Incidence of RNA viruses infecting taro and tannia in East Africa and molecular characterisation of dasheen mosaic virus isolates.

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) plants growing in 25 districts across Ethiopia, Kenya, Tanzania and Uganda were surveyed for four RNA viruses. Leaf samples from 392 plants were tested for cucumber mosaic virus (CMV), dasheen mosaic virus (DsMV), taro vein chlorosis virus (TaVCV) and Colocasia bobone disease-associated virus (CBDaV) by RT-PCR. No samples tested positive for TaVCV or CBDaV, while CMV was only detected in three tannia samples with mosaic symptoms from Uganda. DsMV was detected in 40 samples, including 36 out of 171 from Ethiopia, one out of 94 from Uganda and three out of 41 from Tanzania, while none of the 86 samples from Kenya tested positive for any of the four viruses. The complete genomes of nine DsMV isolates from East Africa were cloned and sequenced. Phylogenetic analyses based on the amino acid sequence of the DsMV CP-coding region revealed two distinct clades. Isolates from Ethiopia were distributed in both clades, while samples from Uganda and Tanzania belong to different clades. Seven possible recombination events were identified from the analysis carried out on the available 15 full-length DsMV isolates. Nucleotide substitution ratio analysis revealed that all the DsMV genes are under strong negative selection pressure.

Infectivity of an Infectious Clone of Banana Streak CA Virus in A-Genome Bananas (Musa acuminata ssp.).

We have characterized the complete genome sequence of an Australian isolate of banana streak CA virus (BSCAV). A greater-than-full-length, cloned copy of the virus genome was assembled and agroinoculated into five tissue-cultured plants of nine different Musa acuminata banana accessions. BSCAV was highly infectious in all nine accessions. All five inoculated plants from eight accessions developed symptoms by 28 weeks post-inoculation, while all five plants of M. acuminata AA subsp. zebrina remained symptomless. Symptoms were mild in six accessions but were severe in Khae Phrae (M. acuminata subsp. siamea) and the East African Highland banana accession Igisahira Gisanzwe. This is the first full-length BSCAV genome sequence reported from Australia and the first report of the infectivity of an infectious clone of banana streak virus.

Molecular characterisation of a putative new polerovirus infecting pumpkin (Cucurbita pepo) in Kenya.

Next-generation sequencing of RNA extracted from a pumpkin plant with mosaic symptoms in Kenya identified the presence of a polerovirus sequence closely related to pepo aphid-borne yellows virus (PABYV). The near-complete polerovirus sequence comprised 5,810 nucleotides and contained seven putative open reading frames (ORFs) with a genome organisation typical of poleroviruses. BLASTp analysis of the translated sequences of ORFs 0, 1 and 2 revealed that their amino acid sequences differed by more than 10% from the corresponding protein sequences of other poleroviruses. These results suggest that this virus is a putative novel member of the genus Polerovirus, which has been provisionally named "pumpkin polerovirus" (PuPV).

Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses.

The genus Badnavirus is characterized by members that are genetically and serologically heterogeneous which presents challenges for their detection and characterization. The presence of integrated badnavirus-like sequences in some host species further complicates detection using PCR-based protocols. To address these challenges, we have assessed and optimized various RCA protocols including random-primed RCA (RP-RCA), primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-RCA) and specific-primed RCA (SP-RCA). Using Dioscorea bacilliform AL virus (DBALV) as an example, we demonstrate that viral DNA amplified using the optimized D-RCA and SP-RCA protocols showed an 85-fold increase in badnavirus NGS reads compared with RP-RCA. The optimized RCA techniques described here were used to detect a range of badnaviruses infecting banana, sugar cane, taro and yam demonstrating the utility of RCA for detection of diverse badnaviruses infecting a variety of host plant species.

Characterization of a novel member of the family Caulimoviridae infecting Dioscorea nummularia in the Pacific, which may represent a new genus of dsDNA plant viruses.

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic analysis, DNUaV appears to be a distinct novel member of the family and may represent a new genus.

Characterization of an Australian isolate of taro bacilliform virus and development of an infectious clone.

The badnavirus taro bacilliform virus (TaBV) has been reported to infect taro (Colocasia esculenta L.) and other edible aroids in several South Pacific island countries, but there are no published reports from Australia. Using PCR and RCA, we identified and characterized an Australian TaBV isolate. A terminally redundant cloned copy of the TaBV genome was generated and shown to be infectious in taro following agro-inoculation. This is the first report of TaBV from Australia and also the first report of an infectious clone for this virus.

The Quest for Golden Bananas: Investigating Carotenoid Regulation in a Fe'i Group Musa Cultivar.

The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fe'i group Musa cultivar, "Asupina", has been examined and compared to that of a low-carotenoid-accumulating cultivar, "Cavendish", to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in "Cavendish" and the conversion of amyloplasts to chromoplasts during fruit ripening in "Asupina". Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in "Cavendish" fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.

Complete genome sequence of a novel zantedeschia mild mosaic virus isolate: the first report from Australia and from Alocasia sp.

The complete genome of an Australian isolate of zantedeschia mild mosaic virus (ZaMMV) causing mosaic symptoms on Alocasia sp. (designated ZaMMV-AU) was cloned and sequenced. The genome comprises 9942 nucleotides (excluding the poly-A tail) and encodes a polyprotein of 3167 amino acids. The sequence is most closely related to a previously reported ZaMMV isolate from Taiwan (ZaMMV-TW), with 82 and 86 % identity at the nucleotide and amino acid level, respectively. Unlike the amino acid sequence of ZaMMV-TW, however, ZaMMV-AU does not contain a polyglutamine stretch at the N-terminus of the coat-protein-coding region upstream of the DAG motif. This is the first report of ZaMMV from Australia and from Alocasia sp.

