New insights into the evolution of host specificity of three Penicillium species and the pathogenicity of P. Italicum involving the infection of Valencia orange (Citrus sinensis)

Blue and green molds, the common phenotypes of post-harvest diseases in fruits, are mainly caused by Penicillium fungal species, including P. italicum, P. digitatum, and P. expansum. We sequenced and assembled the genome of a P. italicum strain, which contains 31,034,623 bp with 361 scaffolds and 627 contigs. The mechanisms underlying the evolution of host specificity among the analyzed Penicillium species were associated with the expansion of protein families, genome restructuring, horizontal gene transfer, and positive selection pressure. A dual-transcriptome analysis following the infection of Valencia orange (Citrus sinensis) by P. italicum resulted in the annotation of 9,307 P. italicum genes and 24,591 Valencia orange genes. The pathogenicity of P. italicum may be due to the activation of effectors, including 51 small secreted cysteine-rich proteins, 110 carbohydrate-active enzymes, and 12 G protein-coupled receptors. Additionally, 211 metabolites related to the interactions between P. italicum and Valencia orange were identified by gas chromatography-time of flight mass spectrography, three of which were further confirmed by ultra-high performance liquid chromatography triple quadrupole mass spectrometry. A metabolomics analysis indicated that P. italicum pathogenicity is associated with the sphingolipid and salicylic acid signaling pathways. Moreover, a correlation analysis between the metabolite contents and gene expression levels suggested that P. italicum induces carbohydrate metabolism in Valencia orange fruits as part of its infection strategy. This study provides useful information regarding the genomic determinants that drive the evolution of host specificity in Penicillium species and clarifies the host-plant specificity during the infection of Valencia orange by P. italicum. P. italicum GL_Gan1, a local strain in Guangzhou, China, was sequenced. Comparison of the genome of P. italicum GL_Gan1 with other pathogenic Penicillium species, P. digitatum and P. expansum, revealed that the expansion of protein families, genome restructuring, HGT, and positive selection pressure were related to the host range expansion of the analyzed Penicillium species. Moreover, gene gains or losses might be associated with the speciation of these Penicillium species. In addition, the molecular basis of host-plant specificity during the infection of Valencia orange (Citrus sinensis) by P. italicum was also elucidated by transcriptomic and metabolomics analysis. The data presented herein may be useful for further elucidating the molecular basis of the evolution of host specificity of Penicillium species and for illustrating the host-plant specificity during the infection of Valencia orange by P. italicum.蓝青霉与绿青霉是果蔬采后病害的常见表型，其致病原主要为青霉属（Penicillium）真菌，涵盖意大利青霉（P. italicum）、指状青霉（P. digitatum）与扩展青霉（P. expansum）三个物种。本研究对一株意大利青霉菌株的基因组进行了测序与组装，该基因组全长31,034,623 bp，包含361个支架序列（scaffold）与627个重叠群（contig）。本研究分析的青霉属物种间宿主特异性进化机制，与蛋白质家族扩张、基因组重排、水平基因转移（horizontal gene transfer, HGT）以及正选择压力密切相关。针对意大利青霉侵染巴伦西亚橙（Citrus sinensis，甜橙）后的样本开展双重转录组分析，共注释得到9,307个意大利青霉基因与24,591个巴伦西亚橙基因。意大利青霉的致病性可能源于效应因子的激活，这类效应因子包括51个小型分泌型富半胱氨酸蛋白、110个碳水化合物活性酶（carbohydrate-active enzymes, CAZymes）以及12个G蛋白偶联受体（G protein-coupled receptors, GPCRs）。此外，本研究通过气相色谱-飞行时间质谱（gas chromatography-time of flight mass spectrography）鉴定出211个与意大利青霉-巴伦西亚橙互作相关的代谢物，其中3个经超高效液相色谱-三重四极杆质谱（ultra-high performance liquid chromatography triple quadrupole mass spectrometry）进一步验证。代谢组学分析结果显示，意大利青霉的致病性与鞘脂信号通路及水杨酸（salicylic acid）信号通路显著相关。此外，代谢物含量与基因表达水平的关联分析表明，意大利青霉可通过诱导巴伦西亚橙果实的碳水化合物代谢，作为其侵染宿主的核心策略之一。本研究为解析青霉属物种宿主特异性进化的基因组决定因子提供了宝贵数据，并阐明了意大利青霉侵染巴伦西亚橙过程中的宿主互作特异性机制。本研究还对中国广州本土菌株意大利青霉GL_Gan1进行了基因组测序；将该菌株的基因组与另外两种致病青霉属物种——指状青霉与扩展青霉的基因组进行比较后发现，蛋白质家族扩张、基因组重排、水平基因转移以及正选择压力，与本研究涉及的青霉属物种的宿主范围扩张紧密相关，而基因的获得与丢失可能与这些青霉属物种的物种形成过程密切关联。此外，本研究通过转录组学与代谢组学分析，进一步阐明了意大利青霉侵染巴伦西亚橙时的宿主特异性分子基础。本文所呈现的研究数据，可为后续解析青霉属物种宿主特异性进化的分子基础，以及阐明意大利青霉侵染巴伦西亚橙的宿主特异性机制提供重要参考。