Transient expression of the full-length glycoprotein from infectious hematopoietic necrosis virus in bean (Phaseolus vulgaris) leaves via agroinfiltration.

The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO4  + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 µg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.

Multi-functionPlantDefensin,AntimicrobialandHeavyMetal Adsorbent Peptide.

BACKGROUND: Defensin peptide isolated from plants are often heterogeneous in length, sequence and structure, but they are mostly small, cationic and amphipathic. Plant defensins exhibit broad-spectrum antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria, fungi and etc. Plant defensins also play an important role in innate immunity, such as heavy metal and some abiotic stresses tolerance. OBJECTIVES: In this paper, in vitro broad-spectrum activities, antimicrobial and heavy metal absorption, of a recombinant plant defensin were studied. MATERIAL AND METHODS: SDmod gene, a modified plant defensin gene, was cloned in pBISN1-IN (EU886197) plasmid, recombinant protein was produced by transient expression via Agroinfiltration method in common bean. The recombinant protein was tested for antibacterial activity against Gram-negative, Gram-positive bacteria and Fusarium sp. the effects of different treatments on heavy metal zinc absorption by this peptide were tested. RESULTS: We confirmed the antibacterial activities of this peptide against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria, and antifungal activities of this peptide against Fusarium spp. (Fusarium oxysporum and Fusarium solani). High metal absorption coefficient for this peptide was also observed. RESULTS: Out of six actinobacterial isolates, VITVAMB 1 possessed the most efficient RO-16 decolorization property. It decolorized 85.6% of RO-16 (250 mg L-1) within 24hrs. Isolate VITVAMB 1 was identified to be Nocardiopsis sp. Maximum dye decolorization occurred at pH 8, temperature 35oC, 3% salt concentration and a dye concentration of 50 mg L-1. CONCLUSIONS: Results suggesting that modified defensin peptide facilitates a broader range of defense activities. dedefensins are an important part of the innate immune system in eukaryotes. These molecules have multidimensional properties that making them promising agents for therapeutic drugs.

Experimental and theoretical studies of cadmium ions absorption by a new reduced recombinant defensin.

Heavy metal pollutants such as Cd, Hg, Pb, As, and Se are considered as both a global problem and a growing threat to the humanity. Being strongly poisonous to the metal-sensitive enzymes and leading to the growth inhibition and death of organisms, these metals have a toxic impact on the plants and animals. Inducing the metal-binding cysteine-rich peptides such as metallothioneins, phytochelatins, and defensins, higher organisms like plants and animals usually react to the heavy metal stress. In this study, a recombinant defensin protein was expressed in bean and its ability in the cadmium absorption was determined. Experimental studies revealed that this protein was able to absorb cadmium ions in reduced form more than oxide one. Molecular dynamics simulations were carried out in order to evaluation of experimental studies, using a model of Cd2+ or Na+ and Cl- ions enclosed in a fully hydrated simulation box with the recombinant defensin. The theoretical results also suggested that the reduced recombinant defensin was more powerful in the absorption of Cd2+ than its oxide form. The present study is the first report of Cd2+ absorption potential of this new reduced recombinant defensin. The results unraveled that this recombinant defensin can be adopted as a molecular switch in the cadmium pollution of the environment and also the important role of sulfur groups in the absorption of cadmium ions.

In silico search and biological validation of microRNAs related to drought response in peach and almond.

Plant responses to drought stress are regulated at the transcriptional and post-transcriptional levels through noncoding endogenous microRNAs. These microRNAs play key roles in gene expression, mainly by down-regulating target mRNAs. In this work, an in silico search and validation for microRNAs related to drought response in peach ('G.H. Hill'), almond ('Sefied') and an interspecific peach-almond hybrid ('GN 15') has been performed. We used qPCR to analyse the gene expression of several miRNAs described as being related to drought response in peach, including miR156, miR159, miR160, miR167, miR171, miR172, miR398, miR403, miR408, miR842 and miR2275 under mild and severe water deficit. These miRNAs were in silico selected on the basis of previous works, their conservation in plants and their drought response. qPCR analysis confirmed the implication of these miRNAs in the dehydration stress response in the three assayed genotypes. Comparison of miRNA expression patterns in the three evaluated genotypes indicated that the hybrid GN 15 showed higher expression levels of specific miRNAs which should be related to the observed drought tolerance. mRNA target transcripts of the miRNAs studied were predicted using the Rose database, which includes transcription factors that regulate plant growth and development. In addition, results showed that the promoter region contains responsive elements to hormone-mediated regulatory elements. Network analysis not only unravelled the interaction between miRNAs and their predicted gene targets but also highlighted the roles of miRNAs in response to drought stress.