Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Clarifying the spatiality and quantity of virus in whitefly vectors can help in understanding begomovirus-whitefly interactions and in the development of novel strategies for controlling serious begomoviral diseases. The advantages of this technique are that we can quickly isolate whitefly tissues, co-localize viral and whitefly proteins, and quantify viruses in whitefly whole bodies and virus-infected plants. At the appropriate experimental time point, after virus acquisition, place ice anesthetized female whiteflies into a drop of PBS on a microscope slide and use a fine acupuncture needle to open the abdomen of the whitefly under a stereo microscope.

Use the needle to extract the midgut and place the midgut into a collection dish of PBS. To collect the PSGs, split the body at the thorax from the dorsal side near the head and fix the whitefly with a needle through the thorax. Use a second needle to shake the whitefly until the two salivary glands are separated from the body and transfer the glands to a new dish of PBS.

To collect the ovaries, completely open the abdomen and use a needle to separate the ovaries from the other tissues. Use a pipette to remove the excess tissue and transfer the ovaries into a new dish of PBS. To collect a hemolymph sample, add 10 microliters of PBS onto a new microscope slide and place a new whitefly into the droplet.

Use a fine acupuncture needle to gently make a hole in the abdomen. Then apply slight pressure to the abdomen to release the hemolymph into the buffer and collect an eight microliter sample of the liquid. To visualize TYLCV localization in harvested whitefly PSG, midgut, or ovarian tissues, transfer 15 to 20 specimens into a glass 3.5 centimeter Petri dish and aspirate the excess buffer.

Fix the tissues in one milliliter of 4%paraformaldehyde for two hours at room temperature before carefully washing the samples three times for two minutes in one milliliter of fresh TBS per wash. After the last wash, permeabilize the samples with one milliliter of 0.5%Triton X-100 for 30 minutes before washing three times as just demonstrated with one milliliter of TBST per wash. After the last wash, block any nonspecific binding with one milliliter of 1%bovine serum albumin for two hours at room temperature followed by three washes in TBST as demonstrated.

After the last wash, label the cells with the primary antibodies of interest overnight at four degrees Celsius protected from light. The next morning, wash the samples three times with TBST before adding the appropriate secondary antibodies for two hours at room temperature protected from light. At the end of the incubation, wash the midguts three times and transfer the specimens to a clean microscope slide.

Remove as much buffer as possible and stain the nuclei with one drop of DAPI before mounting with a coverslip and examining the labeled tissues by confocal microscopy. For TYLCV quantification, wash the harvested whitefly tissue samples two to three times in fresh PBS and transfer 20 midguts, 20 PSGs, or one ovary in five microliters of PBS into individual PCR tubes containing 25 microliters of lysis solution. Add five to seven two millimeter diameter ceramic beads to each tube and grind the samples in a homogenizer.

Add one eight microliter sample of hemolymph into a new PCR tube and add 10 microliters of lysis to the tube with thorough mixing. Incubate all of the samples at 65 degrees Celsius for one hour followed by a 10-minute incubation at 100 degrees Celsius and a brief centrifugation. Next, add 18 microliters of master mix into the vials of quantitative PCR strip tubes and add two microliters of each DNA sample to at least three vials.

When all of the samples have been loaded, close the tubes and centrifuge to sediment the solution at the bottom of each vial. Load the vials onto a real-time thermal cycler for quantitative PCR, selecting cyber as the fluorophore dye and unknown as the sample type. Then export the quantitative data to a spreadsheet for analysis of the relative quantity of viral DNA in each sample.

Here, representative images of TYLCV in DAPI staining in whitefly PSGs, midguts, and ovaries are shown. As observed, TVLCV demonstrates a greater accumulation within the PSGs and midguts than in the ovaries. Quantification of the virus in whitefly PSGs, midguts, hemolymph, and ovaries after feeding on TYLCV-infected tomato for 24, 48, and 72 hours indicates that the quantity of virus increases in different whitefly tissues in direct correlation to the increase in acquisition access period.

It is best to use fresh whiteflies, as dead insects will increase the difficulty of dissection. Following this procedure, other analyses, like western blotting and co-immunoprecipitation, can be performed to answer additional questions about viral protein expression and viral and vector protein interactions.