Iranian Journal of Biotechnology
Volume:18 Issue: 4, Autumn 2020

Wheat dwarf virus (WDV) is a leafhopper-transmitted DNA virus which causes yellowing and stunting in wheat and barley fields leading to considerable crop loss around the world. Mainly, two host-specific forms of WDV have been characterized in wheat and barley (WDV-Wheat and WDV-Barley, respectively).

This study was aimed to amplify, sequence and describe subgenomic DNAs (sgDNAs) associated with WDV infection among wheat and barley plants. The nucleotide sequence of sgDNAs were then compared to that of parental genomic DNAs (gDNAs) and the differences were shown.

A total of 65 symptomatic plants were surveyed for WDV infection using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and polymerase chain reaction (PCR). Rolling circle amplification followed by restriction analysis (RCA-RA) was applied to identify both gDNAs and sgDNAs in the infected wheat and barley plants. Nucleotide sequence of eight full-length WDV genomes and five sgDNAs were determined.

Genomic sequences of WDV-Wheat and WDV-Barley isolates obtained in this study were identified as WDV-Fand WDV-B, respectively, forming a separate cluster in the phylogenetic tree with the highest bootstrap support (100%). Sequence analysis of sgDNA molecules revealed that they have undergone different mutation events including deletions in viral genes, duplication of coding regions, and insertion of host-derived sequences.

The association of different types of sgDNAs were found in WDV-infected wheat and barley plants. The sgDNAs exhibited remarkable changes compared to their parental molecules and they might play a role in symptom severity, host genome evolution and emergence of new virus variants/species.

Rice tungro disease (RTD) is a viral disease mainly affecting rice in Asia. RTD caused by Rice tungro bacilliform virus and Rice tungro spherical virus. To date, there are only 5 RTSV isolates have been reported. In this study, we aimed to report the complete nucleotide sequence of Malaysian isolate of Rice tungro spherical virus Seberang Perai (RTSV-SP) for the first time. RTSV-SP was characterized and its evolutionary relationship with previously reported Indian and Philippines isolates were elucidated. RTSV-SP isolate was isolated from a recent outbreak in a paddy field in Seberang Perai zone of Malaysia. Its complete genome was amplified by RT-PCR, cloned and sequenced. Sequence analysis indicated that the genome of RTSV-SP consisted of 12,173 nucleotides (nt). Comparative analysis of 6 complete genome sequences using Clustal Omega showed that Seberang Perai isolate shared the highest nucleotide identity (96.04%) with Philippine-A isolate, except that the sORF-2 of RTSV-SP is shorter than RTSV Philippine-A by 27 amino acid residues. RTSV-SP found to cluster in Southeast Asia (SEA) group based on the whole genome sequence phylogenetic analysis using MEGA X software. Phylogenetic classification of RTSV isolates based on the complete nucleotide sequences showed more distinctive clustering pattern with the addition of RTSV-SP whole genome to the available isolates. Present study described the isolation and molecular characterization of RTSV-SP.

High antagonistic ability of different Trichoderma species against a diverse range of plant pathogenic fungi has led them to be used as a biological fungicide in agriculture. They can also promote plant growth, fertility, resistance to stress, and absorption of nutrients. They are also opportunistic and symbiotic pathogens, which can lead to the activation of plant defense mechanisms.

The aim of this present study was to investigate possible enhancement of lytic enzymes production and biocontrol activity of T. virens against Rhizoctonia solani through gamma radiation and to find the relationship between changes in lytic enzyme production and antagonistic activity of T. virens.

Dual culture conditions were used to evaluate the antagonistic effect of T. virens and its gamma mutants against R. solani. Then, their chitinase and cellulase activities were measured. For more detailed investigation of enzymes, densitometry pattern of the proteins was extracted from the T. virens wild-type and its mutants were obtained viaSDS-polyacrylamide gel electrophoresis.

