Grain shattering by cell death and fracture in Eragrostis tef.

Abscission, known as shattering in crop species, is a highly regulated process by which plants shed parts. Although shattering has been studied extensively in cereals and a number of regulatory genes have been identified, much diversity in the process remains to be discovered. Teff (Eragrostis tef) is a crop native to Ethiopia that is potentially highly valuable worldwide for its nutritious grain and drought tolerance. Previous work has suggested that grain shattering in Eragrostis might have little in common with other cereals. In this study, we characterize the anatomy, cellular structure, and gene regulatory control of the abscission zone (AZ) in E. tef. We show that the AZ of E. tef is a narrow stalk below the caryopsis, which is common in Eragrostis species. X-ray microscopy, scanning electron microscopy, transmission electron microscopy, and immunolocalization of cell wall components showed that the AZ cells are thin walled and break open along with programmed cell death (PCD) at seed maturity, rather than separating between cells as in other studied species. Knockout of YABBY2/SHATTERING1, documented to control abscission in several cereals, had no effect on abscission or AZ structure in E. tef. RNA sequencing analysis showed that genes related to PCD and cell wall modification are enriched in the AZ at the early seed maturity stage. These data show that E. tef drops its seeds using a unique mechanism. Our results provide the groundwork for understanding grain shattering in Eragrostis and further improvement of shattering in E. tef.

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

CRISPR/Cas9-mediated tetra-allelic mutation of the 'Green Revolution' SEMIDWARF-1 (SD-1) gene confers lodging resistance in tef (Eragrostis tef).

Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.

Cassava shrunken-2 homolog MeAPL3 determines storage root starch and dry matter content and modulates storage root postharvest physiological deterioration.

KEY MESSAGE: Among the five cassava isoforms (MeAPL1-MeAPL5), MeAPL3 is responsible for determining storage root starch content. Degree of storage root postharvest physiological deterioration (PPD) is directly correlated with starch content. AGPase is heterotetramer composed of two small and two large subunits each coded by small gene families in higher plants. Studies in cassava (Manihot esculenta) identified and characterized five isoforms of Manihot esculenta ADP-glucose pyrophosphorylase large subunit (MeAPL1-MeAPL5) and employed virus induced gene silencing (VIGS) to show that MeAPL3 is the key isoform responsible for starch and dry matter accumulation in cassava storage roots. Silencing of MeAPL3 in cassava through stable transgenic lines resulted in plants displaying significant reduction in storage root starch and dry matter content (DMC) and induced a distinct phenotype associated with increased petiole/stem angle, resulting in a droopy leaf phenotype. Plants with reduced starch and DMC also displayed significantly reduced or no postharvest physiological deterioration (PPD) compared to controls and lines with high DMC and starch content. This provides strong evidence for direct relationships between starch/dry matter content and its role in PPD and canopy architecture traits in cassava.

From Traditional Breeding to Genome Editing for Boosting Productivity of the Ancient Grain Tef [Eragrostis tef (Zucc.) Trotter].

Tef (Eragrostis tef (Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population and is currently cultivated in several countries for grain and forage production. It is one of the most nutritious grains, and is also more resilient to marginal soil and climate conditions than major cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during the growing season. Climate change is triggering several biotic and abiotic stresses which are expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative and robust approach in order to improve resilience to diverse types of stresses and increase crop yields. Traditional breeding has been extensively implemented to develop crop varieties with traits of interest, although the technique has several limitations. Currently, genome editing technologies are receiving increased interest among plant biologists as a means of improving key agronomic traits. In this review, the potential application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system. This is expected to improve stress resilience and boost productivity, thereby ensuring food and nutrition security in the region where it is needed the most.

Stacking disease resistance and mineral biofortification in cassava varieties to enhance yields and consumer health.

