Differences in the 3' intergenic region and the V2 protein of two sequence variants of tomato curly stunt virus play an important role in disease pathology in Nicotiana benthamiana.

Tomato production in South Africa is threatened by the emergence of tomato curly stunt virus (ToCSV), a monopartite Begomovirus transmitted by the whitefly vector Bemisia tabaci (Genn.). We investigated the role of sequence differences present in the 3' intergenic region (IR) and the V2 coding region on the differing infectivity of ToCSV sequence variant isolates V30 and V22 in the model host Nicotiana benthamiana. Using virus mutant chimeras, we determined that the development of the upward leaf roll symptom phenotype is mediated by sequence differences present in the 3' IR containing the TATA-associated composite element. Sequence differences present in the V2 coding region are responsible for modulating disease severity and symptom recovery in V22-infected plants. Serine substitution of V22 V2 Val27 resulted in a significant increase in disease severity with reduced recovery, the first study to demonstrate the importance of this V2 residue in disease development. Two putative ORFs, C5 and C6, were identified using in silico analysis and detection of an RNA transcript spanning their coding region suggests that these ORFs may be transcribed during infection. Additional virus-derived RNA transcripts spanning multiple ORFs and crossing the boundaries of recognised polycistronic transcripts, as well as the origin of replication within the IR, were detected in ToCSV-infected plants providing evidence of bidirectional readthrough transcription. From our results, we conclude that the diverse responses of the model host to ToCSV infection is influenced by select sequence differences and our findings provide several avenues for further investigation into the mechanisms behind these responses to infection.

WRKY Transcription Factors in Cassava Contribute to Regulation of Tolerance and Susceptibility to Cassava Mosaic Disease through Stress Responses.

Among the numerous biological constraints that hinder cassava (Manihot esculenta Crantz) production, foremost is cassava mosaic disease (CMD) caused by virus members of the family Geminiviridae, genus Begomovirus. The mechanisms of CMD tolerance and susceptibility are not fully understood; however, CMD susceptible T200 and tolerant TME3 cassava landraces have been shown to exhibit different large-scale transcriptional reprogramming in response to South African cassava mosaic virus (SACMV). Recent identification of 85 MeWRKY transcription factors in cassava demonstrated high orthology with those in Arabidopsis, however, little is known about their roles in virus responses in this non-model crop. Significant differences in MeWRKY expression and regulatory networks between the T200 and TME3 landraces were demonstrated. Overall, WRKY expression and associated hormone and enriched biological processes in both landraces reflect oxidative and other biotic stress responses to SACMV. Notably, MeWRKY11 and MeWRKY81 were uniquely up and downregulated at 12 and 67 days post infection (dpi) respectively in TME3, implicating a role in tolerance and symptom recovery. AtWRKY28 and AtWRKY40 homologs of MeWRKY81 and MeWRKY11, respectively, have been shown to be involved in regulation of jasmonic and salicylic acid signaling in Arabidopsis. AtWRKY28 is an interactor in the RPW8-NBS resistance (R) protein network and downregulation of its homolog MeWRKY81 at 67 dpi in TME3 suggests a negative role for this WRKY in SACMV tolerance. In contrast, in T200, nine MeWRKYs were differentially expressed from early (12 dpi), middle (32 dpi) to late (67 dpi) infection. MeWRKY27 (homolog AtWRKY33) and MeWRKY55 (homolog AtWRKY53) were uniquely up-regulated at 12, 32 and 67 dpi in T200. AtWRKY33 and AtWRKY53 are positive regulators of leaf senescence and oxidative stress in Arabidopsis, suggesting MeWRKY55 and 27 contribute to susceptibility in T200.

Comparative analysis of virus-derived small RNAs within cassava (Manihot esculenta Crantz) infected with cassava brown streak viruses.

Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21-24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5'-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD.

Diversity of dicotyledenous-infecting geminiviruses and their associated DNA molecules in southern Africa, including the South-west Indian ocean islands.

The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-ßs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.

Genetic diversity and geographic distribution of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) genotypes associated with cassava in East Africa.

The genetic variability of whitefly (Bemisia tabaci) species, the vectors of cassava mosaic begomoviruses (CMBs) in cassava growing areas of Kenya, Tanzania, and Uganda, was investigated through comparison of partial sequences of the mitochondria cytochrome oxidase I (mtCOI) DNA in 2010/11. Two distinct species were obtained including sub-Saharan Africa 1 (SSA1), comprising of two sub-clades (I and II), and a South West Indian Ocean Islands (SWIO) species. Among the SSA1, sub-clade I sequences shared a similarity of 97.8-99.7% with the published Uganda 1 genotypes, and diverged by 0.3-2.2%. A pairwise comparison of SSA1 sub-clade II sequences revealed a similarity of 97.2-99.5% with reference southern Africa genotypes, and diverged by 0.5-2.8%. The SSA1 sub-clade I whiteflies were widely distributed in East Africa (EA). In comparison, the SSA1 sub-clade II whiteflies were detected for the first time in the EA region, and occurred predominantly in the coast regions of Kenya, southern and coast Tanzania. They occurred in low abundance in the Lake Victoria Basin of Tanzania and were widespread in all four regions in Uganda. The SWIO species had a sequence similarity of 97.2-97.7% with the published Reunion sequence and diverged by 2.3-2.8%. The SWIO whiteflies occurred in coast Kenya only. The sub-Saharan Africa 2 whitefly species (Ug2) that was associated with the severe CMD pandemic in Uganda was not detected in our study.

Construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific PCR.

RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.