Simultaneous Detection of Plant- and Fungus-Derived Genes Constitutively Expressed in Single Pseudoidium neolycopersici-Inoculated Type I Trichome Cells of Tomato Leaves via Multiplex RT-PCR and Nested PCR

Type I trichomes of tomato leaves (Solanum lycopersicum Mill. cv. Moneymaker), as outgrowths the plant epidermis, are suitable for monitoring infection processes powdery mildew species using a high-fidelity digital microscope (DM) without fungal staining. On trichomes, (Pseudoidium neolycopersici L. Kiss) isolate KTP-03 produced maximum four vigorously elongated hyphae per conidium, which stopped growth approximately 12 days after inoculation. Single trichome cells, invaded by at various stages during 12-day period inoculation single conidia, were cut bases and directly collected with small precision scissors (i.e., microscissors) held manipulator under DM. Subsequently, suc-polymerase chain reaction (PCR) (reverse transcription (RT)-PCR followed nested (N)-PCR) was conducted to explore gene expression in infected trichome. We selected intron-containing genes from tomatoes fungi detection constitutive transcripts, namely plasma membrane H+-ATPase (LHA2) ?-tubulin 2 (TUB2) genes. In suc-PCR, band spliced mRNAs both LHA2 TUB2 detected, suggesting that successfully transcribed KTP-03-infected trichomes. With combined primers (multiplex RT-PCR/N-PCR), two bands detected through amplification intron-spliced Therefore, our single-trichome cell PCR method is effective detecting patterns fungus. Combinations microscopy, microscissors, multiplex RT-PCR/N-PCR techniques will be useful simultaneously analysing molecular interactions between plants level leaf cells. Also, this employed technique benefit other crops, possessing elucidate pathogens.