Delivering the benefits of agricultural biotechnology to smallholder farmers requires that resources be directed towards staple food crops. To achieve effect at scale, beneficial traits must be integrated into multiple, elite farmer-preferred varieties with relevance across geographical regions. The staple root crop cassava (Manihot esculenta) is consumed for dietary calories by more than 800 million people, but its tuberous roots provide insufficient iron and zinc to meet nutritional needs. In Africa, cassava yields are furthermore limited by the virus diseases, cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). In this study, we strove to develop cassava displaying high-level resistance to CBSD and CMD to attain food and economic security for cassava farmers, along with biofortified levels of iron and zinc to enhance consumer health. RNAi-mediated technology was used to achieve resistance to CBSD in two East African and one Nigerian farmer-preferred cultivars that harboured resistance to CMD. The Nigerian cvs. TMS 95/0505 and TMS 91/02324 were modified with T-DNA imparting resistance to CBSD, along with AtIRT1 (major iron transporter) and AtFER1 (ferritin) transgenes to achieve nutritionally significant levels of iron and zinc in cassava storage roots (145 and 40 µg/g dry weight, respectively). The inherent resistance to CMD was maintained in all four disease resistant and mineral enhanced cassava cultivars described here, demonstrating that this technique could be deployed across multiple farmer-preferred varieties to benefit the food and nutritional security of consumers in Africa.

Author Correction: Biofortification of field-grown cassava by engineering expression of an iron transporter and ferritin.

In the version of this article initially published, a relevant work was not cited. The following sentence has been inserted following the sentence ending "Aspergillus phytase" in the third paragraph of the article: "Overexpression of AtIRT1, AtNAS1 and bean FERRITIN in rice resulted in 3.8-fold higher iron and 1.8-fold higher zinc concentrations than in the wild-type control12." A corresponding reference has been added: 12. Boonyaves, K., Wu, T. Y., Gruissem, W. & Bhullar, N. K. Enhanced grain iron levels in rice expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN gene cassette. Front. Plant Sci. 8, 130 (2017). The error has been corrected in the HTML and PDF versions of the article.

Biofortification of field-grown cassava by engineering expression of an iron transporter and ferritin.

Less than 10% of the estimated average requirement (EAR) for iron and zinc is provided by consumption of storage roots of the staple crop cassava (Manihot esculenta Crantz) in West African human populations. We used genetic engineering to improve mineral micronutrient concentrations in cassava. Overexpression of the Arabidopsis thaliana vacuolar iron transporter VIT1 in cassava accumulated three- to seven-times-higher levels of iron in transgenic storage roots than nontransgenic controls in confined field trials in Puerto Rico. Plants engineered to coexpress a mutated A. thaliana iron transporter (IRT1) and A. thaliana ferritin (FER1) accumulated iron levels 7-18 times higher and zinc levels 3-10 times higher than those in nontransgenic controls in the field. Growth parameters and storage-root yields were unaffected by transgenic fortification in our field data. Measures of retention and bioaccessibility of iron and zinc in processed transgenic cassava indicated that IRT1 + FER1 plants could provide 40-50% of the EAR for iron and 60-70% of the EAR for zinc in 1- to 6-year-old children and nonlactating, nonpregnant West African women.

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

Multiple morphogenic culture systems cause loss of resistance to cassava mosaic disease.

BACKGROUND: Morphogenic culture systems are central to crop improvement programs that utilize transgenic and genome editing technologies. We previously reported that CMD2-type cassava (Manihot esculenta) cultivars lose resistance to cassava mosaic disease (CMD) when passed through somatic embryogenesis. As a result, these plants cannot be developed as products for deployment where CMD is endemic such as sub-Saharan Africa or the Indian sub-continent. RESULT: In order to increase understanding of this phenomenon, 21 African cassava cultivars were screened for resistance to CMD after regeneration through somatic embryogenesis. Fifteen cultivars were shown to retain resistance to CMD through somatic embryogenesis, confirming that the existing transformation and gene editing systems can be employed in these genetic backgrounds without compromising resistance to geminivirus infection. CMD2-type cultivars were also subjected to plant regeneration via caulogenesis and meristem tip culture, resulting in 25-36% and 5-10% of regenerated plant lines losing resistance to CMD respectively. CONCLUSIONS: This study provides clear evidence that multiple morphogenic systems can result in loss of resistance to CMD, and that somatic embryogenesis per se is not the underlying cause of this phenomenon. The information described here is critical for interpreting genomic, transcriptomic and epigenomic datasets aimed at understanding CMD resistance mechanisms in cassava.

Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch.