The mutant T.vi M8, T. virens wild-type and mutant T.vi M20 strains showed the maximum antagonistic effects against the pathogen, respectively. Data showed that the mutant T. vi M8 reduced the growth of R. solani by 58 %. The mutants revealed significantly different (p <0.05) protein contents, chitinase and cellulase production (mg.mL-1) and activity (U.mL-1) compared to the wild-type strain. The highest extracellular protein production in the supernatant of chitinase and cellulase TFM was observed for the T.vi M11 and T.vi M17 strains, respectively. The T.vi M12 and wild-type strains secretedchitinase and cellulase significantly more than other strains did. Densitometry of SDS-PAGE gel bands indicated that both the amount and diversity of chitinase related proteins in the selected mutant (T. vi M8) were far higher than those of the wild-type. The diversity of molecular weight of proteins extracted from the T. virens M8 (20 proteins or bands) was very high compared to the wild-type (10 proteins) and mutant T.vi M15 (2 proteins).

Overall, there was a strong link between the diversity of various chitinase proteins and the antagonistic properties of the mutant M8.

Plant and animal cells possess a ubiquitous protein known as heat shock proteins (HSPs). Hsps were originally described in relation to heat shock and against abiotic and biotic stresses. Heat shock protein was classified in other crops on the bases of single classes or all classes but in Citrus sinensis Hsps groups, classes, subfamilies and members were not classified and characterized up to our knowledge. Present study was focused on the identification and grouping of C. sinensis Hsps (CsHsps) classes, members among classes, their phylogenetic relationship, gene structure, conserved motifs and identification of proteins by using bioinformatics tools and analyses. Genomic, Peptide and CDS sequences of CsHsps were downloaded from phytozome. MEGA 7 used for the phylogenetic analysis, GSDS for gene structure, UGENE for the multiple sequence alignment and MEME suite for the conserved motif analysis. The genome size of C. sinensis was 367 Mb, Chromosome number (2n)18, having 151 Hsps with six groups CsHsp10, 20, 40, 60,70 and 90. CsHsp20 was the largest group having 54 members, followed by CsHsp60 and CsHsp70 both having 30 members respectively. CsHsps members within a class shared more similar gene and protein structure. CsHsp 60, CsHsp 70 and CsHsp90 shared more conserved and similar amino acid pattern. Each class had some important proteins such as Cpn in CsHsp10, Hypothetical proteins in CsHsp20 and 40, Dnak in CsHsp60, Molecular chaperone in CsHsp70 and Hsp90 in CsHsp90. These proteins are produced by cells in response to stresses in citrus. Chaperonins and some hypothetical proteins identified in CsHsps, help in ATP synthesis and protein degradation. This is genome wide analysis and classification sets the groundwork for future investigations to fully characterize functionally the Citrus Hsps families and underscores the relevance of Hsps response to abiotic and biotic stresses in Citrus.

MicroRNAs, as small non-coding RNAs, are recently reported to be involved in plant defense system against pathogens including fungi.

In this research, it was intended to investigate candidate susceptible rice (Oryza Sativa) Osa-miRNA expression alteration following the infection by Rhizoctonia solani.

To this aim, literature review suggested eight conserved plant miRNAs that are involved in other plant-pathogen interactions. Then, sixty days old rice plants (Hashemi, susceptible cultivar) were inoculated with R. solani and candidate miRNA expression alterations were investigated 2 hpi (hours post inoculation), 2 dpi (days post inoculation) and 6 dpi.

RT-qPCR analysis suggested four subgroups of candidate miRNAs based on the time of their responses to the pathogenesis of R. solani. While Osa-miR-156 was early-responsive, Osa-miR159 was the last-responsive and Osa-miR167, Osa-miR171, Osa-miR408, and Osa-miR444 were late responsive to R. solani infection. Osa-miR166 and Osa-miR393 were non-responsive to this infection, compared to the mock-inoculated control group. Consistently, Os-SPL3 and Os-MADS known target genes were expressed in reverse correlation to Osa-miR156 and Osa-miR444, respectively.

