苹果炭疽叶枯病菌GcAP1复合体β亚基基因的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2017.08.007

Abstract

Abstract: 【Objective】 The objectives of this study are to determine the function of β subunit of adaptor protein GcAP1 complex in growth and pathogenicity of Glomerella leaf spot of apple pathogen Glomerella cingulata, investigate expression patterns of the GcAP1β in the fungal growth and pathogenicity, decipher whether or not GcAP1β regulate the expression of endopolygalacturonase genes CgPG1 and CgPG2, pectin lyase genes pnl-1 and pnl-2, pectate lyase genes pelA and pelB, and to lay a foundation for further studies of adaptor protein in pathogenic signal transduction pathways of G. cingulata. 【Method】 Based on the GcAP1β deletion vector and GcAP1β-gfp fused expression vector, the Δgcap1β mutant and the GcAP1β complementation strain Δgcap1β-GcAP1β were structured using ATMT, respectively, verified by RT-PCR and Southern blot analysis. Colony growth rate, sporulation, germination rate, appressorial formation rate and pathogenicity of the Δgcap1β mutant and the GcAP1β complementation strain Δgcap1β-GcAP1β were assayed, compared with the wild-type strain W16. GcAP1β subcellular localization was carried out with the bioinformatics softwares ProtComp 9.0 and TMHMM, along with signal observation of GcAP1β-GFP. The GcAP1β expression levels in hyphae, conidia, appressoria and pathogenicity stage were identified by qRT-PCR. Moreover, the expression levels of CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB in the wild-type strain W16 and the Δgcap1β mutant were detected, respectively. 【Result】 GcAP1β is 2 321 bp in length, including 3 introns, which encodes a 720 amino acids. Compared with the wild-type strain W16, the Δgcap1β mutant showed a rill-like fold colony and decreased growth, while sporulation, germination rate and appressorial formation rate were unaffected. Virulence of the Δgcap1β mutant reduced significantly, which induced tiny spots on the leaves. Moreover, the GcAP1β complementation strain Δgcap1β-GcAP1β fully restored the phenotype flaws by reintroducing GcAP1β to the Δgcap1β mutant. Fluorescent signal showed that the fused protein GcAP1β-GFP was distributed to the cytoplasm. qRT-PCR analysis showed that GcAP1β expresses through the lifecycle of G. cingulata, and the highest expression level of GcAP1β occurred at the post-invasion to leaves. Compared with WT, the Δcgap1β mutant showed a drastic reduction of CgPG1 transcripts (20.3%), CgPG2 transcripts (16.5%), pnl-1 transcripts (8.2%), pnl-2 transcripts (14.4%), pelA transcripts (4.4%) and pelB transcripts (0.8%). 【Conclusion】 The adaptor protein GcAP1 complex is distributed to the cytoplasm and is necessary for growth and development of G. cingulata; GcAP1 regulates the expression of CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB and is a vital virulence factor of G. cingulata.

Key words: adaptor protein, pectinase, apple, Colletotrichum, virulence

ZHANG JunXiang, JI ZhiRui, WANG Na, XU ChengNan, CHI FuMei, ZHOU ZongShan. Gene Cloning and Functional Analysis of GcAP1 Complex Beta Subunit in Glomerella cingulata[J].Scientia Agricultura Sinica, 2017, 50(8): 1430-1439.

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Gene Cloning and Functional Analysis of GcAP1 Complex Beta Subunit in Glomerella cingulata

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