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Modulation of Expression of PVY^NTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars.

ABSTRACT: Potato virus Y (PVY) belongs to the genus Potyvirus and is considered to be one of the most harmful and important plant pathogens. Its RNA-dependent RNA polymerase (RdRp) is known as nuclear inclusion protein b (NIb). The recent findings show that the genome of PVY replicates in the cytoplasm of the plant cell by binding the virus replication complex to the membranous structures of different organelles. In some potyviruses, NIb has been found to be localized in the nucleus and associated with the endoplasmic reticulum membranes. Moreover, NIb has been shown to interact with other host proteins that are particularly involved in promoting the virus infection cycle, such as the heat shock proteins (HSPs). HSP70 is the most conserved among the five major HSP families that are known to affect the plant-pathogen interactions. Some plant viruses can induce the production of HSP70 during the development of infection. To understand the molecular mechanisms underlying the interactive response to PVY^NTN (necrotic tuber necrosis strain of PVY), the present study focused on StHSC70-8 and PVY^NTN-NIb gene expression via localization of HSC70 and NIb proteins during compatible (susceptible) and incompatible (hypersensitive) potato-PVY^NTN interactions. Our results demonstrate that NIb and HSC70 are involved in the response to PVY^NTN infections and probably cooperate at some stages of the virus infection cycle. Enhanced deposition of HSC70 proteins during the infection cycle was associated with the dynamic induction of PVY^NTN-NIb gene expression and NIb localization during susceptible infections. In hypersensitive response (HR), a significant increase in HSC70 expression was observed up to 3 days post-inoculation (dpi) in the nucleus and chloroplasts. Thereafter, between 3 and 21 dpi, the deposition of NIb decreased, which can be attributed to a reduction in the levels of both virus accumulation and PVY^NTN-NIb gene expression. Therefore, we postulate that increase in the expression of both StHSC70-8 and PVY^NTN-NIb induces the PVY infection during susceptible infections. In contrast, during HRs, HSC70 cooperates with PVY^NTN only at the early stages of interaction and mediates the defense response signaling pathway at the later stages of infection.

SUBMITTER: Koziel E

PROVIDER: S-EPMC8619674 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHaemaphysalis flava is a hematophagous ectoparasite that acquires the nutrition needed for development and reproduction by sucking blood and digesting the blood meal. During blood-sucking and blood-meal digestion, the prevention of blood coagulation is important for this tick. Previous studies have shown that heat shock cognate 70 (HSC70) protein has certain anticoagulant activities, but its immunogenicity remains unclear. Also, whether the mutation of individual bases of the TKD-like peptide of HSC70 through the overlap extension method can change its anticoagulant activities and immunogenicity remains to be investigated.MethodsThe gene encoding the HSC70 protein was cloned from a complementary DNA library synthesized from H. flava. The coding gene of the TKD-like peptide of HSC70 was mutated into a TKD peptide coding gene (HSC70TKD) using the overlap extension method. Escherichia coli prokaryotic expression plasmids were constructed to obtain the recombinant proteins of HSC70 (rHSC70) and HSC70TKD (rHSC70TKD). The purified rHSC70 and rHSC70TKD were evaluated at different concentrations for anticoagulant activities using four in vitro clotting assays. Emulsifying recombinant proteins with complete and incomplete Freund's adjuvants were subcutaneously immunized in Sprague Dawley rats. The serum antibody titers and serum concentrations of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected using an indirect enzyme-linked immunosorbent assay to assess the immunogenicity of rHSC70 and rHSC70TKD.ResultsThe open reading frame of HSC70 was successfully amplified and found to have a length of 1958 bp. The gene encoding the TKD-like peptide of HSC70 was artificially mutated, with the 1373-position adenine (A) of the original sequence mutated into guanine (G), the 1385-position cytosine (C) mutated into G and the 1386-position G mutated into C. rHSC70 and rHSC70TKD that fused with His-tag were obtained using the expression plasmids pET-28a-HSC70 and pET-28a-HSC70TKD, respectively. rHSC70 and rHSC70TKD prolonged the thrombin time (TT) and reduced the fibrinogen (FIB) content in the plasma, but did not affect the prothrombin time (PT) or activated partial thromboplastin time (APTT) when compared to the negative control. Interestingly, the ability of rHSC70TKD to prolong the TT and reduce the FIB content in the plasma was better than that of rHSC70. The specific antibody titers of both rHSC70 and rHSC70TKD in rat serum reached 1:124,000 14 days after the third immunization. The serum concentration of IFN-γ in the rHSC70TKD group was higher than that in the rHSC70 group. The rHSC70 group has the highest serum concentration of IL-4, and the serum concentration of IL-4 in the rHSC70TKD group was higher than that in the negative group.ConclusionsrHSC70 and rHSC70TKD exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. rHSC70TKD had better anticoagulant activities than rHSC70. Both rHSC70 and rHSC70TKD had good immunogenicity and induced humoral and cellular immunity.

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Modulation of Expression of PVY^NTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars.

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Modulation of Expression of PVYNTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars.

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OmicsDI is part of the ELIXIR infrastructure

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Modulation of Expression of PVY^NTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars.