Small deletions in the potato leafroll virus readthrough protein affect particle morphology, aphid transmission, virus movement and accumulation

Abstract

Potato leafroll virus (PLRV) capsid comprises 180 coat protein (CP) subunits, with some percentage containing a readthrough domain (RTD) extension located on the particle's surface. The RTD N terminus is highly conserved in luteovirids and this study sought to identify biologically active sites within this region of the PLRV RTD. Fourteen three-amino-acid-deletion mutants were generated from a cloned infectious PLRV cDNA and delivered to plants by Agrobacterium inoculations. All mutant viruses accumulated locally in infiltrated tissues and expressed the readthrough protein (RTP) containing the CP and RTD sequences in plant tissues; however, when purified, only three mutant viruses incorporated the RTP into the virion. None of the mutant viruses were aphid transmissible, but the viruses persisted in aphids for a period sufficient to allow for virus transmission. Several mutant viruses were examined further for systemic infection in four host species. All mutant viruses, regardless of RTP incorporation, moved systemically in each host, although they accumulated at different rates in systemically infected tissues. The biological properties of the RTP are sensitive to modifications in both the RTD conserved and variable regions.

Simple and Rapid Detection of Potato leafroll virus by Reverse Transcription Loop-mediated Isothermal Amplification

“…Secondary symptoms in potatoes cultivated from infected tubers are severe with stunting of the plant and rolling of the leaves (Harrison, 1984). PLRV infection and expression in Nicotiana benthamiana and hairy nightshade did not show leaf rolling symptoms, whereas they displayed inter-vein chlorosis (Peter et al, 2008). Like other viruses, PLRV infection sometimes displays no symptom or plants may develop symptoms similar to those caused by different viruses (Webster et al, 2004) or nonpathogenic disorders due to nutritional deficiency, water stresses, insect damages, or pollution.…”

“…PLRV was inoculated using Agrobacterium tumefaciens culture carrying the plasmid pBPY, full-length cDNA of the PLRV-Canada strain (GeneBank No: D13954.1) as described by Lee et al (2005) and Peter et al (2008). A. tumefaciens containing the plasmid pBPY was grown in LB broth with kanamycin (50 µg/ml) for 48 h at 28°C.…”

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

“…Four-to five-week-old N. benthamiana leaves were inoculated with cDNA clones of wild-type (WT) and two PLRV mutants: one that only translates CP and lacks the RTD (⌬RTD) (9) and one that translates a truncated RTP lacking the C-terminal 218 amino acids (⌬RTDCT 499 ) (14). Plants were infected via Agrobacterium tumefaciens-mediated transformation as previously described (31).…”

“…Mutagenesis studies performed with infectious clones have greatly enhanced our understanding of the domains within viral proteins that are responsible for regulating different aspects of the polerovirus life cycle, including virus assembly, systemic movement, phloem retention, and vector transmission (2)(3)(4)(5)(6)(7)(8)(9). However, very few host proteins have been identified as interacting directly with any polerovirus protein (10)(11)(12)(13).…”

“…It is a representative member of a family of economically important plant viruses, and a number of seminal studies using virus mutants have elegantly linked the sequence of the structural proteins to function during plant infection and insect transmission (7)(8)(9)). Yet not a single, specific site of protein interaction has been defined between these proteins and host proteins.…”