From these data, it is suggested that both early (Osa-miR-156) and late (Osa-miR167, Osa-miR171, OsamiR408, Osa-miR444) responsive miRNAs might be involved in R. solani infection in rice plants.

World Health Organization (WHO) reported that more than 80% of people in the world use herbal traditional medicines nowadays. Many endemic medicinal plants, especially Nepeta species, are facing to extinction as a result of high harvesting, limited distribution, and habitat destruction.Tissue culture is a successful method for plant secondary metabolites production. Nepeta binaloudensis is a medicinal plant belonging to family Lamiaceae. Our study was focused on devising an optimum procedure for callus induction and phenolic compounds production in N. binaloudensis. First, we are focused on finding suitable explants and media for callus induction. Then, subsequent experiments were conducted to find an optimal concentration of plant growth regulators (PGRs) and reducedglutathione for maximum biomass production, and phenolic compounds production in calli. In this study, the usage of whole plant grown in Hoagland nutrient solution, were used as a source of explants. Also, different media including, ½ MS, MS, and B5 and different combination of PGRs (NAA and BAP) were used for optimization of calli induction. Based on the results of the first experiment, leaf-originated explants, and macro half strength MS (½ MS) medium were used for the next experiments. The highest FW (Fresh Weight) and DW (Dry Weight) of calli were observed in ½ MS medium, supplemented with 2 μM/L reduced-glutathione, 2 mg.L-1 BAP, and 2 mg.L-1 NAA. The maximum amount of total phenolic, flavonoid, tannin contents and free-radical scavenger were observed in calli which were grown in ½ MS medium supplemented with 2 μM/L reduced-glutathione, 2 mg.L-1 BAP, and 2 mg.L-1 NAA. Our study finds the optimum condition for calli induction and phenolic compounds production in N. binaloudensis.

Plant growth, reproduction and yields are severely damaged under adverse environmental stresses. These stresses can be either biotic or abiotic, and many stress related proteins are expressed in response to these stresses. Among these proteins dehydrins are reported to have a role primarily in the abiotic stresses. Dehydrins are very diverse proteins and a uniform annotation system is needed for their functional characterization in the future research. The aim of the present work is to identify, classify and analyze the expression of dehydrin proteins under different biotic and abiotic stresses in the selected plant species by using different computational tools. Prosite database is used for dehydrin proteins identification, and to conform the location of conserved motifs in selected plant species. The dehydrins extracted from uniprot database were annotated, based on the ensemble plant gene id. Subcellular localization was predicted using PSI predictor tool. Dehydrin expression analyses wereretrieved form the genevestigator tool. Dehydrins were annotated on the basis of dehydrin gene locus and conserved motifs available in different domain databases. Dehydrins were identified and annotated in Arabidopsis thaliana (13), Glycine max (12), Zea mays (05), Oryzasativa (11), Solanum tuberosum (05), Solanum lycopersicum (06), Triticum aestivum (32) and Vitis vinifera (06). It has been proposed that dehydrins are located primarily in cytosol and nucleus. Based on genevestigater expression analyses the plant species selected for this study contain all the classes of dehydrins, namely YnSKn, Kn, SKn, and YnKn; except class KnS. Dehydrins are diverse proteins and a uniform classification is introduced for their better characterization. The distribution of dehydrins in different tissues and developmental stages suggest an important function throughout plantgrowth cycle. It has also been concluded that dehydrins expressed particularly in drought, cold and salt stresses, and may have limited role in heat, anoxia, heavy-metal and biotic stresses as well.

