Deciphering the Cryptic Genome: Genome-wide Analyses of the Rice Pathogen Fusarium fujikuroi Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites

The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.藤仓镰孢菌（Fusarium fujikuroi）可通过产生赤霉素（gibberellins, GAs）引发水稻恶苗病（bakanae disease），同时该菌还会产生有害的真菌毒素（mycotoxins）。然而，其合成全套天然化合物的遗传能力及其在该菌与水稻互作中的作用此前仍未明确。本研究报道了一株藤仓镰孢菌的高质量基因组序列，该序列被组装为12个支架序列（scaffolds），对应该菌已报道的12条染色体。我们结合染色质免疫共沉淀测序（ChIP-seq）、转录组、蛋白质组以及基于高效液相色谱-傅里叶变换质谱（HPLC-FTMS）的代谢组分析手段，鉴定了潜在的次生代谢物生物合成基因簇，并探究了其在氮有效性响应与植物信号刺激下的调控模式。研究结果显示，绝大多数而非全部基因簇的表达水平与蛋白质组和ChIP-seq数据呈显著相关。将藤仓镰孢菌的基因组与其他6种镰孢菌基因组进行比较后发现，仅少量基因簇在这些物种中保守，这为理解镰孢菌属次生代谢的演化分化提供了新的视角。值得注意的是，赤霉素生物合成基因存在于部分近缘物种中，但赤霉素的生物合成仅局限于藤仓镰孢菌，这提示该特性在其侵染偏好宿主水稻的过程中具有选择优势。在所分析的基因组序列中，包含聚酮合酶基因（PKS19）的基因簇以及包含非核糖体肽合成酶基因（NRPS31）的基因簇均为藤仓镰孢菌所特有。通过对过表达该基因簇的工程化藤仓镰孢菌菌株进行HPLC-FTMS分析，我们鉴定了这两个基因簇所对应的代谢产物。植物体内表达实验表明，PKS19所衍生的代谢产物在水稻侵染过程中发挥特定作用。综上，本研究通过整合比较基因组学与全基因组实验分析，鉴定了新型基因与次生代谢产物，这些物质助力藤仓镰孢菌作为水稻病原菌实现演化上的成功。