番茄幼苗的肽饲喂和机械伤害

Lei Wang; Yan  Wang; Georg Felix

doi:10.21769/BioProtoc.3194

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Peer-reviewed

Peptide Feeding and Mechanical Wounding for Tomato Seedlings

番茄幼苗的肽饲喂和机械伤害

LW Lei Wang email

Y Yan Wang

GF Georg Felix

发布: 2019年03月20日第9卷第6期 DOI: 10.21769/BioProtoc.3194 浏览次数: 5390

评审: Amey RedkarHailong GuoMarta Bjornson

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Abstract

Plants need to respond appropriately to wounding and herbivorous insects. Peptide signals have been implicated in local and systemic induction of appropriate plant defense responses. To study these peptide signals and their perception in host plants, it is important to have reproducible bioassays. Several assays, such as treatment of peptide solution via pressure infiltration, have been developed. Here, we provide detailed protocols for peptide feeding and mechanical wounding for tomato seedlings. To directly introduce peptides into tomato seedlings, peptide solution is fed through the excised stem via the transpiration stream. To mimic the wounding caused by insect feeding, leaflets of tomato seedlings are mechanically damaged with a hemostat; and wounded and systemic unwounded leaves are harvested and analyzed separately. Samples from both assays may be further assessed by examining the transcript level of marker genes by quantitative real-time PCR (qRT-PCR).

Keywords: Tomato

Plant-insect interaction

Background

Peptide signaling plays crucial roles in regulating plant growth, development and interaction with the surrounding environment (Tavormina et al., 2015). Perception of the peptide systemin, for example, is critical for resistance against chewing insect larvae (Pearce et al., 1993; Wang et al., 2018). Attack by such larvae and related wounding lead to various immune responses, one of which is the induction of defense-related marker genes. In tomato, the genes encoding proteinase inhibitor 1 (PIN1) and proteinase inhibitor 2 (PIN2) were shown to be highly activated after insect feeding or wounding, and have been extensively used as hallmarks for defense triggered by insects or wounding (Pearce et al., 1991; Ryan and Pearce, 2003). Here we provide a protocol focusing on details of peptide feeding and wounding assays, which can be followed by quantification of PIN1 transcript via qRT-PCR. The protocol has been optimized to minimize variations inherent in these assays and was previously used to assess the function of systemin perception (Wang et al., 2018).

Materials and Reagents

12 mm x 75 mm glass test tubes (VWR, catalog number: 212-0013) (plastic tubes also work)
Latex gloves or nitrile gloves
5 ml pipette tips (Kinesis, catalog number: U696256)
Aluminum foil
Single edged steel razor blades (Carl Roth, catalog number: CK07.1)
9.5 cm round plant pots with a volume of 0.32 L (Gartnereinkauf, catalog number: 410103) (pots with similar size or larger also work)
6.5 cm round plant pots with a volume of 0.1 L (Gartnereinkauf, catalog number: 410066)
Plant seedling propagation trays with clear lids (60 cm x 40 cm x 6 cm) (meyer-shop, catalog number: 749114) (other similar trays also work)
5 ml pipette (Kinesis, catalog number: 2400631)
Tomato seeds
Potting soil (Pikiererde and Topferde ordered from Einheitserde, Germany; mix 1:1) (other potting soil with medium to high nutrient and fine texture also works)
Systemin peptide (peptide sequence: AVQS KPPC KRDP PKMQ TD) (GenScript, catalog number: 88910480001)
Sterile water
70% ethanol
Liquid nitrogen

Equipment

5.5-inch hemostat with serrated jaws (Merck, catalog number: Z168858-1EA)
Long tweezers (Carl Roth, catalog number: PT95.1)
Scissors
Plastic racks for test tubes
-80 °C freezer
-20 °C freezer

Procedure

English

中文翻译

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如何引用

Wang, L., Wang, Y. and Felix, G. (2019). Peptide Feeding and Mechanical Wounding for Tomato Seedlings. Bio-protocol 9(6): e3194. DOI: 10.21769/BioProtoc.3194.