Development of VEGF antagonists, which inhibit its interaction with the receptors, is a widely used strategy for the inhibition of angiogenesis and tumor growth. In the present study, a VEGFR-1 antagonistic peptide was designed and its potential for binding to VEGFR-1 and VEGFR-2 was evaluated by theoretical studies. Based on the X-ray structure of VEGF-B/VEGFR-1 D2 (PDB ID: 2XAC), an antagonistic peptide (known as VGB1) was designed, and its model structure was constructed using homology modeling in the MODELLER, version 9.16. The validity of the modeled structures was estimated employing several web tools. Finally, one model waschosen and molecular dynamics (MD) simulation was applied using the GROMACS package, version 5.1.4, to allow conformational relaxation of the structure. Next, docking process of the peptide with VEGFR-1 and VEGFR-2 was performed by HADDOCK web server and the docking structures were optimized by MD simulation for 20 ns. The far-UV circulardichroism (CD) spectrum of VGB1 was recorded to evaluate the overall structure of the peptide. The far-UV CD spectrum indicated that VGB1 contains α helix structure. The results from docking studies suggested that Van der Waals and nonpolar interactions play the most important role in the peptide binding to VEGFR-1. In addition, our results implicated the relevance of both Van der Waals and electrostatic interactions in the formation of complex between VGB1 and VEGFR-2. In addition to the common binding residues in the corresponding region of VEGF-A and VEGF-B, additional binding residues also were predicted for the interaction of VGB1 with VEGFR-1 and VEGFR-2. The results of MD and molecular docking simulations predicted that VGB1 recognizes both VEGFR-1 and VEGFR-2, which may lead to the prevention of the downstream signaling triggered by these receptors.

Some evidences show that immune infiltration is closely related to the clinical outcomes in cancers such as colorectal cancer. However, previous studies have not explained the diversity of cell types that make up the immune response. In particular, although some studies and reviews have shown that immunotherapy is important for cancer treatment, few studies have elucidated the relationship between prostate cancer (PCa) phenotype and immune infiltration. In this study, we analyzed whether different types of tumor-infiltrating immune cells would affect the clinical phenotypes and survival of PCa based on a deconvolution algorithm and annotated gene expression profiles. The 22 subsets of immune cells inferred by CIBERSORT and the infiltration abundance of 6 immune cells calculated by TIMER were used to determine the associations between them and the PCa traits and survival response. In addition, the survival tree models were constructed to classify PCa patients into four subtypes, and the traits and prognosis were compared among these subtypes. As a result, we found that some PCa patients with high death risk lacking immune infiltration were related to the poor prognosis. For the cell subsets studied and subtypes analysis, a low proportion of mast resting cells and T-cells follicularhelper exhibited the obvious association with poor outcome. In summary, our study suggested the differences in the cellular composition of the immune infiltrate in PCa, and these differences might be important determinants for PCa traits and prognosis.

Microalgal biotechnology has gained much attention previously. Monoculture algae cultivation has been carried out extensively in the last decades. However, although the mixed microalgae cultivation has some advantageous over pure cultures, there is still a lack of knowledge about the performance of mixed cultures.

In this study, it has been tried to investigate all growth aspects of marine and freshwater microalgal species in a mixed culture and their biological effects on biomass growth and composition based on wastewater nutrient consumption.

Three algal species of Chlorella vulgaris, Scenedesmus obliquus, and Nannochloropsis sp. were cultivated in saline wastewater individually, then the effects of mixing the three strains on biomass productivity, nutrient removal efficiency, chlorophyll, carotenoid, and lipid content were investigated.

The obtained results revealed that the mixed culture of three strains showed the highest biomass productivity of 191 mg. L-1.d-1. Also, while there were no significant differences between the performance of mono and mixed culture of algal species in the removal efficiency of wastewater nutrients, the three-strain microalgal mixed culture showed the highest values of 3.5 mg.L-1.d-1 and 5.75 mg.L-1.d-1 in the removal rate of phosphate and nitrate, respectively. In terms of total chlorophyll and carotenoid per produced biomass, however, the mixed culture of three species showed the lowest values of4.08 and 0.6 mg. g biomass-1, respectively.

The finding proves the potential of attractive and economically feasible mixed microalgae cultivation for high percentage nutrient removal and microalgal biomass production.

