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

Shota Iwasaki,Koji Kakutani,Naoko Okada,Yutaka Kimura,Yoshihiro Takikawa,Teruo Nonomura,Tomoko Suzuki,Yuling Bai,Yoshinori Matsuda

doi:10.3390/agriculture12020254

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.

Highlights

Biotic stresses such as fungi, bacteria and viruses affect the growth and development of plants [1]
All conidia of KTP-03 germinated on the trichomes at 3–5 h (Figure 2—3), and produced primary appressoria at 6–10 h after inoculation (Figure 2—4)
The success rate of multiplex reverse transcription (RT)-polymerase chain reaction (PCR)/N-PCR amplification was approximately 98.2% for LHA2 transcripts in 220 trichomes and 95.0% for tubulin 2 (TUB2) transcripts in 200 conidia inoculated onto trichomes (20 samples at each fungal infection stage), in hyphae-removed trichomes that possessed a haustorium and 3–5 haustoria, respectively (Table 2). These results indicated that intron-spliced mRNAs were simultaneously amplified with multiplex RT-PCR/N-PCR of single P. neolycopersici-noninoculated/inoculated leaf type I trichome cells, and from single trichome cells that possessed only powdery mildew haustoria

Summary

Introduction

Biotic stresses such as fungi, bacteria and viruses affect the growth and development of plants [1]. The PR proteins are studied and coded by the host plant under biotic stress [2], and are accumulated in the infected leaves, but are produced systemically in association with the development of systemic acquired resistance (SAR). Thaumatin-like proteins (TLPs) produced in plants are associated with developmental processes and defense against phytopathogens and elicitors [4]. On the other hand, regarding the effects of fungi on plants, Manghwar et al [9] evaluated the expression of important PR protein genes in resistant and susceptible varieties of wheat and maize under Bipolaris sorokiniana fungal stress, and described that these PR genes can be further over-expressed in transgenic plants to create resistance against various pathogens. Manghwar et al [11] studied the behavior of important PR genes in different wheat varieties, identifying which genes could be used for cloning into wheat and other transgenic crops to create resistance to Fusarium equiseti

Objectives

Methods

Results

Discussion

Conclusion