Short interfering RNA accumulation correlates with host recovery in DNA virus-infected hosts, and gene silencing targets specific viral sequences. (33/222)

Viruses are both inducers and targets of posttranscriptional gene silencing (PTGS), a natural defense mechanism in plants. Here we report molecular evidence of the ability of single-stranded DNA (ssDNA) viruses to induce PTGS in infected plants irrespective of the severity of or recovery from the symptoms. Our results reveal that five distinct species of cassava-infecting geminiviruses were capable of triggering PTGS by producing two classes of virus-specific short interfering RNAs (siRNAs) of 21 to 26 nucleotides in two plant hosts, tobacco (Nicotiana benthamiana) and cassava (Manihot esculenta, Crantz). However, the efficacy of virus-induced PTGS varied depending on the intrinsic features of the virus and its interaction with the plant host. We found that symptom recovery over time in plants infected with the isolates of African cassava mosaic virus (ACMV-[CM]) or Sri Lankan cassava mosaic virus was associated with a much higher level of virus-derived siRNA accumulation compared to plants infected with viruses that do not show symptom recovery. Furthermore, we determined that the C terminus of AC1 that overlaps with the N terminus of AC2 early viral genes involved in virus replication were the primary targets for ACMV-[CM]-induced PTGS, whereas the C terminus of BC1 was targeted for the East African cassava mosaic Cameroon virus. In addition, our results reveal the possibility for double-stranded RNA formation during transcription in ssDNA viruses, which explains in part how these viruses can trigger PTGS in plants.  (+info)

Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing. (34/222)

Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by approximately 8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by approximately 8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.  (+info)

Response of cassava leaf area expansion to water deficit: cell proliferation, cell expansion and delayed development. (35/222)

BACKGROUND AND AIMS: Cassava (Manihot esculenta) is an important food crop in the tropics that has a high growth rate in optimal conditions, but also performs well in drought-prone climates. The objectives of this work were to determine the effects of water deficit and rewatering on the rate of expansion of leaves at different developmental stages and to evaluate the extent to which decreases in cell proliferation, expansion, and delay in development are responsible for reduced growth. METHODS: Glasshouse-grown cassava plants were subjected to 8 d of water deficit followed by rewatering. Leaves at 15 developmental stages from nearly full size to meristematic were sampled, and epidermal cell size and number were measured on leaves at four developmental stages. KEY RESULTS: Leaf expansion and development were nearly halted during stress but resumed vigorously after rewatering. In advanced-stage leaves (Group 1) in which development was solely by cell expansion, expansion resumed after rewatering, but not sufficiently for cell size to equal that of controls at maturity. In Group 2 (cell proliferation), relative expansion rate and cell proliferation were delayed until rewatering, but then recovered partially, so that loss of leaf area was due to decreased cell numbers per leaf. In Group 3 (early meristematic development) final leaf area was not affected by stress, but development was delayed by 4-6 d. On a plant basis, the proportion of loss of leaf area over 26 d attributed to leaves at each developmental stage was 29, 50 and 21 % in Group 1, 2 and 3, respectively. CONCLUSIONS: Although cell growth processes were sensitive to mild water deficit, they recovered to a large extent, and much of the reduction in leaf area was caused by developmental delay and a reduction in cell division in the youngest, meristematic leaves.  (+info)

Determination of total antioxidant activity in three types of local vegetables shoots and the cytotoxic effect of their ethanolic extracts against different cancer cell lines. (36/222)

