Iranian Journal of Biotechnology
Volume:13 Issue: 4, Autumn 2015

A recently published article describing the draft genome of Erwiniamallotivora BT-Mardi (1), the causal pathogen of papaya dieback infection in Peninsular Malaysia, hassignificant potential to overcome and reduce the effect of this vulnerable crop (2). The authors found that the draft genome sequenceis approximately 4824 kbp and the G+C content of the genomewas 52-54%, which is very similarto the reference genomes of other Erwinia species. They concluded that the draft genome would be useful for understanding host-pathogen interaction and determiningthe mechanism of infection of the disease.In addition to this, we believe that there are some furtherdirectionsthat should be considered in the future. Firstly, the comprehensive analysis of pathogenicity genes in E. mallotivora, whichwould enable the comparison of genes important in pathogenicitybetween species in the genusErwinia, and highlight any differences. These include the most important and well-studied pathogenicity system in gram negative bacteria: the Type III secretion (TTSS). Investigation of genes important in the pathogenesis of E. mallotivorawill decipher the function of these genes andunravel the complex interaction of the bacteria and host, and this will enable the understanding of their capabilities to enter plant cells,overcome host resistance and induce disease. This in-depth understanding will potentially allow the identification and application of new sources of biocontrolfor this disease. Secondly, the elucidation and identification of the potential pathogen-inducible and defence-related genes in papaya. The papaya genome is available onlineto enable the identification of the putative immunity related genes (IRG) and it will be greatly useful for revealing the blueprint of host genes under pathogen stress. Wee at al. (3) reported the expression analysis for four possible defence related genes in the E. mallotivara host (zinc finger protein, leucine rich protein, aquaporin and peroxidase), in order to understand the defence mechanisms in papaya. This information will be valuable for strategies in generating host resistance and understanding host-pathogen interaction. Thirdly,the discovery of papaya microRNAs (miRNAs) is also important. Previous studies have shown that miRNAs could act in wide range of biological processes as well as in the plant’s defence system towards biotic and abiotic stresses. Therefore, profiling and functional analysis of miRNAs would be important to generatenovel possibilities in improving resistance to dieback. The high potential application of miRNAs in the host defence mechanism indicates that they should be used or manipulated to confer pathogen resistance in papaya. Finally, thedevelopment of resistant plants using a genetic engineering approach is also an important tool in the control of papaya dieback disease. Currently, there is no chemical controltreatment available and no resistant papaya varieties offeredin order to overcome this severe disease, and generating resistant plants using a biotechnology approach could be the solution. The strategy of disruption of bacterial virulence factors, improvement of plant defence responses and the expression of antimicrobial proteins of non-plant origincould be applied in papaya to confer resistance. The draft genome of E. mallotivora BT Mardi is the first step and the initialkey to the understanding of the host-microbe interaction and mechanism of infection of this severedisease. This could be fundamental for plant pathologists, molecular biologists and botanists in Malaysia - it could help them to focus on the ultimate goal of control of papaya dieback disease.

Gossypium arboreum is resistant to Cotton leaf curl Burewala virus and its cognate Cotton leaf curl Multan betasatellite (CLCuBuV and CLCuMB). However, the G. arboreum wax deficient mutant (GaWM3) is susceptible to CLCuV. Therefore, epicuticular wax was characterized both quantitatively and qualitatively for its role as physical barrier against whitefly mediated viral transmission and co-related with the titer of each viral component (DNA-A, alphasatellite and betasatellite) in plants. The hypothesis was the CLCuV titer in cotton is dependent on the amount of wax laid down on plant surface and the wax composition. Analysis of the presence of viral genes, namely alphasatellite, betasatellite and DNA-A, via real-time PCR in cotton species indicated that these genes are detectable in G. hirsutum, G. harknessii and GaWM3, whereas no particle was detected in G. arboreum. Quantitative wax analysis revealed that G. arboreum contained 183 µg/cm2 as compared to GaWM3 with only 95 µg/cm2. G. hirsutum and G. harknessii had 130 µg/cm2 and 146 µg/cm2, respectively. The GC-MS results depicted that Lanceol, cis was 45% in G. harknessii. Heptadecanoic acid was dominant in G. arboreum with 25.6%. GaWM3 had 18% 1,2,-Benenedicarboxylic acid. G. hirsutum contained 25% diisooctyl ester. The whitefly feeding assay with Nile Blue dye showed no color in whiteflies gut fed on G. arboreum. In contrast, color was observed in the rest of whiteflies. From results, it was concluded that reduced quantity as well as absence of (1) 3-trifluoroacetoxytetradecane, (2) 2-piperidinone,n-|4-bromo-n-butyl|, (3) 4-heptafluorobutyroxypentadecane, (4) Silane, trichlorodocosyl-, (5) 6-Octadecenoicacid, methyl ester, and (6) Heptadecanoicacid,16-methyl-,methyl ester in wax could make plants susceptible to CLCuV, infested by whiteflies.

