Post-transcriptional Silencing of the Tomatinase Gene in Fusarium oxysporum f. sp lycopersici
Post-transcriptional Silencing of the Tomatinase Gene in Fusarium oxysporum f. sp lycopersici
复制标题
尖孢镰刀菌番茄酶基因的转录后沉默。
DOI：
--
复制
发表时间：
2002
期刊：
影响因子：
--
通讯作者：
M. Kameya
中科院分区：
文献类型：
--
作者：
S. Ito;H. Takahara;T. Kawaguchi;Shuhei Tanaka;M. Kameya
香蕉枯萎番茄属植物产生一种胞外酶番茄碱酶，已知该酶能使α-番茄碱解毒，α-番茄碱是番茄植物中真菌生长的预先形成的抑制剂。为了了解番茄碱酶在致病性中的作用，我们在F. oxysporum F.番茄叶锈菌获得的突变体在体外对α-番茄碱的敏感性增加，番茄碱酶活性降低。接种试验表明，突变体对番茄的致病性降低，表明番茄碱酶在该菌致病性中起重要作用。然而，Southern印迹分析表明，突变体的常驻番茄碱酶基因没有被破坏，但基因置换载体异位整合到突变体的基因组中。为了阐明所涉及的机制，我们研究了一个异位插入突变体在DNA和RNA水平。未检测到番茄碱酶基因甲基化。北方印迹分析表明番茄碱酶基因在异位突变体中表达水平较低，并且存在反义RNA。因此，转录后基因沉默似乎参与了番茄碱酶在异位突变体中的抑制。
Fusarium oxysporum f. sp. lycopersici produces an extracellular enzyme tomatinase, which has been known to detoxify α-tomatine, a preformed inhibitor of fungal growth in tomato plants. To understand the role of the tomatinase in pathogenicity, we performed transformation-mediated disruption of the tomatinase gene in F. oxysporum f. sp. lycopersici. Mutants obtained showed increase in sensitivity to α-tomatine and decrease in tomatinase activity in vitro. Inoculation assay showed reduced pathogenicity of the mutants on tomato plants, suggesting an important role of tomatinase in pathogenicity of this fungus. However, Southern blot analysis revealed that the resident tomatinase gene of the mutants was not disrupted but the gene replacement vector was ectopically integrated into the genome of the mutants. To clarify the mechanisms involved, we examined one of the ectopic insertion mutants at both DNA and RNA levels. No methylation of the resident tomatinase gene was detected. Northern blot analysis revealed low level of the tomatinase gene expression and also the presence of antisense RNA in the ectopic mutant. Thus, post-transcriptional gene silencing seems to be involved in the suppression of tomatinase in the ectopic mutant.