RNAseq, virulence, and phylogenetics studies of the gene easR of Metarhizium brunneum

Ergot alkaloid synthesis (eas) gene clusters found in several fungi encode
biosynthesis of agriculturally and pharmaceutically important ergot
alkaloids. Whereas the biosynthetic genes of the ergot alkaloid pathway
have been well characterized, regulation of those genes is unknown. We
characterized a gene with sequence similarity to a putative transcription
factor and that was found adjacent to the eas cluster of
Metarhizium brunneum, a plant symbiont and insect pathogen. The function
of the novel gene, easR, was explored by CRISPR-Cas9-derived gene
knockouts. To maximize potential for ergot alkaloid accumulation, strains
of M. brunneum were injected into larvae of the insect Galleria
mellonella.  Larvae infected with the wild type contained
abundant ergot alkaloids, but those infected with easR knockouts lacked
detectable ergot alkaloids.  The easR knockout strains had
significantly reduced or no detectable mRNA from eas cluster genes in
RNAseq and qualitative RT-PCR analyses, whereas the wild-type strain
contained abundant mRNA from all eas genes. These data demonstrate that
the product of easR is required for ergot alkaloid accumulation
and provide evidence it has a role in expression of ergot alkaloid
biosynthesis genes. Larvae infected with an easR knockout survived
significantly longer than those infected with the wild type (P <
0.0001), indicating a role for EasR, and indirectly confirming a role for
ergot alkaloids, in virulence of M. brunneum to insects. Homologs
of easR were found associated with eas clusters of at least 15 other ergot
alkaloid-producing fungi, indicating EasR homologs may contribute to
regulation of ergot alkaloid synthesis in additional fungi.

麦角生物碱合成（ergot alkaloid synthesis, eas）基因簇广泛存在于多种真菌中，其编码产物参与农业与医药领域重要麦角生物碱的生物合成过程。尽管麦角生物碱合成通路的生物合成基因已得到充分解析，但其调控机制仍不明晰。本研究针对一株与假定转录因子序列具有相似性的基因展开表征分析，该基因紧邻植物共生菌、昆虫病原真菌布氏绿僵菌（Metarhizium brunneum）的eas基因簇。本研究通过CRISPR-Cas9介导的基因敲除技术，对该新型基因easR的功能开展了探究。为最大化麦角生物碱的积累潜力，我们将布氏绿僵菌菌株接种至大蜡螟（Galleria mellonella）幼虫体内。感染野生型菌株的幼虫体内可检测到大量麦角生物碱，而感染easR敲除菌株的幼虫则无法检出麦角生物碱。转录组测序（RNAseq）与定性逆转录聚合酶链式反应（RT-PCR）分析结果显示，easR敲除菌株的eas簇基因mRNA水平显著降低，甚至无法检测到，而野生型菌株的所有eas基因均能转录产生丰富的mRNA。上述实验数据证实，easR的编码产物是麦角生物碱积累所必需的，并证明其可调控麦角生物碱生物合成基因的表达。感染easR敲除菌株的幼虫存活时间显著长于感染野生型菌株的幼虫（P < 0.0001），这表明EasR在布氏绿僵菌对昆虫的毒力中发挥作用，同时间接证实了麦角生物碱在该过程中的功能。研究还发现，至少15种其他产麦角生物碱真菌的eas基因簇旁侧均存在easR的同源基因，这提示EasR同源蛋白可能在其他真菌的麦角生物碱合成调控中发挥类似功能。