Endogenous small-noncoding RNAs and their roles in chilling response and stress acclimation in Cassava

BACKGROUND: Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs. RESULTS: In order to understand sncRNAs’ roles in plant chilling response and stress acclimation, we performed a comprehensive study of miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate chilling stresses, that underwent further severe chilling stress after chilling acclimation at moderate stress, and that grew under the normal condition. We also included castor bean (Ricinus communis) in our study to understand conservation of sncRNAs. In addition to known miRNAs, we identified 32 (22 and 10) novel miRNAs as well as 47 (26 and 21) putative secondary siRNA-yielding and 8 (7 and 1) nat-siRNA-yielding candidate loci in Cassava and castor bean, respectively. Among the expressed sncRNAs, 114 miRNAs, 12 ta-siRNAs and 2 nat-siRNAs showed significant expression changes under chilling stresses. CONCLUSION: Systematic and computational analysis of microRNAome and experimental validation collectively showed that miRNAs, ta-siRNAs, and possibly nat-siRNAs play important roles in chilling response and chilling acclimation in Cassava by regulating stress-related pathways, e.g. Auxin signal transduction. The conservation of these sncRNA might shed lights on the role of sncRNA-mediated pathways affected by chilling stress and stress acclimation in Euphorbiaceous plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-634) contains supplementary material, which is available to authorized users

Recent Biotechnological Advances in the Improvement of Cassava

Cassava (Manihot esculenta Crantz) is the fourth most important source of carbohydrates for human consumption in the tropics and thus occupies a uniquely important position as a food security crop for smallholder farmers. Consequently, cassava improvement is of high priority to most national agricultural research institutions in the tropics. With advances in functional genomics and genome editing approaches in this post genomics era, there are unprecedented opportunities and potential to accelerate the improvement of this important crop. These new technologies will need to be directed toward addressing major cassava production constraints, notably virus resistance, protein content, tolerance to drought and reduction of hydrogen cyanide content. Here, we discuss the important role novel functional genomics and genome editing technologies have and will continue to play in cassava improvement efforts. These approaches, including artificial miRNA (amiRNA), trans-acting small interfering RNA (tasiRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9), and Targeting Induced Local Lesions IN Genomes (TILLING), have been shown to be effective in addressing major crop production constraints. In addition to reviewing specific applications of these technologies in cassava improvement, this chapter discusses specific examples being deployed in the amelioration of cassava or of other crops that could be applied to cassava in future

Caracterización de miRNAs involucrados en respuesta a infección por bacterias del género Xanthomonas en arroz y yuca

Los microRNAs (miRNAs) son moléculas de RNAs pequeñas que juegan un papel importante en la regulación de la expresión génica en eucariotas mediante el silenciamiento de mRNAs complementarios (targets). En este trabajo se estudió su papel en interacciones con bacterias del género Xanthomonas en arroz y yuca. Mediante análisis bioinformático de librerías de RNAs pequeños se identificaron y cuantificaron miRNAs, y se estudió la expresión de los posibles targets. Se encontró que en yuca la respuesta mediada por miRNAs está caracterizada por la inducción de miRNAs que reprimen señalización por auxinas y por la represión de miRNAs involucrados en el control de genes de resistencia. Mientras que en arroz los miRNAs no parecen tener un papel crucial en defensa aunque la expresión de miRNAs varía en respuesta a distintas cepas bacterianas. Y se identificaron también posibles mecanismos de regulación transcripcional de miRNAs mediados por factores de transcripción vegetales y bacterianos.Abstract. microRNAs (miRNAs) are small RNA molecules involved in the control of gene expression in eukaryotes through the silencing of complementary mRNAs (targets). This work addresses their role in interactions with bacteria from the genus Xanthomonas in rice and cassava. miRNAs were identified and quantified through the bioinformatic analyses of sRNA libraries and the expression of the possible targets was also studied. It was found that in cassava the response mediated by miRNAs is characterized mainly by the induction of miRNAs that repress auxin silencing and by the repression of miRNAs involved in the control of resistance genes. While in rice miRNAs seemed not to have a crucial role in defense even though the expression of miRNAs was variable in response to different bacterial strains. This work also addresses the identification of possible mechanisms of transcriptional regulation of miRNAs mediated by vegetal and bacterial transcription factors.Maestrí

