![]() Protoplasts were isolated following previously described protocol. Preparation of Kenaf Protoplasts for Fluorescent in Situ Hybridization (FISH) This will also contribute to the understanding of virus long distance movement and symptom development. This study is aimed to address this question and to uncover any additional function(s) of p23, based on mutations of its basic amino acids. Since p23 is essential for HCRSV replication and it is a putative transcription factor, whether HCRSV infection can be affected by mutations of the basic amino acids is not known. Basic amino acids K, R, and H represent the mutation sites in the nuclear localization signal (NLS) of p23.įor the p23 NLS, any mutation to the three basic amino acids lysine (K), arginine (R) and histidine (H) ( Figure 1) will abolish its nuclear localization. The 3-dimensional rectangles below represent the corresponding predicted mature proteins. The dotted vertical line represents a readthrough codon UAG. Untranslated region (UTR) putative transcription factor (PTF) RNA-dependent RNA polymerases (RdRps) movement protein (mp) pathogenesis-related gene (prg) coat protein (CP). The upper rectangles represent open reading frames. Organization of HCRSV genomic RNA and its corresponding open reading frames and predicted proteins (not drawn to scale). In addition, the p23 possesses a novel nuclear localization signal (NLS) which interacts with importin α and facilitates HCRSV RNA genome to enter nucleus. Different from other Carmoviruses, HCRSV contains a novel ORF (p23) which is a putative transcription factor and it is indispensable for host-specific replication. ![]() In addition, p27 and its other in-frame isoforms (p25 and p22.5) affect symptom expression and potentiate Carmoviruses movement in kenaf ( Hibiscus cannabinus L.). The HCRSV CP (p38) is a gene silencing suppressor. A biologically active cDNA clone of HCRSV p223 has been obtained previously. HCRSV genome contains 3911 nucleotides with seven ORFs ( Figure 1). In addition, coat protein (CP) is also involved in virus movement for TCV. The p8 and p9, which are translated from subgenomic (sg) RNA1, are involved in cell-to-cell movement. In general, the two 5′ proximal open reading frames (ORFs) of Carmoviruses encode a p28 and a readthrough p81, which are thought to be involved in virus replication –. These include Carnation mottle virus, Cowpea mottle virus, Hibiscus chlorotic ringspot virus (HCRSV), Melon necrotic spot virus, Pelargonium flower break virus, Saguaro cactus virus and Turnip crinkle virus. A few of them are studied more extensively. We conclude that the p23 protein of HCRSV is required for virus long distance movement.Īmong positive-sense single-stranded plant RNA viruses, there are 19 reported members in the genus Carmovirus, family Tombusviridae (International Committee on Taxonomy of Viruses, version = 2012). This study demonstrates that when p23 is prevented from entering the nucleus, it results in restriction of virus long distance movement which in turn abrogates symptom expression in the newly emerged leaves. Both the p23 and the CP genes of HCRSV were detected in the newly emerged leaves of infected plants but CP was not detected by Western blot and no symptom was observed on those leaves at 19 days post inoculation. Results showed that the two mutants were able to replicate in protoplasts but unable to move from inoculated leaves to newly emerged leaves. ![]() The effect of p23 was further confirmed by using artificial microRNA inoculation-mediated silencing. Virus replication and its coat protein expression were detected by fluorescent in situ hybridization and Western blot, respectively. Primer-specific reverse transcription-PCR was conducted to detect gene transcript level of p23 and viral coat protein separately. To investigate additional function(s) of p23, mutations of basic amino acids lysine (K), arginine (R) and histidine (H) that abolish its nuclear localization, were introduced into a biologically active full-length cDNA clone p223 of HCRSV for testing its effects on virus replication and virus movement in vivo. Our previous results showed that the p23 is indispensable for host-specific replication and is localized in the nucleus with a novel nuclear localization signal. The p23 is a unique protein in the Hibiscus chlorotic ringspot virus which belongs to Family Tombusviridae Genus Carmovirus.
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