Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida

Abstract

Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro. Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-␤-D-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. IMPORTANCELimited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere with the CTV binding to its insect vector to block the transmission.T he survival of a virus is dependent on its ability to move from host to host, which for many plant viruses requires an insect vector (1). The mechanisms by which viruses are transmitted by these insects have been classified into two general groups: circulative and noncirculative. More than half of the viruses with a described mode of transmission fall into the latter category and are defined by attachment to sites within the vector's stylet, cibarium, or foregut (2, 3). There are also differences in both the acquisition and retention of noncirculative viruses (4), previously described as nonpersistent versus semipersistent. While there is no clear demarcation between the two, nonpersistent viruses can be acquired and disseminated through probing and salivation within a matter of minutes, while most semipersistent viruses may only be acquired and subsequently transmitted through deep phloem feeding, which generally requires hours for acquisition, and vectors remain vi...

Sequence variation in two genes determines the efficacy of transmission of citrus tristeza virus by the brown citrus aphid

“…It is not presently known how this diversity, particularly in the p27, p65 and p61 genes, affects transmission efficacy of individual isolates. It has recently been demonstrated [10] that this virus uses three proteins to interact with the lining of the cibarium of the aphid: (1) p27, the minor coat protein, which has been reported to be involved in vector interaction in other characterized closteroviruses [11,12], (2) p65, a HSP70like molecular chaperone, and (3) p61, a HSP90-like molecular chaperone, both of which are required for virion assembly [13].…”

“…Having determined that FS577 was transmitted at significantly greater frequency than T36, or its infectious clone, we constructed a series of hybrid infectious clones between the two isolates. As previous research had shown that the p65, p61, and p27 genes are involved in the interaction between virus and aphid [10], we focused on these genes. First, a single replacement from within p6 to within p18 (bases 11661 to 17300) was constructed through amplification (Table 1) of a fragment from FS577-1-8 cDNA, and substituted into the T36 infectious clone [15] using PmeI-PstI restriction sites.…”

“…While these two isolates differ by only *2 % at the nucleotide level, these genetic differences translate into phenotypic differences. Most importantly, T36 contains nine amino acid substitutions in p61 and seven in p65, two genes previously shown to be involved in aphid transmission of CTV [10] that are not found in FS577 or other extant members of that strain.…”

“…Following the recent discovery that CTV uses three proteins, p27, p65, and p61, to interact with its aphid vector [10], we asked what effect polymorphism within these genes has on transmissibility. We focused on the poorly transmissible T36 isolate, which has been graft-propagated in greenhouse conditions for over 30 years (S. Garnsey, personal communication), and compared it to FS577, a highly transmissible member of the same genetic lineage.…”

Direct and indirect influences of virus–insect vector–plant interactions on non-circulative, semi-persistent virus transmission

“…Understanding the viral determinant (s) and specific virion retention sites underlying the mechanism of LIYV transmission has provided insights into the NCSP transmission processes and paved the way for similar investigations of other NCSP criniviruses (Ng, unpublished) [10]. Recently, Killiny et al [11] performed VR assays in which aphid vectors were fed purified CTV virions tagged with the green fluorescent protein (GFP) and found GFP signals retained within the vectors' foreguts. In in vitro competitive binding assays where the dissected foreguts of aphid vectors were co-incubated with specific CTV proteins and the GFP-tagged virions, three of the capsid proteins, CPm, P61 and P65, significantly reduced the virion-associated GFP fluorescence in the vectors' foreguts, suggesting that these proteins played a role in mediating specific virion retention in the aphid vector.…”