Abstract
BackgroundAlthough the canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. However, CP functions of Apple stem pitting virus (ASPV) has not been comprehensively documented. This study aimed to characterize the functions of ASPV CP and any functional diversification caused by sequence diversity of six ASPV CP variants and studied their biological, serological, pathogenic and viral suppressor of RNA silencing (VSR) functions.MethodsSix ASPV CP variants that have previously been shown to belong to different subgroups were selected here to study their diversity functions. Agrobacterium mediated infiltration (Agroinfiltration) was used to express YFP-ASPV-CPs in Nicotiana. benthamiana and infect Nicotiana. occidental with PVX-ASPV-CPs in. Confocal microscopy was used to detect YFP-ASPV-CPs florescence. CPs expressed in Escherichia coli BL21 (DE3) were induced by IPTG.ResultsIn this study, we showed that recombinant CPs expressed in Escherichia coli BL21 (DE3) had different levels of serological reactivity to three anti-ASPV antibodies used to detect ASPV. Furthermore, fusion CPs with YFP (YFP-CPs) expressed in N. benthamiana cells differed in their ability to form aggregates. We also showed that ASPV isolates that harbour these CPs induced different biological symptoms on its herbaceous host N. occidentalis. At the same time, we found that all six CPs when expressed in PVX vector showed similar VSR activity and produced similar symptoms in N. occidentalis, despite their differences in amino acids.ConclusionsDifferent ASPV isolates induced different symptoms in N. occidentalis, however, ASPV CP variants expressed in PVX vector showed the same symptoms in N. occidentalis plants. Also, we showed that ASPV CP variants has the same level of VSR activity, but they have different abilities to aggregate in N. benthamiana.
Highlights
The canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle
Different symptoms induced in N. occidentalis by Apple stem pitting virus (ASPV) isolates ASPV induces yellow vein leaves (Fig. 1a left) and stone fruit on pear but does not induce symptoms on apple leaves or fruit
ASPV CPs different in their propensity to aggregate To elucidate whether genetic diversity of ASPV CPs affects their subcellular localization in plant cell, we examined subcellular localization of CP variants by transiently expressing CPs fused with YFP at its N-terminus (YFP-ASPV-CPs (HB-HN1–3/HB-HN9–3/HB-HN6–8/ HB-HN7–18/YN-MRS-17/LN-AP1–1)) in N. benthamiana, which is widely used to express protein transiently [25]
Summary
The canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. Apple stem pitting virus (ASPV) is the type species of the Foveavirus genus in the Betaflexiviridae family [1] It possesses a single stranded positive RNA (+ssRNA) genome comprising of approximately 9300 nucleotides (nts), which encodes five open reading frames (ORFs, ORF1-ORF5) as well as the 5′ untranslated region (UTR) and 3’ UTR. Variants from pear isolates could be divided into six subgroups (subgroup A-F), and CP variants from a different subgroup have a different CP size because of amino acid insertions or deletions in the N terminal portion of CP [13] These previous observations might imply host-driven adaptations have affected genetic diversification of ASPV CP variants
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