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Biology

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

Published: May 14th, 2021

DOI:

10.3791/62605

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences

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.

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.

Most described plant viruses are transmitted by insects from the order Hemiptera that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips1,2. 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 propagative3,4,....

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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 .......

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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 phalloidin10,18. The laser scanning confocal micrograph in Figure 2 sh.......

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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.......

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This work was supported by grants from the National Natural Science Foundation of China (31630058 to X.W. and 31772134 to W.L.).

....

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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

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