香蕉枯萎病菌myosin-1基因的功能分析及外施dsRNA介导的抗性评估

 香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)引起的香蕉毁灭性土传病害,其中 4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。SMART在线软件分析myosin-1基因具有肌球蛋白马达蛋白(myosin motor domain, MMD),肌动蛋白尾结构TH1(myosin tail)和 Src家族同源结构域SH3(src homology domain 3),与禾谷镰刀菌中氰烯菌酯靶标基因myosin-5具高度的蛋白同源性,相似性高达83%。利用Split-marker基因重组技术获得Foc4的myosin-1基因敲除突变体,Δmyosin-1突变株丧失了对氰烯菌酯的敏感性,菌丝生长缓慢,产孢量减少且孢子畸形,对香蕉致病力严重下降,证实myosin-1是氰烯菌酯在Foc4中的作用靶标基因。外施靶向myosin-1体外转录的dsRNA,能抑制菌丝的生长,降低菌丝活性;菌丝膨胀扭曲分枝增多,出现典型的球状结构,与氰烯菌酯处理后的表型一致。在盆栽活体人工接种实验中,体外施用dsRNA可以明显抑制枯萎病外部症状的发展,推迟发病时间,赋予寄主抗性,结果说明体外施用dsRNA可以作为新型杀菌剂防治香蕉枯萎病。综上,myosin-1基因作为氰烯菌酯在Foc4中的靶标基因具有高度的序列保守,在调控菌丝生长发育,产孢以及致病力等方面发挥重要作用,而外施dsRNA具有防治香蕉枯萎菌的巨大潜力。

Functional analysis of myosin-1 in Fusarium oxysporum f. sp. cubense race 4 and evaluation of resistance mediated by exogenous dsRNA

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc race 4 (Foc4) is currently known as a major concern in global banana production. The protein domain analysis using a web resource SMART, revealed that myosin-1 gene contains myosin motor domain (MMD), myosin tail (TH1) and src homology domain 3 (SH3), which have high similarity to the myosin-5 gene in Fusarium graminearum. The split-marker strategy was applied to knockout the myosin-1 gene through PEG-mediated protoplast transformation with the recombinant fragments. The positive candidate mutants were obtained by PCR screening with four pairs of primers. The results demonstrated that the knockout mutants of myosin-1 showed slow growth, hyphal deformity, decreased conidia production, and significantly decreased pathogenicity to banana, while the Δmyosin-1 mutant lost its sensitivity to phenamacril. Foc4 mycelia showed growth retardation and reduction in cell viability in vitro cultures, while the mycelia exhibited multiple branching and typical conglobate structure 24 h post-treatment with dsRNA targeting the myosin-1 gene, which remained similar to the phenotype treated with phenamacril in vitro. The exogenous dsRNA mediated resistance to Foc4 infection was assessed through pre-treatment with dsRNA before artificial inoculation with Foc4 spores to pot culture banana plantlet. The dsRNA targeting myosin-1 can inhibit the external symptoms of Fusarium wilt disease, delay the time of plant death and confer plant resistance to Foc4. Taken together, myosin-1 gene might play an important role in the growth, sporulation and pathogenicity of Foc4, and externally applied dsRNA has potential to protect bananas against Foc4 infection.