图1 细胞骨架荧光标记载体原理图 A：利用Lifeact-EGFP标记微丝骨架原理图,LA：Lifeact编码序列,PgpdA：构巢曲霉3-磷酸甘油醛脱氢酶基因的启动子,TtrpC：构巢曲霉色氨酸合成基因trpC的终止子,HPT：潮霉素磷酸转移酶抗性基因;B：利用MBD-EGFP标记微丝骨架原理图,MBD：Microtubule binding domain;C：利用同源重组原理在CgTUB1基因下游敲入EGFP编码序列的原理图,WT：野生型菌株,Mutant：突变体,Flanking region：CgTUB1的下游序列

Fig. 1 Diagram of fluorescent labeled cytoskeleton vectors A: The diagram of microfilament labeling vector through Lifeact-EGFP; LA: coding sequence of Lifeact; PgpdA: promoter of glyceraldehyde-3-phosphate dehydrogenase in Aspergillus nidulans; TtrpC: terminator of tryptophan synthesis gene in Aspergillus nidulans; HPT：Hygromycin phosphate transferase resistance gene. B: The diagram of microtubule labeling vector through MBD-EGFP; MBD：microtubule binding domain. C: The diagram of inserting EGFP coding sequence behind gene CgTUB1 through homologous recombination; WT: wild type; Mutant: mutant strain; Flanking region: downstream of gene CgTUB1

图2 细胞骨架荧光标记重组菌株的PCR鉴定 A：Lifeact-EGFP标记微丝骨架重组菌株的PCR检测结果;B：MBD-EGFP标记微管骨架重组菌株的PCR检测结果;C：EGFP敲入CgTUB1基因位点重组菌株上游重组片段PCR检测结果;D：EGFP敲入CgTUB1基因位点重组菌株下游重组片段PCR检测结果;M：DNA marker DM5000

Fig. 2 PCR diagnosis of cytoskeleton labeling recombinant strains A: PCR diagnosis of microfilament labeling strains by Lifeact-EGFP. B：PCR diagnosis of microtubule labeling strains by MBD-EGFP. C: PCR diagnosis of the upstream recombinant fragment of CgTUB1-EGFP labeling strains. D: PCR diagnosis of the downstream recombinant fragment of CgTUB1-EGFP labeling strains. M: DNA marker DM5000

图3 荧光标记重组菌株孢子中的细胞骨架结构 CK：表达EGFP的对照菌株;Lifeact-EGFP：微丝骨架标记重组菌株;CgTUB1-EGFP：微管骨架标记重组菌株;Z Projection：Z轴切片图像的叠加图;Scale bar：5 μm。下同

Fig. 3 Cytoskeleton structure in conidiophores of fluorescent labeled recombinant strains CK: Control strain expressing EGFP; Lifeact-EGFP: microfilament labeled recombinant strain; CgTUB1-EGFP: microtubule labeled recombinant strain; Z Projection：stacked image of Z-series slices; scale bar：5 μm. The same below

图4 荧光标记重组菌株菌丝中的细胞骨架结构

Fig. 4 Cytoskeleton structure in the hypha of fluorescent labeled recombinant strains

图5 野生型菌株与重组菌株菌落直径及产孢量 A：在PDA培养基上培养时不同菌株的菌落直径;B：在PDB液体培养基中培养时不同菌株的孢子产量;WT：野生型菌株

Fig. 5 Clony diameters and conidiations of WT and mutant strains A: Clony diameter of WT and mutant strains on PDA media; B: conidiation of WT and mutant strains on PDB liquid media; WT: wild type strain

图6 接种野生型菌株与重组菌株后橡胶树叶片的发病情况及病斑直径 A：接种病原菌3 d后橡胶树叶片的发病情况;B：接种病原菌2、3 d后橡胶树叶片的病斑直径

Fig. 6 Virulence assay on rubber tree leaves and lesion diameter after inoculation with WT and recom-binant strains A: Disease symptoms of rubber tree leaves at day 3 after inoculation (dpi); B: Mean lesion diameters of rubber tree leaves at day 2 and 3 (dpi).