Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Summary

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Abstract

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

Introduction

Most described plant viruses are transmitted by insects from the order Hemiptera that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips^1,2. The piercing-sucking mouthparts of hemipteran insects pierce the plant tissue for feeding and secreting saliva, concomitantly efficiently transmitting the virus². Different transmission mechanisms of plant viruses by vector insects have been described. These include nonpersistent, semipersistent and persistent. The persistent type is either non-propagative or propagative^3,4,....

Protocol

1. Nonviruliferous insect rearing

1. Collect WBPHs from rice fields and rear with rice seedlings in 1 L glass beakers covered with insect-proof net in an incubator at 28 °C with 16 h light and 8 h dark. Because SRBSDV is not transmitted via eggs, newly hatched nymphs are not viruliferous.
2. With a brush pen, gently brush insects from the beaker rearing insects into a new beaker of fresh rice seedlings each week until WBPH nymphs have hatched. Continue rearing these hatched nonviruliferous nymphs to 2- or 3-instar.
   ​NOTE: Brush carefully to avoid WBPHs flying from the beaker or damaging them.

2. Virus ....

Results

Figure 1 illustrates all steps in this protocol: insect rearing, virus acquisition, excision of the gut, immunofluorescent labeling, and making the slide.

Excised WBPH guts from adults were fixed in 4% (m/v) paraformaldehyde, permeabilized with 2% (v/v) Triton X-100, and then incubated with Dylight 633 phalloidin^10,18. The laser scanning confocal micrograph in Figure 2 shows t.......

Discussion

For best results, a few key points should be considered. First, a high ratio of viruliferous insects among the total population is necessary. Although the minimum AAP for SRBSDV by WBPH nymphs and adults is 5 min¹⁷, the insects should be allowed to feed on fresh SRBSDV-infected rice plants for 2 d to achieve an acquisition efficiency of up to 80%. Since the SRBSDV virions can be detected in 80% of the midguts¹⁸, we excised and labeled the viruliferous insects at 2 d after a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3% Bull serum albumin (BSA)	Coolaber	SL1331	Dilute antibodies
Cover glass	Solarbio	YA0771-18*18mm	For slide making
Dissecting microscope	Beitja	XTL-7045B1	For insect dissection
Laser scanning confocal microscope	Zeiss	Zeiss LSM880	Observe fluorescence signal
Microscope slides	Solarbio	ZBP-7105	For slide making
Mounting medium with 4'6-diamidino-2-phenylindole (DAPI)	Abcam	AB104139	Label cell necleus
Paraformaldehyde	Sigma	158127	For tissues fixation
Phalloidin	Invitrogen	A22284	Label actin of midgut epithiels
Triton X-100	Amresco	0290C484	For tissues permeation
Tweezers (5-SA)	AsOne	6-7905-40	For insect dissection