Project description:Agrobacterium-mediated cassava transformation via friable embryogenic callus (FEC) has allowed the robust production of transgenic cassava. So far, the transformation has been performed mostly for 60444 (the model cassava variety for transformation) and African varieties due to the high ability of callus induction and good regeneration capacity from embryogenic tissues. It is important to develop the transformation methods for elite cassava varieties in Asian area which is one of main cassava production area in the world, however, the suitable transformation method for Asian elite variety via FEC has not been reported. Here, we developed the transformation method in Kasetsart 50 (KU50) which has the highest planting area in Thailand and Vietnam. In cassava transformation, the preparation of FEC is considered as a key step. The ability of FEC induction from KU50 was improved efficiently by the use of media with reduced nutrients and excess vitamin B1, even if the FEC-inducible efficiency was 5 times lower compared with cv. 60444. The optimal concentration of NAA for regeneration from FEC to cotyledon was 1.0 mg/L and the optimal concentration of BAP for shoot formation from cotyledon was 0.4 mg/L. The transformation efficiency was estimated as 45% for 60444 and 22% for KU50. Transcriptome analysis indicated that KU50 FEC is out of balance between cell wall production and assembly, because the expression of the genes related to loosing cell wall was upregulated in the FEC from KU50 compared with 60444. We hope that the developed technology will contribute to molecular breeding of useful cassava plants in Asia by the simultaneous use of genome-editing technology.
Project description:microRNAs can play a crucial role in stress response in plants, including biotic stress. Some miRNAs are known to respond to bacterial infection. This work has addressed the role of miRNAs in Manihot esculenta (cassava)-Xanthomonas axonopodis pv. manihotis (Xam) interaction. Illumina sequencing was used for analyzing small RNA libraries from cassava tissue infected and non-infected with Xam. Cassava variety MBRA685 (resistant to Xam-CIO151) Six-week-old plants were inoculated with 36h-old cultures of the aggressive Xanthomonas axonopodis pv. manihotis strain CIO151 in both leaves and stems.
Project description:Cassava is the most important root crop in the tropics but rapid post-harvest physiological root deterioration (PPD) is a major constraint to commercial cassava production. We used label-free quantitative proteomics to generate an extensive cassava root and PPD proteome. Over 2400 unique proteins were identified in the cassava root and nearly 300 proteins showed significant abundance regulation during PPD. A candidate gene for reducing PPD was identified from the regulated proteins with enzymatic assays and afterwards verified with a transgene approach. This demonstrates the relevance of proteomics approach for crop improvements.
Project description:Analysis of transcriptional response of virus-infected cassava and identification of putative sources of resistance for cassava brown streak disease transcriptome analysis of two varieties of cassava that differ in their level of resistance to cassava brown streak virus.
Project description:Cassava Anthracnose Disease (CAD) that caused by the fungus Colletotorichum anthracnose is a serious disease of cassava in worldwide. In this study, we aim to establish the cassava oligo-DNA microarray representing approximately 30,000 cassava genes and apply it to investigate the molecular mechanisms against fungal infection using two cassava cultivars; Huay Bong 60 (HB60, resistant line for CAD) and Hanatee (HN, sensitive line for CAD). Based on expression profiling, we showed that the expression of various biotic stress-inducible genes, such as detoxification enzyme related genes is higher in HB60 under the treated conditions and non-treated condition, compared with HN. These results show that stress-inducible signaling pathways including ROS detoxification are constitutively activated in HB60 even under normal growth conditions without stress. These results suggest that our microarray is a useful tool for analyzing the cassava transcriptome and add new insight into the host responses of cassava against fungal infection.
Project description:Analysis of transcriptional response of virus-infected cassava and identification of putative sources of resistance for cassava brown streak disease
Project description:Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. It has been reported that landraces lose CMD resistance after regeneration through de novo morphogenesis. We sequenced WGBS data of 14 samples.
Project description:Cassava mosaic disease caused by cassava begomoviruses is the most serious disease of cassava in Africa. However, the molecular mechanisms leading to symptom development of infected cassava plants are poorly understood. Here a high throughput digital gene expression profiling (DGE) based on Illumina Solexa sequencing technology was used to investigate the global transcriptional response of cassava to the African cassava mosaic virus infection. Results showed that 3,210 genes were differentially expressed in virus-infected cassava leaves. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that photosynthesis related genes were most affected, which was consistent with the chlorotic symptom on the infected leaves. The upregulation of chlorophyll degradation genes, e.g. the genes encoding chlorophyllase and pheophorbide a oxygenase, as well as the downregulation of the major apoproteins genes in light harvesting complex II (LHCII) identified by the DGE analysis were confirmed by qRT-PCR. Together with the reduction of chlorophyll b content and fewer grana stacks in the infected leaf cells, this study reveals that the degradation of chlorophyll plays an important role during ACMV symptom development for the first time. Meanwhile, we believe that the non-lethal effect on photosystem is a trick for virus to avoid fierce host immune response and a result of the long-term co-evolution. This study will provide a road map for future investigations into virus symptom development.
Project description:Cassava mosaic disease caused by cassava begomoviruses is the most serious disease of cassava in Africa. However, the molecular mechanisms leading to symptom development of infected cassava plants are poorly understood. Here a high throughput digital gene expression profiling (DGE) based on Illumina Solexa sequencing technology was used to investigate the global transcriptional response of cassava to the African cassava mosaic virus infection. Results showed that 3,210 genes were differentially expressed in virus-infected cassava leaves. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that photosynthesis related genes were most affected, which was consistent with the chlorotic symptom on the infected leaves. The upregulation of chlorophyll degradation genes, e.g. the genes encoding chlorophyllase and pheophorbide a oxygenase, as well as the downregulation of the major apoproteins genes in light harvesting complex II (LHCII) identified by the DGE analysis were confirmed by qRT-PCR. Together with the reduction of chlorophyll b content and fewer grana stacks in the infected leaf cells, this study reveals that the degradation of chlorophyll plays an important role during ACMV symptom development for the first time. Meanwhile, we believe that the non-lethal effect on photosystem is a trick for virus to avoid fierce host immune response and a result of the long-term co-evolution. This study will provide a road map for future investigations into virus symptom development. ACMV-infected cassava leaves mixture from three independent replicates were collected for RNA extractions at 20 dpi. Control samples were harvested from empty agrobacteria treated leaves incubated under the same conditions.