Antimicrobial peptides play crucial roles in organisms as the first line of defense against invading pathogens. To isolate the hepcidin (hepc1) gene from the liver of turbot (Scophthalmus maximus) challenged with Vibrio anguillarum (GenBank accession number: AM113708), characterize it, and assess its expression level in various tissues. The DNA sequence of hepcidin from S. maximus was determined from the total RNA extracted and reverse transcribed from this fish. The expression levels of tissue-specific hepcidin transcripts were determined using reverse-transcriptase polymerase chain reactions. Hepcidin levels increased in the livers, head kidneys and spleens of the fish. The transcriptional increase was especially noticeable in the liver after bacterial infection commencement. The presence of hepcidin and interleukin-beta (IL-1β) in blood leukocytes was compared at the transcription level and hepcidin transcripts were detected earlier than IL-1β transcripts after infection, indicating that hepcidin might serve as the first line of defense to kill bacteria and may also play a more direct and effective role than that of IL-1β during the initial stage of the innate immune response when turbot are exposed to bacteria invasion. Hepcidin might serve as the first line of defense to kill bacteria and may also play a more direct and effective role than that of IL-1β during the initial stage of the innate immune response when turbot are exposed to bacteria invasion.

IP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors. Based on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1–604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated. In the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1μs) was applied. As expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction. It seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Honey has been known as a traditional medicine for centuries with its antibacterial properties. It is considered one of the most enduring substances used in wound management.

This study aimed to: (i) evaluate the effects of Malaysian Trigona honey on bacterial structure and (ii) assess the anti-virulence potential of this honey by examining their impacts on the expression of selected genes (involved in stress survival and biofilm formation) in a test organism.

Trigona honey’s impacts on the bacterial structure (cell morphology) and the expression profiles of select Pseudomonas aeruginosa and Streptococcus pyogenes genes were examined using scanning electron microscopy(SEM) and real-time PCR (RT-qPCR) analysis, respectively.

SEM showed that the decreased cell density deformed, disrupted, and damaged cells for both bacteria. RT-qPCR showed that the expression of fleN, fleQ, and fleR genes of P.aeruginosa were decreased, 4.26-fold, 3.80-fold and 2.66- fold respectively. In addition, scpA, ftsY, and emm13 of S.pyogenes were decreased, 2.87-fold, 3.24-fold, and 4.65-fold respectively.

Our results indicate that Trigona honey may be an effective inhibitor and virulence modulator of P. aeruginosa and S. pyogenes via multiple molecular targets. This deduction needs to be investigated in vivo.

Reteplase, the recombinant form of tissue plasminogen activator, is a thrombolytic drug with outstanding characteristics, while demonstrating limited solubility and reduced plasminogen activation. Previously, we in silico designed a variant of Reteplase with positively supercharged surface, which showed promising stability, solubility and activity. This study was devoted to evaluation of the utility of supercharging technique for enhancing these characteristics in Reteplase. To test the hypothesis that reinforced surface charge of a rationally-designed Reteplase variant will not compromise its stability, will increase its solubility, and will enhance its plasminogen cleavage activity. Supercharged Reteplase coding sequence was cloned in pDest527 vector and expressed in E. coli BL21 (DE3). The expressed protein was extracted by cell disruption. Inclusion bodies were solubilized using guanidine hydrochloride, followed by dialysis for protein refolding. After confirmation with SDS-PAGE and western blotting, extracted proteins were assayed for solubility and tested for bioactivity. SDS-PAGE and western blot analysis confirmed the successful expression of Reteplase. Western blot experiments showed most of Reteplase expressed in the insoluble form. Plasminogen cleavage assay showed significantly higher activityof the supercharged variant than the wild type protein (P < 0.001). The stability of the supercharged variant was also comparable to the wild type. Our findings, i.e. the contribution of the surface supercharging technique to retained stability, enhanced plasminogen cleavage activity, while inefficiently changed solubility of Reteplase, contain implications for future designs of soluble variants of this fibrinolytic protein drug.