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A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus.

ABSTRACT: BACKGROUND:The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava's recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L. METHODS:Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey's HSD test and the 2-??CTstatistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model. RESULTS:The differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces. CONCLUSIONS:This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

SUBMITTER: Chatukuta P

PROVIDER: S-EPMC7685591 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus.

Chatukuta Patience P Rey Marie Emma Christine MEC

Virology journal 20201123 1

<h4>Background</h4>The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava's recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the ...[more]

PMID: 33228712

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Project description:In susceptible plant hosts, co-evolution has favoured viral strategies to evade host defenses and utilize resources to their own benefit. The degree of manipulation of host gene expression is dependent on host-virus specificity and certain abiotic factors. In order to gain insight into global transcriptomic changes for a geminivirus pathosystem, South African cassava mosaic virus [ZA:99] (SACMV-ZA:99]) and Arabidopsis thaliana, 4 x 44K Agilent microarrays were adopted. After normalization, a 2-fold change filtering of data (p<0.05) identified 1,820 differentially expressed genes in apical leaf tissue. A significant increase in differential gene expression over time (451 genes at 14 dpi, 742 genes at 24 dpi, and 1011 genes at 36 dpi) was observed. This increase in expression, correlated with an increase in SACMV accumulation as virus copies were 5-fold higher at 24 dpi and 6-fold higher at 36 dpi than at 14 dpi (1.1x104 virus copies present at 14 dpi, 5.7x104 copies at 24 dpi, and 6.3x104 copies at 36 dpi). Many 2-fold genes were primarily involved in stress and defense responses, phytohormone signalling pathways, cellular transport, cell-cycle regulation, transcription, oxidation-reduction, and other metabolic processes. Forty-one genes (2.3%) were shown to be continuously expressed across the infection period, indicating that the majority of genes were transient and unique to a particular time point. Plant signalling networks were disrupted and manipulated by SACMV-[ZA:99] in order to affect homeostasis and antagonize host’s defense responses. At the same time, an adaptive response was initiated to reprogramme metabolism and divert energy from growth-related processes to defense, all leading to disruption of normal biological host processes. Comparisons between SACMV-[ZA:99] with plant-infecting RNA and DNA viruses revealed similarities and differences in expression patterns among viruses, showing either general defense or virus-specific responses. Within the Geminiviridae family in particular, similarities in cell-cycle regulation and gene expression patterns correlated between SACMV-[ZA:99] and Cabbage leaf curl virus (CaLCuV) but differences were also evident. For instance, CaLCuV showed antagonistic interactions between Salicyclic Acid (SA) and Jasmonic Acid (JA) pathways, whereas SACMV displayed synergism. Differences in gene induction, repression and outcome between the two geminiviruses clearly demonstrated host-specific interactions with SACMV-[ZA:99] leading to infection. To our knowledge this is the first geminivirus study identifying differentially expressed transcripts across 3 time points

Project description:In susceptible plant hosts, co-evolution has favoured viral strategies to evade host defenses and utilize resources to their own benefit. The degree of manipulation of host gene expression is dependent on host-virus specificity and certain abiotic factors. In order to gain insight into global transcriptomic changes for a geminivirus pathosystem, South African cassava mosaic virus [ZA:99] (SACMV-ZA:99]) and Arabidopsis thaliana, 4 x 44K Agilent microarrays were adopted. After normalization, a 2-fold change filtering of data (p<0.05) identified 1,820 differentially expressed genes in apical leaf tissue. A significant increase in differential gene expression over time (451 genes at 14 dpi, 742 genes at 24 dpi, and 1011 genes at 36 dpi) was observed. This increase in expression, correlated with an increase in SACMV accumulation as virus copies were 5-fold higher at 24 dpi and 6-fold higher at 36 dpi than at 14 dpi (1.1x104 virus copies present at 14 dpi, 5.7x104 copies at 24 dpi, and 6.3x104 copies at 36 dpi). Many 2-fold genes were primarily involved in stress and defense responses, phytohormone signalling pathways, cellular transport, cell-cycle regulation, transcription, oxidation-reduction, and other metabolic processes. Forty-one genes (2.3%) were shown to be continuously expressed across the infection period, indicating that the majority of genes were transient and unique to a particular time point. Plant signalling networks were disrupted and manipulated by SACMV-[ZA:99] in order to affect homeostasis and antagonize hostM-bM-^@M-^Ys defense responses. At the same time, an adaptive response was initiated to reprogramme metabolism and divert energy from growth-related processes to defense, all leading to disruption of normal biological host processes. Comparisons between SACMV-[ZA:99] with plant-infecting RNA and DNA viruses revealed similarities and differences in expression patterns among viruses, showing either general defense or virus-specific responses. Within the Geminiviridae family in particular, similarities in cell-cycle regulation and gene expression patterns correlated between SACMV-[ZA:99] and Cabbage leaf curl virus (CaLCuV) but differences were also evident. For instance, CaLCuV showed antagonistic interactions between Salicyclic Acid (SA) and Jasmonic Acid (JA) pathways, whereas SACMV displayed synergism. Differences in gene induction, repression and outcome between the two geminiviruses clearly demonstrated host-specific interactions with SACMV-[ZA:99] leading to infection. To our knowledge this is the first geminivirus study identifying differentially expressed transcripts across 3 time points A three time-point (14, 24, and 36 dpi) study was carried out to identify differentially expressed genes in SACMV-[ZA:99] infected Arabidopsis leaf cells using a direct comparison design against mock-inoculated controls. Three biological replicates and 1 technical replicate for both SACMV-[ZA:99]-infected and mock-inoculated controls were conducted at each time point.

