CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5′ and or downstream of the 3′ targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6–8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.

Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (ΔCP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length ΔCP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718-005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Cross-protection against CBSV by siRNAs generated from the full-length UCBSV ΔCP confirms a previous report in tobacco. The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease.

The banana Xanthomonas wilt disease (BXW) has threatened the livelihood of millions of farmers in East Africa. Use of resistant varieties is the most cost-effective method of managing this bacterial disease. A reliable and rapid screening method is needed to select resistant banana varieties. An in vitro screening method was developed for early evaluation of Xanthomonas wilt resistance using small tissue culture-grown plantlets. Eight cultivars of banana were screened with sixteen isolates of Xanthomonas campestris pv. musacearum using this method. There were significant differences (P<0.0001) in susceptibility among the various banana cultivars tested, whereas no significant difference (P=0.92) in pathogenicity was observed between the pathogen isolates. The cv. Pisang Awak (Kayinja) was found to be highly susceptible and Musa balbisiana resistant. Nakitembe was found to be moderately resistant while cvs Mpologoma, Mbwazirume, Sukali Ndiizi, FHIA-17 and FHIA-25 were susceptible. The susceptibility of these cultivars was further tested in vivo by artificial inoculation of potted plants with similar results. This study shows that an in vitro screening test can serve as a convenient, cheap and rapid screening technique to discriminate BXW-resistant from BXW-susceptible banana cultivars.

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.

Cassava brown streak disease (CBSD) is currently considered the most important disease of cassava in East Africa and a significant threat to food security in the region. 1 CBSD is caused by two distinct virus species: Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), both of which belong to the genus Ipomovirus, family Potyviridae, and possess a ssRNA genome of messenger sense.2,3 Both viruses are transmitted by whiteflies (Bemisia tabaci), resulting in CBSDsymptomatic plants carrying infections by one or both viral species. Since its re-emergence in Uganda in 2004, 4 CBSD has become prevalent in East Africa, with new outbreaks of the disease resulting from dissemination of infected cassava planting materials and high whitefly vector populations. 4,5 Genetically engineered resistance to CBSD was reported recently in tobacco and cassava plants under greenhouse conditions 6,7 and in cassava in a confined field trial (CFT) in Uganda. 8 Two RNAi constructs targeting UCBSV were tested in the field: p718 consisting of an 894 bp inverted repeat sequence (nts 208-1101) of a truncated version of the full-length UCBSV coat protein (CP), and p719 consisting of a 397 bp portion (nts 208-604) of the UCBSV CP N-terminal.7 Tissue culturederived cassava plants of cv 60444 shown to be accumulating transgenically derived UCBSV CP specific siRNAs, were planted in the field and proved to be highly resistant to whitefly transmitted UCBSV. Fourteen transgenic lines were tested, seven of each of the two constructs. Across all 14 lines, greater than 98% of the plants (n = 60) remained free of UCBSV 11 months after planting (MAP), as determined by RT-PCR. Protection against the non-targeted CBSV was observed in two transgenic lines of the p718 construct (carrying the full length CP sequence). This was most striking in line p718-001 in which 54 out of 60 plants (90%) remained CBSD symptom free and RT-PCR negative for Keywords: cassava brown streak disease, RNAi-mediated resistance, cassava, transgenic, confined field trial, UgandaAbbreviations: CBSD, Cassava brown streak disease; CBSV, Cassava brown streak virus; CFT, confined field trial; CMD, cassava mosaic disease; CP, coat protein; MAP, months after planting; NaCRRI, National Crops Resources Research Institute; RT-PCR, reverse transcription polymerase chain reaction; siRNA, small interfering RNA; ssRNA, single-stranded RNA; UCBSV, Uganda Cassava brown streak virus a confined field trial was established to determine durability of RNai-mediated resistance to cassava brown streak disease (cBsD). stem cuttings were obtained from field-grown cassava plants of cv 60444 transgenic for construct p718, consisting of an 894 bp inverted repeat sequence from the Ugandan cassava brown streak virus (UcBsV) coat protein.Plants were established from three transgenic lines previously shown to provide complete resistance to UcBsV and differing levels of protection to the non-homologous virus species cassava brown streak virus (cBsV), and grown for 11 months. cBsD sympto...

Endogenous FLOWERING LOCUS T homolog MeFT1 was transgenically overexpressed under control of a strong constitutive promoter in cassava cultivar 60444 to determine its role in regulation of flowering and as a potential tool to accelerate cassava breeding. Early profuse flowering was recorded in-vitro in all ten transgenic plant lines recovered, causing eight lines to die within 21 days of culture. The two surviving transgenic plant lines flowered early and profusely commencing as soon as 14 days after establishment in soil in the greenhouse. Both transgenic lines sustained early flowering across the vegetative propagation cycle, with first flowering recorded 30-50 days after planting stakes compared to 90 days for non-transgenic controls. Transgenic plant lines completed five flowering cycles within 200 days in the greenhouse as opposed to twice flowering event in the controls. Constitutive overexpression of MeFT1 generated fully mature male and female flowers and produced a bushy phenotype due to significantly increased flowering-induced branching. Flower induction by MeFT1 overexpression was not graft-transmissible and negatively affected storage root development. Accelerated flowering in transgenic plants was associated with significantly increased mRNA levels of MeFT1 and the three floral meristem identity genes MeAP1, MeLFY and MeSOC1 in shoot apical tissues. These findings imply that MeFT1 encodes flower induction and triggers flowering by recruiting downstream floral meristem identity genes. OPEN ACCESS Citation: Odipio J, Getu B, Chauhan RD, Alicai T, Bart R, Nusinow DA, et al. (2020) Transgenic overexpression of endogenous FLOWERING LOCUS T-like gene MeFT1 produces early flowering in cassava. PLoS ONE 15(1): e0227199.

The Random Amplification of Polymorphic DNA (RAPD) analysis was used to detect the genetic diversity among Ugandan isolates of Xanthomonas campestris pv. musacearum (Xcm), the causal agent of banana Xanthomonas wilt (BXW) disease. Seven random primers were used because of their ability to amplify reproducible and reliable fingerprints generated between 6-12 amplicons each from the Xcm isolates obtained from central core of pseudostems, peduncles, fruit peelings, sap, nectar, insects' bodies and bacterial oozes. Regardless of the source and geographical origin, similar fingerprints were generated from the tested isolates. Using a similarity coefficient of 58%, the unweighted pair group method with arithmetic averaging (UPGMA) analysis did not reveal any significant differences in clustering, with exception of a single isolate that had unique fingerprints. Prior to the genetic analysis, all the isolates compared showed no significant difference (P = 0.92) with regard to incubation period for appearance of symptoms and the severity of symptoms in pathogenicity test. Thus, our data indicates that the population of Xcm in Uganda is clonal, that is, one uniform population being spread fast and efficiently, suggesting that there is a low likelihood of the current population to rapidly evolve, in the near future, into more virulent strains to overcome any resistance deployed.