Fusarium oxysporum genes expressed in planta

1)香蕉枯萎从mRNA和蛋白质水平检测了黄瓜木质部汁液中特异表达的Foc基因。对通过抑制性消减杂交分离的29个Foc cDNA克隆进行测序，并对所得核苷酸序列进行同源性搜索。进一步研究了与NIPSNAP基因和阳离子转运蛋白基因同源性较高的两个cDNA克隆在植物中的表达。根据这两个序列设计的特异性引物进行RT-PCR分析，结果表明它们对应的基因在寄主植物中确实表达。采用双向SDS聚丙烯酰胺凝胶电泳技术分析了黄瓜离体木质部汁液中积累的FOC蛋白。从凝胶中提取22个蛋白质点，并进行肽质量指纹分析。序列比对结果表明，数据库中没有与所获得的肽质量指纹图谱相匹配的蛋白质。选择已知在植物中表达的番茄专化型（sp. lycopersici）作为基因破坏的靶基因。获得的18株突变体对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的致病性降低，表明番茄碱酶在该菌致病性中起重要作用。Northem杂交分析表明，其中一株突变株在α-番茄碱诱导下表达了Fotom 1的反义RNA，表明转录后基因沉默至少在该突变株中参与了番茄碱酶活性的抑制。

1) Fusarium oxysporum f. sp. cucumerinum (Foc) genes expressed specifically in cucumber xylem sap were examined at mRNA and protein levels. Twenty-nine Foc cDNA clones isolated by suppression subtractive hybridization were sequenced and the resultant nucleotide sequences were subjected to homology searches. Two cDNA clones showing high homology with NIPSNAP gene and putative cation transporter gene were further examined for their expression in planta. RT-PCR analysis with specific primers designed from the two sequences revealed that their corresponding genes are actually expressed in host plants. Foc proteins accumulated in cucumber xylem sap in vitro were analyzed by two-dimensional SDS polyacrylamide gel electrophoresis. Twenty-two protein spots were extracted from the gels and subjected to peptide mass fingerprinting analysis. Sequence alignment searches showed that no proteins in databases were matched to the peptide mass fingerprintings obtained.2) Tomatinase gene (Fotoml) of Fusarium oxysporum f. sp. lycopersici which has been known to be expressed in planta was chosen as a target gene for gene disruption. Eighteen mutants obtained showed increase in sensitivity to α-tomatine and decrease in tomatinase activity. Inoculation assay showed reduced pathogenicity of the mutants, suggesting an important role of tomatinase in pathogenicity of this fungus. Northem blot analysis showed that antisense RNA of Fotoml was expressed when one of the mutants was induced by α-tomatine, suggesting posttranscreptional gene silencing is involved in the suppression of tomatinase activity at least in this mutant strain.