Iron absorption in raw and cooked bananas: a field study using stable isotopes in women.

BACKGROUND: Banana is a staple food in many regions with high iron deficiency and may be a potential vehicle for iron fortification. However, iron absorption from bananas is not known. OBJECTIVE: The objective of this study was to evaluate total iron absorption from raw and cooked bananas. DESIGN: Thirty women (34.9±6.6 years) from rural Mexico were randomly assigned to one of two groups each consuming: 1) 480 g/day of raw banana for 6 days, or 2) 500 g/day of cooked banana for 4 days. Iron absorption was measured after extrinsically labeling with 2 mg of (58)Fe and a reference dose of 6 mg (57)Fe; analysis was done using ICP-MS. RESULTS: Iron content in cooked bananas was significantly higher than raw bananas (0.53 mg/100 g bananas vs. 0.33 mg/100 mg bananas, respectively) (p<0.001). Percent iron absorption was significantly higher in raw bananas (49.3±21.3%) compared with cooked banana (33.9±16.2%) (p=0.035). Total amount of iron absorbed from raw and cooked bananas was similar (0.77±0.33 mg vs. 0.86±0.41 mg, respectively). CONCLUSION: Total amount of absorbed iron is similar between cooked and raw bananas. The banana matrix does not affect iron absorption and is therefore a potential effective target for genetic modification for iron biofortification.

The extremophile Nicotiana benthamiana has traded viral defence for early vigour.

A single lineage of Nicotiana benthamiana is widely used as a model plant(1) and has been instrumental in making revolutionary discoveries about RNA interference (RNAi), viral defence and vaccine production. It is peerless in its susceptibility to viruses and its amenability in transiently expressing transgenes(2,3). These unparalleled characteristics have been associated both positively and negatively with a disruptive insertion in the RNA-dependent RNA polymerase 1 gene, Rdr1(4-6). For a plant so routinely used in research, the origin, diversity and evolution of the species, and the basis of its unusual abilities, have been relatively unexplored. Here, by comparison with wild accessions from across the spectrum of the species' natural distribution, we show that the laboratory strain of N. benthamiana is an extremophile originating from a population that has retained a mutation in Rdr1 for ∼0.8 Myr and thereby traded its defence capacity for early vigour and survival in the extreme habitat of central Australia. Reconstituting Rdr1 activity in this isolate provided protection. Silencing the functional allele in a wild strain rendered it hypersusceptible and was associated with a doubling of seed size and enhanced early growth rate. These findings open the way to a deeper understanding of the delicate balance between protection and vigour.

Updates in inducible transgene expression using viral vectors: from transient to stable expression.

The prospect of economically producing useful biologics in plants has greatly increased with the advent of viral vectors. The ability of viral vectors to amplify transgene expression has seen them develop into robust transient platforms for the high-level, rapid production of recombinant proteins. To adapt these systems to stably transformed plants, new ways of deconstructing the virus machinery and linking its expression and replication to chemically controlled promoters have been developed. The more advanced of these stable, inducible hyper-expression vectors provide both activated and amplified heterologous transgene expression. Such systems could be deployed in broad acre crops and provide a pathway to fully exploit the advantages of plants as a platform for the manufacture of a wide spectrum of products.

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse.

BACKGROUND: The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. RESULTS: Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. CONCLUSIONS: The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane.

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

Improved molecular tools for sugar cane biotechnology.

Sugar cane is a major source of food and fuel worldwide. Biotechnology has the potential to improve economically-important traits in sugar cane as well as diversify sugar cane beyond traditional applications such as sucrose production. High levels of transgene expression are key to the success of improving crops through biotechnology. Here we describe new molecular tools that both expand and improve gene expression capabilities in sugar cane. We have identified promoters that can be used to drive high levels of gene expression in the leaf and stem of transgenic sugar cane. One of these promoters, derived from the Cestrum yellow leaf curling virus, drives levels of constitutive transgene expression that are significantly higher than those achieved by the historical benchmark maize polyubiquitin-1 (Zm-Ubi1) promoter. A second promoter, the maize phosphonenolpyruvate carboxylate promoter, was found to be a strong, leaf-preferred promoter that enables levels of expression comparable to Zm-Ubi1 in this organ. Transgene expression was increased approximately 50-fold by gene modification, which included optimising the codon usage of the coding sequence to better suit sugar cane. We also describe a novel dual transcriptional enhancer that increased gene expression from different promoters, boosting expression from Zm-Ubi1 over eightfold. These molecular tools will be extremely valuable for the improvement of sugar cane through biotechnology.

An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane.

Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures.

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5L (15-25FPU/g glucan) was in the following order: glycerol/HCl>HCl>H2SO4>NaOH, with the glycerol system achieving ≈ 100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6-6%) obtained with mixtures of endo-1,4-ß-glucanase (EG) and ß-glucosidase (ßG) for the NaOH substrate were 2-6 times that of acid substrates. However, glucan conversions (15-60%) obtained with mixtures of cellobiohydrolase (CBH I) and ßG on acidified glycerol substrate were 10-30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.

In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants.

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the ß-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes.

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

Apoptosis-related genes confer resistance to Fusarium wilt in transgenic 'Lady Finger' bananas.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.