There is a growing demand for mass production of shikalkin (a natural pigment consisted of shikonin and alkannin) due to its increasing applications in cosmetics, pharmaceutical and nutrition industries. The root of Iranian Arnebia euchroma produces shikalkin. The promising capability of this plant for shikalkin production has already been demonstrated in cell culture studies. Elicitation effect of Rhizoctonia solani (R. solani) in comparison with the effects of Cu2+, methyl jasmonate (MJ), and salicylic acid (SA) on the shikalkin production was investigated in A. euchroma callus. The calli from different origins (leaf, collar and root) were proliferated on a modified Linsmaier-Skoog (mLS) medium and were subsequently transferred onto the pigment production medium containing various amounts of the desirable elicitor. Observations were quantified and the pigment production was precisely measured spectrophotometrically. Pigment biosynthesis was induced on White medium containing IAA (1 mM) and kinetin (10 mM) in dark at 25°C. Use of R. solani increased the pigment production by 7 fold greater than normal White medium. Cu2+ only doubled the shikalkin production. MJ and SA showed enhancing effects comparable to that of Cu2+. It is assumed that upon binding of the polysaccharides of the fungal cells to the plant cell surface, a cascade of signaling is initiated that led to expression of genes involving in the biosynthesis of shikalkin.

Chitin is an abundant natural polysaccharide found in fungi, algae, and exoskeleton of insects. Several bacterial species are capable of utilizing chitin as their carbon source. These bacteria produce chitinases for degradation of chitin into N-acetyl-D-glucosamine. So far, regulation of the chitinase encoding genes has been studied in different bacterial species. Among Bacillus species, B. pumilus strain SG2 encodes two chitinases, ChiS and ChiL. The promoter region of chiSL genes (PchiS) is mainly regulated by the general carbon catabolite repression (CCR) system in B. subtilis due to the presence of a catabolite responsive element (cre). Use of PchiS in constructing an inducible expression system in B. subtilis was investigated. In the first step, complete and shortened versions of PchiS were inserted upstream of the lacZ on a pBS72/pUC18 shuttle plasmid. The b-galactosidase activity of B. subtilis carrying one of the relevant plasmids was measured in the presence of different carbon sources. An expression system based on the chitinase promoter of B. pumilus SG2 was established. Modification of PchiS and the culture medium resulted in production of b-galactosidase in B. subtilis up to 1,800 MU activity. The chitinase promoter developed in this study, has potential to be used in an expression vector that could be induced by chitin. In addition, compared to the other inducers like IPTG and lactose, chitin is definitely cheaper and more available as an inducer.

Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore,targeting TIM-1 could be a potential therapeutic approach against such diseases. In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific

Detection of antibodies against HCV is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and it has been widely used in commercial Enzyme Immunoassays (EIA) for HCV diagnosis. Furthermore, since NS4B is a key protein in the virus replication, it is an alternative target for antiviral therapy. Hence, rapid, high yield, and economical production of recombinant HCV NS4B is an obligation for producing HCV diagnostic kits and developing anti-HCV drugs. In the current study, we aimed to develop a new method for high-level expression and purification of NS4B coding region. Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. Coli BL21. Induction was performed by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunogenicity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzyme-linked immunosorbent assay (ELISA). The accuracy of the construct was confirmed by colony PCR and sequencing. SDS-PAGE analysis showed successful expression of the recombinant protein. ELISA and western blotting confirmed the immunoreactivity of the recombinant NS4B. The directional TOPO cloning provides an efficient and easy method for cloning and expression of recombinant HCV NS4B. The directional TOPO cloning should be evaluated for production of other viral proteins.

