewer ATP-binding cassette family transporters. In addition, C. piceae contained more Drug:H⁺ Antiporter-2, Pleiotropic Drug Resistance and Multidrug Resistance Exporter transporters, while V. mali contained less Drug:H⁺ Antiporter-1 transporters. Gene Ontology functional classification indicated that the genes of all Cytospora species concentrated on hydrolase activity, V. mali has the highest number of the genes of this class, and the biological processes were mainly related to carbohydrate metabolism, pectin catabolic and oxidation reduction processes. Among the secondary metabolism core genes, C. piceae had fewer polyketide synthases genes than V. mali and V. pyri. Among the four C. piceae specific secondary metabolites genes, three were nonribosomal peptide synthases genes.[Conclusion]] The carbohydrate active enzymes of four Cytospora species were similar in number, which showed strong pectin degradation ability. A complex pattern of presence or absence of nonribosomal peptide synthases genes in the secondary metabolites core genes, and the expanded multidrug transporters of four Cytospora species were observed, which indicated that they are likely to play an important role in host selection of Cytospora species.