Antioxidants play an important role in inhibiting and scavenging radicals, thus providing protection to humans against infections and degenerative diseases. Literature shows that the antioxidant activity is high on herbal and vegetable plants. Realizing the fact, this research was carried out to determine total antioxidant activity and the potential anticancer properties in three types of selected local vegetable shoots such as Diplazium esculentum (paku shoot), Manihot utillissima (tapioca shoot) and Sauropous androgynus (cekur manis). The research was also done to determine the effect of boiling, on total antioxidant activity whereby samples of fresh shoots are compared with samples of boiled shoots. In every case, antioxidant activity is compared to alpha-tocopherol and two methods of extraction used are the organic and the aqueous methods. Besides that, two research methods used were the ferric thiocyanate (FTC) and thiobarbituric acid (TBA) with absorbance of 500nm and 532nm respectively. Oneway ANOVA test at P<0.05 determines significant differences between various samples. In the cytotoxic study, the ethanolic extract and several cell lines i.e. breast cancer (MDA-MB-231 and MCF-7), colon cancer (Caco-2), liver cancer (HepG2) and normal liver (Chang liver) were used. The IC(50)-value was determined by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The antioxidant study found that all the samples in both aqueous and organic extraction were significantly different. The total antioxidant activity values of aqueous extract in descending order are as follows: M. utilissima (fresh) >D. esculentum (fresh) >S.androgynus (fresh) > M.utilissima (boiled) > D. esculentum (boiled) > S.androgynus (boiled). It also was found that S.androgynus shoots ethanolic extract was able to inhibit the viability of the breast cancer cell lines, MDA-MB-231 with the IC50 value of 53.33 micrograms/ml. However, S.androgynus shoots and D. esculentum shoots ethanolic extracts did not inhibit the viability of MDA-MB-231 cell line. While, the tapioca shoot ethanolic extract was able to inhibit the viability of MCF-7 cell line with the IC(50) value of 52.49 micrograms/ml. S.androgynus shoots and D.esculentum shoots ethanolic extracts did not give an IC(50) value against the MCF-7 cell line. S.androgynus, tapioca and D.esculentum shoots ethanolic extracts did not show cytotoxic effect against the Caco-2 and HepG2. There was no IC(50)-value from any sample against Chang Liver cell line. In conclusion, the antioxidant activity of both fresh and boiled samples were higher than alpha-tocopherol, although fresh vegetable shoots were found to be higher in antioxidant activity compared to boiled shoots. This study also suggested that S.androgynus shoots and tapioca shoots have potential as an anticancer agent against certain breast tumours.  (+info)

Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes. (37/222)

Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.  (+info)

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses. (38/222)

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.  (+info)

Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues. (39/222)

BACKGROUND: Plant viral diseases present major constraints to crop production. Effective sampling of the viruses infecting plants is required to facilitate their molecular study and is essential for the development of crop protection and improvement programs. Retaining integrity of viral pathogens within sampled plant tissues is often a limiting factor in this process, most especially when sample sizes are large and when operating in developing counties and regions remote from laboratory facilities. FTA is a paper-based system designed to fix and store nucleic acids directly from fresh tissues pressed into the treated paper. We report here the use of FTA as an effective technology for sampling and retrieval of DNA and RNA viruses from plant tissues and their subsequent molecular analysis. RESULTS: DNA and RNA viruses were successfully recovered from leaf tissues of maize, cassava, tomato and tobacco pressed into FTA Classic Cards. Viral nucleic acids eluted from FTA cards were found to be suitable for diagnostic molecular analysis by PCR-based techniques and restriction analysis, and for cloning and nucleotide sequencing in a manner equivalent to that offered by tradition isolation methods. Efficacy of the technology was demonstrated both from sampled greenhouse-grown plants and from leaf presses taken from crop plants growing in farmer's fields in East Africa. In addition, FTA technology was shown to be suitable for recovery of viral-derived transgene sequences integrated into the plant genome. CONCLUSION: Results demonstrate that FTA is a practical, economical and sensitive method for sampling, storage and retrieval of viral pathogens and plant genomic sequences, when working under controlled conditions and in the field. Application of this technology has the potential to significantly increase ability to bring modern analytical techniques to bear on the viral pathogens infecting crop plants.  (+info)

Epistasis in the expression of relevant traits in cassava (Manihot esculenta Crantz) for subhumid conditions. (40/222)

There is limited knowledge on the inheritance of agronomic traits in cassava and the importance of epistasis for most crops. A nine-parent diallel study was conducted in subhumid environments. Thirty clones were obtained from each F1 cross. Each clone was represented by six plants, which were distributed in three replications at two locations. Therefore the same 30 genotypes of each F1 cross were planted in the three replications at the two locations. Analysis of variance suggested significant genetic effects for all variables analyzed (reaction to thrips, fresh root and foliage yields, harvest index, dry matter content, and root dry matter yield). Significant epistatic effects were observed for all variables, except harvest index. Dominance variance was always significant, except for dry matter content and dry matter yield. Additive variance was significant only for reaction to thrips. Results suggested that dominance plays an important role in complex traits such as root yield. The significance of epistasis can help us understand the difficulties of quantitative genetics models and QTLs in satisfactorily explaining phenotypic variation in traits with complex inheritance. Significant epistasis would justify the production of inbred parental lines to fix favorable allele combinations in the production of hybrid cassava cultivars.  (+info)