Dataset Information

Assessment of RNAi-induced silencing in banana (Musa spp.).

ABSTRACT:

Background

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana.

Results

Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt).

Conclusions

RNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

SUBMITTER: Dang TV

PROVIDER: S-EPMC4177175 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Publications

Assessment of RNAi-induced silencing in banana (Musa spp.).

Dang Tuong Vi T TV Windelinckx Saskia S Henry Isabelle M IM De Coninck Barbara B Cammue Bruno P A BP Swennen Rony R Remy Serge S

BMC research notes 20140918

<h4>Background</h4>In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful ...[more]

PMID: 25230584

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Project description:BackgroundBananas (Musa spp.) are a globally significant crop and are severely afflicted by diseases for which there are no effective chemical controls. Banana microbiomes may provide novel solutions to these constraints but are difficult to manage due to their high diversity and variability between locations. Hence 'common core' taxa, which are a subset of the microbiome that frequent all, or most, individuals of a host species, represent logical targets for the development of microbiome management approaches. Here, we first performed a pot experiment to characterise the effects of two factors that are likely to differ between farms (viz. edaphic conditions and host genotype) on bacterial diversity in bulk soil and seven plant compartments. From this experiment, we created shortlisted core 'candidates' that were then refined using a survey of 52 field-grown Musa spp. We confirmed the importance of the core through network analysis and by comparing the sequences of our core taxa with those reported in 22 previous studies.ResultsDiversity was found to differ between plant compartments and soils, but not genotypes. Therefore, we identified populations that were frequent across most plants irrespective of the soil in which they were grown. This led to the selection of 36 'common core' bacteria, that represented 65-95% of the dominant taxa in field-grown plants and were identified as highly interconnected 'hubs' using network analysis - a characteristic shown to be indicative of microbes that influence host fitness in studies of other plants. Lastly, we demonstrated that the core taxa are closely related to banana-associated bacteria observed on five other continents.ConclusionsOur study provides a robust list of common core bacterial taxa for Musa spp. Further research may now focus on how changes in the frequencies and activities of these most persistent taxa influence host fitness. Notably, for several of our core taxa, highly similar populations have already been isolated in previous studies and may be amenable to such experimentation. This contribution should help to accelerate the development of effective Musa spp. microbiome management practices.

Project description:The "Cavendish" and "Prata" subgroups represent respectively 47% and 24% of the world banana production. Compared to world average progressing from 10.6 to 20.6 t ha-1 between 1961 and 2016, and despite sustained domestic demand and the introduction of new cultivars, banana yield in Brazil has stagnated around 14.5 t ha-1 mainly due to nutrient and water mismanagement. "Prata" is now the dominant subgroup in N-E Brazil and is fertigated at high costs. Nutrient balances computed as isometric log-ratios (ilr) provide a comprehensive understanding of nutrient relationships in the diagnostic leaf at high yield level by combining raw concentration data. Although the most appropriate method for multivariate analysis of compositional balances may be less efficient due to non-normal data distribution and limited nutrient mobility in the plant, robustness of the nutrient balance approach could be improved using Box-Cox exponents assigned to raw foliar concentrations. Our objective was to evaluate the accuracy of nutrient balances to diagnose fertigated "Prata" orchards. The dataset comprised 609 observations on fruit yields and leaf tissue compositions collected from 2010 to 2016 in Ceará state, N-E Brazil. Raw nutrient concentration ranges were ineffective as diagnostic tool due to considerable overlapping of concentration ranges for low- and high-yielding subpopulations at cutoff yield of 40 Mg ha-1. Nutrient concentrations were combined into isometric log-ratios (ilr) and normalized by Box-Cox corrections between 0 and 1 which may also account for restricted nutrient transfer from leaf to fruit. Despite reduced ilr skewness, Box-Cox coefficients did not improve model robustness measured as the accuracy of the Cate-Nelson partition between yield and the multivariate distance across ilr values. Sensitivity was 94%, indicating that low yields are attributable primarily to nutrient imbalance. There were 148 false-positive specimens (high yield despite nutrient imbalance) likely due to suboptimal nutrition, contamination, or luxury consumption. The profitability of "Prata" orchards could be enhanced by rebalancing nutrients using ilr standards with no need for Box-Cox correction.

Dataset Information

Assessment of RNAi-induced silencing in banana (Musa spp.).

Background

Results

Conclusions

Publications

Assessment of RNAi-induced silencing in banana (Musa spp.).

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