Tobacco mosaic virus hijacks its coat protein-interacting protein IP-L to inhibit NbCML30, a calmodulin-like protein, to enhance its infection.

Calcium is an important plant immune signal that is essential for activating host resistance, but how RNA viruses manipulate calcium signals to promote their infections remains largely unknown. Here, we demonstrated that tobacco mosaic virus (TMV) coat protein (CP)-interacting protein L (IP-L) associates with calmodulin-like protein 30 (NbCML30) in the cytoplasm and nucleus, and can suppress its expression at the nucleic acid and protein levels. NbCML30, which lacks the EF-hand conserved domain and cannot bind to Ca2+ , was located in the cytoplasm and nucleus and was downregulated by TMV infection. NbCML30 silencing promoted TMV infection, while its overexpression inhibited TMV infection by activating Ca2+ -dependent oxidative stress in plants. NbCML30-mediated resistance to TMV mainly depends on IP-L regulation as the facilitation of TMV infection by silencing NbCML30 was canceled by co-silencing NbCML30 and IP-L. Overall, these findings indicate that in the absence of any reported silencing suppressor activity, TMV CP manipulates IP-L to inhibit NbCML30, influencing its Ca2+ -dependent role in the oxidative stress response. These results lay a theoretical foundation that will enable us to engineer tobacco (Nicotiana spp.) with improved TMV resistance in the future.

Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway.

Asparagine synthetase is a key enzyme that catalyses the conversion of amide groups from glutamine or ammonium to aspartate, which leads to the generation of asparagine. However, the role of asparagine synthetase in plant immunity remains largely unknown. Here, we identified a Nicotiana benthamiana asparagine synthetase B (NbAS-B) that associates with tomato mosaic virus coat protein-interacting protein L (IP-L) using the yeast two-hybrid assay and examined its role in tobacco mosaic virus (TMV) resistance. The association of IP-L with NbAS-B was further confirmed by in vivo co-immunoprecipitation, luciferase complementation imaging, and bimolecular fluorescence complementation assays. IP-L and NbAS-B interact in the nucleus and cytosol and IP-L apparently stabilizes NbAS-B, thus enhancing its accumulation. The expressions of IP-L and NbAS-B are continuously induced on TMV-green fluorescent protein (GFP) infection. Co-silencing of IP-L and NbAS-B facilitates TMV-GFP infection. Overexpression of NbAS-B in tobacco reduces TMV-GFP infection by significantly improving the synthesis of asparagine. Furthermore, the external application of asparagine significantly inhibits the infection of TMV-GFP by activating the salicylic acid signalling pathway. These findings hold the potential for the future application of asparagine in the control of TMV.

Overexpression of a pathogenesis-related gene NbHIN1 confers resistance to Tobacco Mosaic Virus in Nicotiana benthamiana by potentially activating the jasmonic acid signaling pathway.

Harpin proteins secreted by plant-pathogenic gram-negative bacteria induce diverse plant defenses against different pathogens. Harpin-induced 1 (HIN1) gene highly induced in tobacco after application of Harpin protein is involved in a common plant defense pathway. However, the role of HIN1 against Tobacco mosaic virus (TMV) remains unknown. In this study, we functionally characterized the Nicotiana benthamiana HIN1 (NbHIN1) gene and generated the transgenic tobacco overexpressing the NbHIN1 gene. In a subcellular localization experiment, we found that NbHIN1 localized in the plasma membrane and cytosol. Overexpression of NbHIN1 did not lead to observed phenotype compared to wild type tobacco plant. However, the NbHIN1 overexpressing tobacco plant exhibited significantly enhanced resistance to TMV infection. Moreover, RNA-sequencing revealed the transcriptomic profiling of NbHIN1 overexpression and highlighted the primary effects on the genes in the processes related to biosynthesis of amino acids, plant-pathogen interaction and RNA transport. We also found that overexpression of NbHIN1 highly induced the expression of NbRAB11, suggesting that jasmonic acid signaling pathway might be involved in TMV resistance. Taken together, for the first time we demonstrated that overexpressing a pathogenesis-related gene NbHIN1 in N. benthamiana significantly enhances the TMV resistance, providing a potential mechanism that will enable us to engineer tobacco with improved TMV resistance in the future.

Molecular characterization and expression analysis of Hsp90 in Schizothorax prenanti.