Dermatophytosis is the common cutaneous infections in humans and animals, which is caused by the keratinophylic fungus called dermatophytes. In recent years, drug-resistance in pathogenic fungi, including dermatophytes to the current antifungal have been increased. The aim of this study was to evaluate the antifungal efficacy of AgNPs against Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum. The antifungal susceptibility of nano-silver particles (AgNPs) compared with Griseofulvin (GR). Its efficacy was investigated against three strains of dermatophytes by both agar dilution and broth microdilution test (BMD). The average minimum inhibitory concentration (MIC) of Ag-NPs on M. canis, T. mentagrophytes and M. gypseum were 200, 180, and 170 μg/ml, respectively. Whereas these strains showed MIC of 25, 100 and 50 μg/ml for Griseofulvin (GR). Our finding indicated that the AgNPs was less active than Griseofulvin (GR) but it had anti-dermatophytic effect.

Multi-drug resistance strains of Pseudomonas aeruginosa which are particularly resistant to imipenem are considered as a new problem for treatment in the medical settings, especially in burnt units of the hospitals. This study was conducted to determine and evaluate the antimicrobial effect of ferrous oxide nanoparticles alone and functionalized with imipenem on Pseudomonas aeruginosa starins producing Metallo β-lactamases (MBL). A clinical sample of above noted bacterium resistant to commonly used antibiotics and concurrently resistant to imipenem antibiotic was diagnosed and isolated by disk diffusion method. The minimum inhibitory concentration (MIC) of bacteria was calculated in the presence of ferrous oxide nanoparticle. The antimicrobial effect of nanoparticles functionalized with antibiotic was also determined. Standard strain of P. aeruginosa ATCC: 27853 was used as control. The clinical sample was resistant to imipenem up to the concentration of 128 μg/ml. Similarly, MIC of the isolate for nanoparticle was 160 μg/ml. In course, it was observed that the combination of 16 μg of antibiotic with 80 μg/ml of Ferrous oxide nanoparticles were able to inhibit the growth of the isolate. Ferrous oxide nanoparticles can impair antibiotic resistance mechanisms of bacteria; moreover, it can make the imipenem resistant bacterium more susceptible to weaker concentrations of antibiotic. It also has its own antibacterial effect in certain concentrations

The advent of recombinant DNA technology has facilitated heterologous expression of proteins from various sources in different host systems inclucding Escherichia coli. If a plant virus coat protein is expressed in the bacterium it can be used as the antigen for antibody preparation. Such a recombinant antigen preparation can be particularly useful where equipment such as ultracentrifuge is unavailable to purify virus particles to use as the antigen for conventional antibody preparation. Heterologous protein expression and purification of the full length Potato virus Y (PVY) coat protein (CP) from isolate pot187 (an affiliate of strain N) to be used as an antigen was the aim of the study. Reverse transcription Polymerase Chain Reaction (RT-PCR) was carried out to amplify an 801 bp fragment of the CP gene from PVY-infected potato leaves. The amplicon was cloned into pGEM-T Easy. The cloned fragment was restricted by BamHI + SacI and the purified fragment was cloned into restricted expression vector pET21a(+) with the same enzymes. The generated plasmid was introduced to E. coli strain RosettaTM. The expression was induced with isopropyl–β-D-thiogalactopyranoside (IPTG) and its protein content was subjected to SDS-PAGE and western blotting. SDS-PAGE analysis of protein from the induced bacteria showed a ~35 KDa protein corresponding to PVY CP. The expression of recombinant protein was confirmed by anti-His anitibody. The full-length cDNA of PVY-CP was amplified from the infected potato leaves. The cDNA was heterologously expressed in E. coli. The produced recombinant CP can be used as an antigen to generate polyclonal antibody.