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, 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.