Storage roots of cassava (Manihot esculenta Crantz), a major subsistence crop of sub-Saharan Africa, are calorie rich but deficient in essential micronutrients, including provitamin A ß-carotene. In this study, ß-carotene concentrations in cassava storage roots were enhanced by co-expression of transgenes for deoxy-d-xylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin-type 1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 µg/g DW, 15- to 20-fold increases relative to roots from nontransgenic plants. Approximately 85%-90% of these carotenoids accumulated as all-trans-ß-carotene, the most nutritionally efficacious carotenoid. ß-Carotene-accumulating storage roots displayed delayed onset of postharvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in ß-carotene-enhanced storage roots. Most significantly, an inverse correlation was observed between ß-carotene and dry matter content, with reductions of 50%-60% of dry matter content in the highest carotenoid-accumulating storage roots of different cultivars. Further analysis confirmed a concomitant reduction in starch content and increased levels of total fatty acids, triacylglycerols, soluble sugars and abscisic acid. Potato engineered to co-express DXS and crtB displayed a similar correlation between ß-carotene accumulation, reduced dry matter and starch content and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed a reduced expression of genes involved in starch biosynthesis including ADP-glucose pyrophosphorylase genes in transgenic, carotene-accumulating cassava roots relative to nontransgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.

A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease.

BACKGROUND: Cassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12-18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12-22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus-induced gene silencing (VIGS) of an endogenous cassava gene. RESULTS: A VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV-K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA-A genomic component and used to inoculate cassava plants by Helios® Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1-VIGS resulted in shoot-tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2-4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1-VIGS. Significantly higher virus titers were detected in CMD-susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants. CONCLUSIONS: A rapid VIGS-based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2-4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for screening transgenic, gene edited and breeding lines under controlled growth conditions.

Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations.

Cassava brown streak disease (CBSD) presents a serious threat to cassava production in East and Central Africa. Currently, no cultivars with high levels of resistance to CBSD are available to farmers. Transgenic RNAi technology was employed to combat CBSD by fusing coat protein (CP) sequences from Ugandan cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) to create an inverted repeat construct (p5001) driven by the constitutive Cassava vein mosaic virus promoter. Twenty-five plant lines of cultivar TME 204 expressing varying levels of small interfering RNAs (siRNAs) were established in confined field trials (CFTs) in Uganda and Kenya. Within an initial CFT at Namulonge, Uganda, non-transgenic TME 204 plants developed foliar and storage root CBSD incidences at 96-100% by 12 months after planting. In contrast, 16 of the 25 p5001 transgenic lines showed no foliar symptoms and had less than 8% of their storage roots symptomatic for CBSD. A direct positive correlation was seen between levels of resistance to CBSD and expression of transgenic CP-derived siRNAs. A subsequent CFT was established at Namulonge using stem cuttings from the initial trial. All transgenic lines established remained asymptomatic for CBSD, while 98% of the non-transgenic TME 204 stake-derived plants developed storage roots symptomatic for CBSD. Similarly, very high levels of resistance to CBSD were demonstrated by TME 204 p5001 RNAi lines grown within a CFT over a full cropping cycle at Mtwapa, coastal Kenya. Sequence analysis of CBSD causal viruses present at the trial sites showed that the transgenic lines were exposed to both CBSV and UCBSV, and that the sequenced isolates shared >90% CP identity with transgenic CP sequences expressed by the p5001 inverted repeat expression cassette. These results demonstrate very high levels of field resistance to CBSD conferred by the p5001 RNAi construct at diverse agro-ecological locations, and across the vegetative cropping cycle.

A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease.

Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD.

Loss of CMD2-mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis.

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer-preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)-mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild-type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2-type varieties TME 3 and TME 7, but the CMD1-type cultivar TMS 30572 and the CMD3-type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2-mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field-level resistance in CMD2-type cultivars presently grown by farmers in East Africa, where CMD pressure is high.

CG gene body DNA methylation changes and evolution of duplicated genes in cassava.

DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

Overexpression of Arabidopsis VIT1 increases accumulation of iron in cassava roots and stems.