The role of small RNAs in susceptibility and tolerance to cassava mosaic disease

A dissertation presented by Sarah Jane Rogans to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. 2016Cassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.LG201

RNA Interference and CRISPR/Cas Gene Editing for Crop Improvement: Paradigm Shift towards Sustainable Agriculture

With the rapid population growth, there is an urgent need for innovative crop improvement approaches to meet the increasing demand for food. Classical crop improvement approaches involve, however, a backbreaking process that cannot equipoise with increasing crop demand. RNA-based approaches i.e., RNAi-mediated gene regulation and the site-specific nuclease-based CRISPR/Cas9 system for gene editing has made advances in the efficient targeted modification in many crops for the higher yield and resistance to diseases and different stresses. In functional genomics, RNA interference (RNAi) is a propitious gene regulatory approach that plays a significant role in crop improvement by permitting the downregulation of gene expression by small molecules of interfering RNA without affecting the expression of other genes. Gene editing technologies viz. the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) have appeared prominently as a powerful tool for precise targeted modification of nearly all crops’ genome sequences to generate variation and accelerate breeding efforts. In this regard, the review highlights the diverse roles and applications of RNAi and CRISPR/Cas9 system as powerful technologies to improve agronomically important plants to enhance crop yields and increase tolerance to environmental stress (biotic or abiotic). Ultimately, these technologies can prove to be important in view of global food security and sustainable agriculture

Comprehensive mechanism of gene silencing and its role in plant growth and development

Gene silencing is a negative feedback mechanism that regulates gene expression to define cell fate and also regulates metabolism and gene expression throughout the life of an organism. In plants, gene silencing occurs via transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). TGS obscures transcription via the methylation of 5′ untranslated region (5′UTR), whereas PTGS causes the methylation of a coding region to result in transcript degradation. In this review, we summarized the history and molecular mechanisms of gene silencing and underlined its specific role in plant growth and crop production

Transference of RXam2 and Bs2 genes to confer resistance against cassava bacterial blight (CBB)

Abstract. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease in all regions where cassava is cultivated. To generate broad spectrum and durable CBB resistance it is necessary pyramiding several R genes. We have identified two candidate resistance genes, named RXam1 and RXam2 which colocalize with QTLs associated to resistance against Xam. RXam1 codes for a RLK (Receptor-Like Kinase) protein. We generated several transgenic cassava lines overexpressing RXam1. In vitro plants of three lines showed reduced symptoms and reduced bacterial growth after Xam infection with strain CIO136 compared to empty vector transgenic plants. The second cassava gene, RXam2, codes for a NB-LRR protein. Using RNAi we generated RXam2-silenced transgenic plants, which were more susceptible to several Xam strains than non-transformed plants. On the other hand, plants overexpressing RXam2 showed reduced symptoms to Xam strains. These data collectively suggest that RXam2 is a resistance gene against Xam. In addition, an autoactive version mutated in the MHD motif (NB domain) of RXam2 was generated through site directed mutagenesis and was able to generate a Hypersensitive Response (HR) by transient agroinfiltration in cassava and tobacco leaves. The autoactive version of RXam2 was cloned under a TALE1Xam-inducible promoter and transient expression in tobacco showed a strong HR when co-infiltrated with a plasmid containing the TALE1Xam gene. Several independent transgenic stable lines are being evaluated to assess TALE1Xam inducibility. Finally, several cassava transgenic plants overexpressing Bs2 from pepper were obtained and showed constitutive, typical immune responses.Doctorad