β-1,6葡聚糖酶前体编码基因Ssa4p对甘蔗黑穗病菌分泌蛋白组的影响

Impact of Ssa4p,a Gene Encodingβ-1,6 Glucan Precursor,on the Secretome of Sugarcane Smut Pathogen Sporisorium scitamineum

甘蔗(Saccharum spp.)是世界也是中国主要的糖料作物。由甘蔗黑穗病菌(Sporisorium scitamineum)引起的甘蔗黑穗病是中国甘蔗生产上最主要的病害,每年造成重大损失。分泌蛋白在真菌侵染寄主植物与获取营养及病理过程起重要作用。本实验室在分析甘蔗黑穗病菌基因组与转录组数据时,发现1个与真菌细胞壁降解有关的β-1,6葡聚糖酶前体的基因Ssa4p。为了揭示Ssa4p对甘蔗黑穗病菌致病性和分泌蛋白组的影响,本研究利用CRISPR/Cas9和T-DNA双元载体所构建的甘蔗黑穗菌基因敲除系统,成功获得了Ssa4p基因的失活突变株。ΔSsa4p突变株(Δ35-Ssa4p)与野生型菌株JG36的配合能力降低且致病性减弱。用iTRAQ方法对Δ35-Ssa4p和野生型菌株的分泌蛋白组进行比较分析,共鉴定到蛋白质1430个,其中上调差异表达蛋白质685个,下调差异表达蛋白质745个。GO功能富集分析及KEGG分析表明,差异蛋白主要定位于无膜细胞器和核糖体,其功能包括参与核糖体、氨基糖和核苷酸糖代谢等生物学过程。本研究发现β-1,6葡聚糖前体影响植物病原真菌的分泌蛋白,加深了对真菌致病性调控机制的认识。

Sugarcane is the main sugar crop in China as well as the world.Sugarcane smut caused by Sporisorium scitamineum is the most important disease in sugarcane production in China and causes great losses every year.Secreted proteins play an important role in establishment of infection and acquisition of nutrients for the pathogen.In the previous study of the genome and transcriptome of S.scitamineum,we identified aβ-1,6 glucanase precursor(encoded by Ssa4p)associated with fungal cell wall degradation.In order to reveal the effects of Ssa4p on the pathogenicity and secretory proteome of Sporisorium scitamineum,in this study,a gene knockout system was constructed by using CRISPR/Cas9 and T-DNA binary vector.The inactivated mutant of Ssa4p gene was successfully obtained.The Ssa4p mutant(Δ35-Ssa4p)showed reduced coordination ability and pathogenicity with the wild-type strain JG36.By comparison of secretomes ofΔ35-Ssa4p and its parental wide-type strain JG35 by iTRAQ,1430 differentially expressed proteins(685 up-regulated and 745 down-regulated)were identified.GO and KEGG analysis revealed that the differentially expressed proteins were mainly located in ribosomes and non-membrane-bounded organelles,which were mainly involved in pathways such as ribosome metabolism,amino sugar and nucleotide sugar metabolism.This study found aβ-1,6 glucan precursor-encoding gene was found to impact a large proportion of secretory proteins in a phytopathogenic fungus,broadening our understanding of pathogenicity regulation in pathogenic fungi.