Iron is extremely abundant in the soil, but its uptake in plants is limited due to low solubility in neutral or alkaline soils. Plants can rely on rhizosphere acidification to increase iron solubility. AtVIT1 was previously found to be involved in mediating vacuolar sequestration of iron, which indicates a potential application for iron biofortification in crop plants. Here, we have overexpressed AtVIT1 in the starchy root crop cassava using a patatin promoter. Under greenhouse conditions, iron levels in mature cassava storage roots showed 3-4 times higher values when compared with wild-type plants. Significantly, the expression of AtVIT1 showed a positive correlation with the increase in iron concentration of storage roots. Conversely, young leaves of AtVIT1 transgenic plants exhibit characteristics of iron deficiency such as interveinal chlorosis of leaves (yellowing) and lower iron concentration when compared with the wild type plants. Interestingly, the AtVIT1 transgenic plants showed 4 and 16 times higher values of iron concentration in the young stem and stem base tissues, respectively. AtVIT1 transgenic plants also showed 2-4 times higher values of iron content when compared with wild-type plants, with altered partitioning of iron between source and sink tissues. These results demonstrate vacuolar iron sequestration as a viable transgenic strategy to biofortify crops and to help eliminate micronutrient malnutrition in at-risk human populations.

Efficient transmission of cassava brown streak disease viral pathogens by chip bud grafting.

BACKGROUND: Techniques to study plant viral diseases under controlled growth conditions are required to fully understand their biology and investigate host resistance. Cassava brown streak disease (CBSD) presents a major threat to cassava production in East Africa. No infectious clones of the causal viruses, Cassava brown streak virus (CBSV) or Ugandan cassava brown streak virus (UCBSV) are available, and mechanical transmission to cassava is not effective. An improved method for transmission of the viruses, both singly and as co-infections has been developed using bud grafts. FINDINGS: Axillary buds from CBSD symptomatic plants infected with virulent isolates of CBSV and UCBSV were excised and grafted onto 6-8 week old greenhouse-grown, disease-free cassava plants of cultivars Ebwanateraka, TME204 and 60444. Plants were assessed visually for development of CBSD symptoms and by RT-PCR for presence of the viruses in leaf and storage root tissues. Across replicated experiments, 70-100% of plants inoculated with CBSV developed CBSD leaf and stem symptoms 2-6 weeks after bud grafting. Infected plants showed typical, severe necrotic lesions in storage roots at harvest 12-14 weeks after graft inoculation. Sequential grafting of buds from plants infected with UCBSV followed 10-14 days later by buds carrying CBSV, onto the same test plant, resulted in 100% of the rootstocks becoming co-infected with both pathogens. This dual transmission rate was greater than that achieved by simultaneous grafting with UCBSV and CBSV (67%), or when grafting first with CBSV followed by UCBSV (17%). CONCLUSIONS: The bud grafting method described presents an improved tool for screening cassava germplasm for resistance to CBSD causal viruses, and for studying pathogenicity of this important disease. Bud grafting provides new opportunities compared to previously reported top and side grafting systems. Test plants can be inoculated as young, uniform plants of a size easily handled in a small greenhouse or large growth chamber and can be inoculated in a controlled manner with CBSV and UCBSV, either singly or together. Disease symptoms develop rapidly, allowing better studies of interactions between these viral pathogens, their movement within shoot and root systems, and how they induce their destructive disease symptoms.

Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors.

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the ß-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator.

The potential of using vector-free minimal gene cassettes (MGCs) with a double terminator for the enhancement and stabilization of transgene expression was tested in sugarcane biolistic transformation. The MGC system used consisted of the enhanced yellow fluorescent protein (EYFP) reporter gene driven by the maize ubiquitin-1 (Ubi) promoter and a single or double terminator from nopaline synthase (Tnos) or/and Cauliflower mosaic virus 35S (35ST). Transient EYFP expression from Tnos or 35ST single terminator MGC was very low and unstable, typically peaking early (8-16 h) and diminishing rapidly (48-72 h) after bombardment. Addition of a ~260 bp vector sequence (VS) to the single MGC downstream of Tnos (Tnos + VS) or 35ST (35ST + VS) enhanced EYFP expression by 1.25- to 25-fold. However, a much more significant increase in EYFP expression was achieved when the VS in 35ST + VS was replaced by Tnos to generate a 35ST-Tnos double terminator MGC, reaching its maximum at 24 h post-bombardment. The enhanced EYFP expression from the double terminator MGC was maintained for a long period of time (168 h), resulting in an overall increase of 5- to 65-fold and 10- to 160-fold as compared to the 35ST and Tnos single terminator MGCs, respectively. The efficiency of the double terminator MGC in enhancing EYFP expression was also demonstrated in sorghum and tobacco, suggesting that the underlying mechanism is highly conserved among monocots and dicots. Our results also suggest the involvement of posttranscriptional gene silencing in the reduced and unstable transgene expression from single terminator MGCs in plants.