细胞分裂蛋白激酶PlCdc15 调控荔枝霜疫霉致病力的机制研究

Mechanism of the cell division protein kinase PlCdc15 regulates the pathogenicity of Peronophythora litchii

摘要

荔枝霜疫霉（Peronophythora litchii）引起的荔枝霜疫病在荔枝生产及采后储运过程中造成产量损失及品质下降等问题，严重威胁荔枝产业的健康发展。荔枝霜疫霉隶属二倍体病原卵菌，目前关于其致病机制的研究鲜有报道。在细胞分裂过程中各信号途径的协同调控对于病原菌的生长发育及其侵染寄主的能力有重要影响。本研究鉴定到了荔枝霜疫霉在细胞分裂过程中的关键蛋白激酶 PlCdc15，功能域预测分析发现 PlCdc15 包含保守的 STKc 激酶功能域。通过 CRISPR/Cas9 基因编辑技术，获得 PlCdc15 的 N 端 STKc 功能域端缺失突变体 ΔPlcdc15-Stk 和 C 端缺失突变体 ΔPlcdc15-Back，发现这两个突变体的营养菌丝生长速率显著降低；孢子囊的产生能力和侵染寄主的能力均显著下降。DAPI 染色观察发现这两个突变体单位菌丝长度内的细胞核显著减少，细胞分裂过程紊乱。对产生的孢子囊进行低温诱导后，发现 ΔPlcdc15-Stk 无法割裂，且丧失了产生卵孢子的能力。本研究结果表明 PlCdc15 的 N 端（激酶功能域端）及其 C 端（无明确功能域端）在荔枝霜疫霉生长发育及侵染寄主过程中均发挥重要功能。本研究结果可为病原卵菌相关研究提供新的理论基础。

Abstract

Peronophythora litchii causes the most destructive disease on litchi, litchi downy blight. PlCdc15, a key protein kinase involved in the cell division process of P. litchi, was identified. Domain prediction analysis revealed that PlCdc15 contains a conserved STKc kinase domain. The CRISPR-Cas9 system, a genome editing tool, was used to produce the N-terminal STKc kinase domain-deletion mutant ΔPlcdc15-Stk and the C-terminal deletion mutant ΔPlcdc15-Back. Both mutants exhibited significant reduction in vegetative growth rate, sporulation capacity, and pathogenicity. DAPI staining revealed disrupted cell division processes in both mutants. After induced at low temperature, ΔPlcdc15-Stk failed to undergo cleavage and lost the ability to produce oospores. All the results highlights the essential roles of both the N-terminal kinase domain and the C-terminal region of PlCdc15 in the growth, development, and pathogenicity ofP. litchii. These findings provide new theoretical insights for research on pathogenic oomycetes.

关键词

荔枝 / 霜疫霉 / 细胞分裂 / 激酶 / PlCdc15

Key words

Peronophythora litchii / cell division / kinase / PlCdc15 / pathogenicity

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基金资助

海南省自然科学基金青年基金项目(322QN237)

海南大学科研启动基金项目(KYQD(ZR)-22090)

国家自然科学基金地区基金项目(32160614)

海南省荔枝产业技术体系项目(HNARS-08)