Abstract
Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor)-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV) virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs), into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605) for biotin-conjugated anti-LIYV IgG (B-αIgG). IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated approx. seven- to eight-fold slower than that of Alexa Fluor.
Highlights
Affiliates of the genus Crinivirus infect diverse plant species (Wisler et al, 1998), and share common features, such as an exclusive tropism for phloem tissues and formation of filamentous virions that are transmitted in a semi-persistent manner by specific phloem-feeding whiteflies of the Bemisia tabaci species complex (Brown et al, 2000; Ng and Falk, 2006b; Dinsdale et al, 2010)
In a recent study in which immunofluorescent localization was used to analyze whiteflies that were sequentially fed Lettuce infectious yellows virus (LIYV) virions, anti-LIYV virion IgG, and an organic fluorophore (Alexa Fluor 488)-conjugated goat anti-rabbit IgG, we found that upon uptake, LIYV virions were retained within the anterior foregut or cibarium of its specific vector B. tabaci biotype A, but not within that of the non-vector B. tabaci biotype B (Chen et al, 2011)
BIOTINYLATION OF ANTI-LIYV IgG AND LABELING WITH STREPTAVIDIN-quantum dots (QDs) CONJUGATE To obtain biotinylated IgG produced against LIYV virions, antiLIYV IgG was first purified from polyclonal anti-LIYV antiserum using ammonium sulfate precipitation and DE52 (Whatman, England) anion exchange chromatography
Summary
Affiliates of the genus Crinivirus (family Closteroviridae) infect diverse plant species (Wisler et al, 1998), and share common features, such as an exclusive tropism for phloem tissues and formation of filamentous virions that are transmitted in a semi-persistent manner by specific phloem-feeding whiteflies of the Bemisia tabaci species complex (Brown et al, 2000; Ng and Falk, 2006b; Dinsdale et al, 2010). These studies have benefited from the use of membrane feeding, a procedure that allows insects with piercing and sucking mouthparts to ingest virion-augmented artificial liquid diet sandwiched between a pair of stretched parafilm Results from these studies provided concrete evidence that the whitefly B. tabaci biotype A can acquire and transmit LIYV virions purified from various sources, including cesium sulfate-sucrose density gradient-purified virions prepared from infected plants, and partially purified virions prepared from tobacco protoplasts inoculated with either virion RNAs or in vitro transcripts produced from cloned cDNAs corresponding to the viral genomic RNAs (Tian et al, 1999; Ng et al, 2004; Ng and Falk, 2006a). Results from these studies suggested that transmission determinants of LIYV reside on the virion itself (Tian et al, 1999; Ng et al, 2004; Ng and Falk, 2006a), which contrasts with the aphid transmission of Cauliflower mosaic virus (CaMV) and viruses in the genus Potyvirus, where additional viral encoded www.frontiersin.org
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