Biosafety of RNA silencing and genome editing technologies in crop plants: Malaysian and Australian research perspectives

Research in agricultural biotechnology can produce novel solutions to address the ever growing demand for food, feed, renewable materials and renewable energy using increasingly limited resources. Yet research is expensive with long timelines before implementation can disseminate the benefits to society, so there is a need to maximise co-operation and communication between scientists, stakeholders and their governments, to optimise research, its development and the implementation of research outcomes, into mainstream applications. Recognising the impacts of regulations on biosafety, biosecurity and intellectual property policy on strategies for research, senior and early career researchers from two research intensive universities in Malaysia and Australia, held a workshop to identify and to deliberate over two key areas of technology that offer much promise for agriculture, namely RNA silencing and genome editing. A major focus of the workshop was the regulation of new breeding technologies, and how the regulations need to take into account these new technologies. Themes discussed were the need for harmonisation of international legal frameworks and careful use of terminology, standards and guidelines; and the need for good communication and consensus within and between groups of stakeholders and law-makers. This mini-review highlights the deliberations and recommendations from the workshop

Synthesis of a soluble flag-tagged single chain variable fragment (scFv) antibody targeting Cucumber Mosaic Virus (CMV) coat protein

Cucumber mosaic virus (CMV) is a serious pathogen of many economically important commercial fruit and vegetable crops worldwide. It is a particular problem in warmer climates, where plants are not grown under cover thus necessitating undesirably high use of agrochemicals for the control of insect vectors. Efforts towards controlling of this virus would include the development of improved methods of virus detection including the ability to produce cost effective and specific reagents. In this study the production of recombinant antibodies provides one such approach. A single chain variable fragment (scFv) antibody, targeted to CMV coat protein, was constructed with mRNA from the spleen of a CMV coat protein-immunized mouse. The nucleotide sequence of the variable heavy (VH) and variable light (VL) framework regions of the mouse spleen cDNA were used to design and construct primers for scFv library construction via RT-PCR. Three rounds of panning of the scFv library with the coat protein of a local isolate of a chilli strain CMV resulted in the cloning of a novel soluble Flag-tagged scFv antibody that suitable for use as a diagnostic reagent with the further potential of in situ application in the development of transgenic plants with novel resistance

Assessment of marker-trait associations for drought and heat tolerance in bread wheat

Abiotic stresses are major constraints to wheat productivity in many parts of the world. Tolerance to abiotic stresses can be achieved indirectly by selection for morpho-physiological traits. Physiological trait based breeding has been associated with improved performance under stress; and hence can combat and adapt wheat to drought and heat stress. Therefore, in the present study, phenotyping was carried out for agro-physiological traits in 52 diverse wheat germplasm lines under timely sown, rainfed and late sown environments for two years. Mean yield of the genotypes over the six environments were positively correlated with NDVI, days to maturity and negatively correlated with canopy temperature. The phenotypic data validated marker-trait associations of a number of meta-QTLs identified earlier for different physiological and agronomic traits. Six and seven meta-QTL genomic regions were found to be consistent in their expression for two years under rainfed/restricted irrigation and late sown environments, respectively. Expression analysis of the underlying candidate gene AK248593.1 in meta-QTL26 region revealed two folds higher expression in the NILs carrying the co-localized SSR markers. The linked markers of the thirteen meta-QTL regions associated with different traits can be used for effective transfer of the QTLs through marker assisted selection in wheat breeding programmes

Evaluation of banana germplasm and genetic analysis of an F 1 population for resistance to Fusarium oxysporum f. sp. cubense race 1

Open Access Article; Published online: 23 Sept 2019Fusarium wilt of bananas (Musa spp.), caused by Fusarium oxysporum f. sp. cubense (Foc) causes up to 100% yield loss in bananas. Foc race 1 in particular is very devastating to dessert bananas in Uganda. One of the effective control strategies for the disease is the development of resistant cultivars through breeding. The objectives of this study were to identify suitable banana germplasm for generating a segregating population for resistance to Foc race 1 and understand the mode of inheritance of resistance to Foc race 1. Twenty-two banana accessions sourced from the National Agricultural Research Organisation in Uganda were challenged with Foc race 1 in a screen house experiment. Monyet, resistant to Foc race 1 and Kokopo, susceptible, were selected and crossed to generate 142 F1 genotypes. These F1 genotypes were also challenged with Foc race 1 in a screen house experiment. Data were collected on rhizome discoloration index (RDI), leaf symptom index (LSI) and pseudo-stem splitting (PSS), and analysed for variability. The banana accessions evaluated showed varying degrees of resistance to Foc race 1. Segregation ratios for resistant versus susceptible progenies fitted 13:3 (χ2 = 0.12, P = 0.73) for RDI and 11:5 (χ2 = 3.04, P = 0.08) for PSS. Estimated broad sense heritability was 27.8% for RDI, 13.9% for LSI and 14.7% for PSS. The results suggest that resistance to Foc race 1 in banana is controlled by at least two dominant genes with epistatic interaction and that heritability of resistance to Foc race 1 is low in Musa spp

Neutralization of bacterial yoeBspn toxicity and enhanced plant growth in Arabidopsis thaliana via co-expression of the toxin-antitoxin genes

Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP.Interestingly,theinducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells

Direct cloning approach for expression of an anti-cucumber mosaic virus single-chain variable fragment in plant

An anti Cucumber Mosaic Virus (anti-CMV) single chain variable fragment (scFv) was constructed into a plant expression vector using self-designed primers. Initially, sequence information of the scFv was obtained through automated sequencing. Based on the sequence information, two primers with appropriate restriction endonuclease sites were designed to facilitate the clone construction. This technique allows insertion of the desired gene to replace the GUS second exon in pCAMBIA 1301, without the need to form a complete cassette including promoter and termination signal prior to cloning into the vector. After insertion of the construct into the plant system through Agroinfection, the foreign protein, anti-CMV scFv antibody was expressed in tobacco plants

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

Boesenbergia rotunda, a herb in the ginger family, contains numerous beneficial compounds, such as flavonoids, flavones and cyclohexenyl chalcone derivatives, that have great potential for pharmaceutical applications. However, the low concentration of the bioactive compounds limits their commercial application. In this study, a simple and reliable Agrobacterium-mediated transformation protocol for B. rotunda cell suspension culture was successfully developed. The minimal inhibitory concentration and natural tolerance of the selective agent, hygromycin, against the cells were 20 mg l−l and 30 mg l−l in liquid media and solid media, respectively. The highest number of transformed regenerants (18 ± 0.00 per ml settled cell volume) was recorded when cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA1304 for 10 min and co-cultivated for 2 days. Prolonged infection time (> 10 min) and co-cultivation period (> 2 days), however, did not increase the transformation efficiencies. The results clearly show that infection and co-cultivation periods strongly influenced the transformation efficiency in ginger. The transformed cells were recovered and showed green fluorescent signals under ultraviolet excitation. An intense blue colour was observed in the transformed cells after β-glucuronidase (GUS) histochemical staining, further confirming the functionality of the GUS enzymes in the regenerants. Polymerase chain reaction analysis of 3-, 6-, 9- and 12-month-old transformed cells confirmed that the protocol enabled stable integration of the mgfp5 gene. Moreover, the comparatively high number of transformed regenerants in this study made it possible to generate a large number of transgenic cells in a short period, which would be useful for high-throughput functional screening of enzymes involved in the biosynthetic pathways of bioactive compounds

Genetic Regulation of the yefM-yoeBSpn Toxin-Antitoxin Locus of Streptococcus pneumoniae

Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae there are at least eight putative TAS, one of them being the yefM-yoeB(Spn) operon studied here. Through footprinting analyses, we showed that purified YefM(Spn) antitoxin and the YefM-YoeB(Spn) TA protein complex bind to a palindrome sequence encompassing the -35 region of the main promoter (P(yefM2)) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P(yefM2) as YefM(Spn) behaved as a weak repressor with YoeB(Spn) as a co-repressor. Interestingly, a BOX element, composed of a single copy each of boxA and boxC sub-elements was found upstream of promoter P(yefM2). BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation and genetic competence. In the yefM-yoeB(Spn) locus, the boxAC element provided an additional weak promoter, P(yefM1), upstream of P(yefM2) which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P(yefM1) was constitutive albeit weaker than P(yefM2). Intriguingly, the coupling of the boxAC element to P(yefM1) and yefM(Spn) in cis (but not in trans) led to transcriptional activation indicating that the regulation of the yefM-yoeB(Spn) locus differ somewhat from other TA loci and may involve as-yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB(Spn) locus suggested that its presence may provide a selective advantage to the bacterium

Genetic Regulation of the yefM-yoeB Toxin-Antitoxin Locus of Streptococcus pneumoniae

14 páginas, 6 figuras, 2 tablas -- PAGS nros. 4612-4625Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae, there are at least eight putative TAS, one of them being the yefM-yoeBSpn operon studied here. Through footprinting analyses, we showed that purified YefMSpn antitoxin and the YefM-YoeBSpn TA protein complex bind to a palindrome sequence encompassing the −35 region of the main promoter (PyefM2) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to PyefM2, since YefMSpn behaved as a weak repressor with YoeBSpn as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter PyefM2. BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeBSpn locus, the boxAC element provided an additional weak promoter, PyefM1, upstream of PyefM2 which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that PyefM1 was constitutive albeit weaker than PyefM2. Intriguingly, the coupling of the boxAC element to PyefM1 and yefMSpn in cis (but not in trans) led to transcriptional activation, indicating that the regulation of the yefM-yoeBSpn locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeBSpn locus suggested that its presence may provide a selective advantage to the bacteriumThis research was financed by the Spanish Ministry of Science and Innovation (grants CSD2008-00013, INTERMODS, and BFU2010-19597, to M.E.), the European Union (grant EU-CP223111, CAREPNEUMO, to M.E.), Malaysian Ministry of Science, Technology and Innovation Science Fund grant 02-02-05-SF0026 and Malaysian Academy of Sciences grant SAGA/MUST/MZA/YCC-1 (M18) to C.C.Y., a Perdana scholarship (to W.T.C.), a Universiti Malaya fellowship (to W.T.C.), the University of Malaya [IPPP/UPDiT/Geran (PPP)/PS039/2007C and PS150-2009A, to W.T.C.], and also a European Molecular Biology Organization (EMBO) short-term fellowship (to W.T.C.)Peer reviewe