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

Abstract

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