Aquatic animals suffer from various environmental stresses because the aquatic environment is a very complex system. To monitor the health status of fish, Hsp90 a potential early warning marker was determined in Schizothorax prenanti after infection with a bacterium. In this study, we cloned Hsp90 from S. prenanti for the first time. The full-length cDNA sequence of SpHsp90 was 2663 bp, contains an open reading frame of 2181 bp, and has a gene encoding 726 amino acids, an estimated molecular mass of 83.38 kDa, and a theoretical isoelectric point of 4.91. The SpHsp90 amino acid sequence has five conserved HSP90 family signatures and shares 87.0-95.5 % identity with other vertebrates. Phylogenetic analysis and structure comparison indicated that SpHsp90 should be a ß isoform of the HSP90 family. SpHsp90 was ubiquitously expressed in all examined tissues, and the highest level of expression was in the kidney. After Streptococcus agalactiae infection, the level of SpHsp90 expression had significant changes (P < 0.05) in the hepatopancreas, spleen, kidney, and blood. The expression increased to the highest level at 6 h in the blood and at 24 h in the hepatopancreas, spleen, and kidney. The results suggested that the SpHsp90 gene could be induced by S. agalactiae in S. prenanti and that SpHsp90 may be involved in resistance to bacterial infection and provide an early warning information. The kidney is the most suitable for detecting SpHsp90 after bacterial infection.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

Molecular cloning, expression analysis, and appetite regulatory effect of peptide YY in Siberian sturgeon (Acipenser baerii).

Peptide YY (PYY) is an anorectic brain-gut peptide involved in feeding regulation and well characterized in mammals. However, the functional role of PYY in the appetite regulatory of fish is not clear. In this study, we characterized a high conservation of PYY cDNA and found high expression levels of PYY mRNA in the brain and digestive tract of Siberian sturgeon. Then, we examined preprandial (pre- and post-feeding) changes of PYY mRNA expression in the brain that showed a significantly increased in 3h post-feeding, suggesting an anorectic possible function of PYY in Siberian sturgeon. Next, we examined the expression of PYY mRNA during 15 days fasting and refeed after fasting. The SsPYY mRNA expression of unfed fish had a significant 2.4, 1.7, 2.0, 2.2, and 2.1-fold decrease compared to 1-, 3-, 6-, 10- and 15-day ad libitum fed animals, respectively. After refeed, SsPYY mRNA significantly increased 1.9 and 4.1-fold above that of the 15-day fed and unfed fish control group (P<0.01). Furthermore, a single intraperitoneal injection of 10, 100 and 200 ng/g BW SsPYY1-36 caused a reduction in the next feeding and no significant reduction in food intake was observed in fish injected with a 1 ng/g BW. Overall, PYY has a potentially role in food intake attenuation of Siberian sturgeon.

Molecular and physiological evidences for the role in appetite regulation of apelin and its receptor APJ in Ya-fish (Schizothorax prenanti).

Apelin is a recently discovered peptide produced by several tissues with diverse physiological actions mediated by its receptor APJ. In order to better understand the role of apelin in the regulation of appetite in fish, we cloned the cDNAs encoding apelin and APJ, and investigated their mRNA distributions in Ya-fish (Schizothorax prenanti) tissues. We also assessed the effects of different nutritional status on apelin and APJ mRNAs abundance. Apelin and APJ mRNAs were ubiquitously expressed in all tissues tested, relatively high expression levels were detected in the heart, spleen, hypothalamus and kidney. Short-term fasting significant increased APJ mRNA expression, but no significant difference between fasted fish and fed control on 5- and 7-day. Meanwhile, apelin mRNA expression consistently increased during the 7-day food deprivation. In order to further characterize apelin in fish, we performed intraperitoneal (i.p.) injection of apelin-13 and examined food intake of the injected fish. Apelin injected at a dose of 100 ng/g body weight induced a significant increase in food intake compared to saline injected fish. Our results suggest that apelin acts as an orexigenic factor in Ya-fish. Their widespread distributions also suggest that apelin and APJ might play multiple physiological regulating roles in fish.

Molecular characterization and tissue expression of peptide YY in Schizothorax prenanti: effects of periprandial changes and fasting on expression in the hypothalamus.

Peptide YY (PYY) is a potent anorectic neuropeptide implicated in feeding regulation in mammals. However, the involvement of PYY in the feeding behavior of teleosts has not been well understood. In this study, we employed molecular, real-time quantitative PCR and physiological studies to characterize the structure, distribution, and appetite regulatory effects of PYY in Schizothorax prenanti (S. prenanti). A very high conservation in PYY sequences was found in teleosts. PYY is widely expressed, with the highest levels of expression in telencephalon, medulla oblongata, pituitary and hypothalamus of S. prenanti. The PYY mRNA expression in the hypothalamus was highly elevated after a meal, suggesting a satiety signal role for PYY in S. prenanti. In addition, PYY gene expression in the hypothalamus was decreased after fasting and increased sharply after refeeding, which suggested that PYY might be involved in the central regulation of appetite in S. prenanti. Overall, our result provides basis for further investigation into the regulation of feeding in S. prenanti.

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti).

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg-Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.