苹果黑腐皮壳菌CAP超家族蛋白VmPR1c的降解功能域和降解途径

真菌与寄主互作过程中常分泌多种效应蛋白,但植物病原真菌CAP超家族蛋白是否参与致病过程尚不明确。基于苹果黑腐皮壳菌Valsa mali基因组CAP同源基因研究发现VmPR1c敲除后突变体致病性明显下降,但VmPR1c蛋白在表达过程中被降解。本研究借助BLAST、NCBI CDD web server、SignalP 4.1、TMHMM 2.0等进行序列分析,利用缺失突变法及在蛋白表达过程中加蛋白酶抑制剂分别对该蛋白的降解功能域和降解途径进行了探索。C端区段缺失突变结果显示,突变涉及CAP结构域中的α-helix结构和CBM区域时,Western blot条带呈现明显加深;涉及CTE区域及CAP结构域中的半胱氨酸时,Western blot条带则呈现不同程度地减弱。替换VmPR1c的信号肽或突变其信号肽切割位点序列,Western blot条带均有不同程度地加深。分别对CTE和NTE中的脯氨酸进行点突变后,蛋白降解更加严重。在蛋白表达过程中加入蛋白酶体抑制剂MG132和溶酶体抑制剂chloroquine,与对照VmPR1c ^△SP-GFP相比,Western blot结果无明显变化,表明VmPR1c蛋白序列C端区域中的CTE、信号肽及其切割位点序列以及CAP结构域中的α-helix结构介导其降解,其中CTE和NTE中的脯氨酸对该蛋白具保护作用。此外,VmPR1c的降解不通过蛋白酶体和溶酶体途径。

Functional degradation domain and potential degradation pathway of the CAP superfamily protein VmPR1c from Valsa mali

Fungi are known to secrete multiple effector proteins during their infection of host plants, whereas, the function of the secreted CAP superfamily proteins has been undefined in fungi. Based on CAP homologue analysis, the gene VmPR1c was identified in the genome of Valsa mali, the causal agent of apple valsa canker. Deletion of VmPR1c greatly attenuated V. mali virulence, however, VmPR1c was degraded when transiently expressed in Nicotiana benthamiana. In order to reveal the degradation mechanism of VmPR1c, we determined the functional degradation domain and degradation pathway of VmPR1c. Sequence analysis were performed using BLAST, NCBI CDD web server, SignalP 4.1, and TMHMM 2.0. Sequence truncation and site-directed mutagenesis were used to explore the degradation domain of VmPR1c. To study the degradation pathway of VmPR1c, various protease inhibitors were added during protein expression. Sequence truncation assays showed that, when the α-helix structure or the CBM region of CAP domain was deleted, VmPR1c protein could be easily detected by Western blotting. When the cysteine residues in CAP domain or the CTE region was mutated, VmPR1c protein bands were variously weakened. When the signal peptide of VmPR1c or its cleavage site was substituted, VmPR1c protein bands were differently deepened. When the proline residue in CTE or NTE region was mutated, VmPR1c degraded more evidently. When the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine was added during transient expression of VmPR1c in N. benthamiana, VmPR1c could not be detected by Western blotting. The conclusion is that the CTE region, signal peptide and the cleavage site of VmPR1c, as well as the α-helix structure of the CAP domain, mediate the degradation of VmPR1c. The proline in CTE and NTE could potentially protect VmPR1c from degradation. The degradation of VmPR1c is not dependent on the proteasome and lysosomal pathways.