Project description:In susceptible plant hosts, co-evolution has favoured viral strategies to evade host defenses and utilize resources to their own benefit. The degree of manipulation of host gene expression is dependent on host-virus specificity and certain abiotic factors. In order to gain insight into global transcriptome changes for a geminivirus pathosystem, South African cassava mosaic virus [ZA:99] and Arabidopsis thaliana, 4×44K Agilent microarrays were adopted. After normalization, a log2 fold change filtering of data (p<0.05) identified 1,743 differentially expressed genes in apical leaf tissue. A significant increase in differential gene expression over time correlated with an increase in SACMV accumulation, as virus copies were 5-fold higher at 24 dpi and 6-fold higher at 36 dpi than at 14 dpi. Many altered transcripts were primarily involved in stress and defense responses, phytohormone signalling pathways, cellular transport, cell-cycle regulation, transcription, oxidation-reduction, and other metabolic processes. Only forty-one genes (2.3%) were shown to be continuously expressed across the infection period, indicating that the majority of genes were transient and unique to a particular time point during infection. A significant number of pathogen-responsive genes were suppressed during the late stages of pathogenesis, while during active systemic infection (14 to 24 dpi), there was an increase in up-regulated genes in several GO functional categories. An adaptive response was initiated to divert energy from growth-related processes to defense, leading to disruption of normal biological host processes. Similarities in cell-cycle regulation correlated between SACMV and Cabbage leaf curl virus (CaLCuV), but differences were also evident. Differences in gene expression between the two geminiviruses clearly demonstrated that, while some global transcriptome responses are generally common in plant virus infections, temporal host-specific interactions are required for successful geminivirus infection. To our knowledge this is the first geminivirus microarray study identifying global differentially expressed transcripts at 3 time points.

Project description:BackgroundCassava mosaic disease is caused by several distinct geminivirus species, including South African cassava mosaic virus-[South Africa:99] (SACMV). To date, there is limited gene regulation information on viral stress responses in cassava, and global transcriptome profiling in SACMV-infected cassava represents an important step towards understanding natural host responses to plant geminiviruses.ResultsA RNA-seq time course (12, 32 and 67 dpi) study, monitoring gene expression in SACMV-challenged susceptible (T200) and tolerant (TME3) cassava landraces, was performed using the Applied Biosystems (ABI) SOLiD next-generation sequencing platform. The multiplexed paired end sequencing run produced a total of 523 MB and 693 MB of paired-end reads for SACMV-infected susceptible and tolerant cDNA libraries, respectively. Of these, approximately 50.7% of the T200 reads and 55.06% of TME3 reads mapped to the cassava reference genome available in phytozome. Using a log2 fold cut-off (p<0.05), comparative analysis between the six normalized cDNA libraries showed that 4181 and 1008 transcripts in total were differentially expressed in T200 and TME3, respectively, across 12, 32 and 67 days post infection, compared to mock-inoculated. The number of responsive transcripts increased dramatically from 12 to 32 dpi in both cultivars, but in contrast, in T200 the levels did not change significantly at 67 dpi, while in TME3 they declined. GOslim functional groups illustrated that differentially expressed genes in T200 and TME3 were overrepresented in the cellular component category for stress-related genes, plasma membrane and nucleus. Alterations in the expression of other interesting genes such as transcription factors, resistance (R) genes, and histone/DNA methylation-associated genes, were observed. KEGG pathway analysis uncovered important altered metabolic pathways, including phenylpropanoid biosynthesis, sucrose and starch metabolism, and plant hormone signalling.ConclusionsMolecular mechanisms for TME3 tolerance are proposed, and differences in patterns and levels of transcriptome profiling between T200 and TME3 with susceptible and tolerant phenotypes, respectively, support the hypothesis that viruses rearrange their molecular interactions in adapting to hosts with different genetic backgrounds.

Dataset Information

A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus.

Publications

A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus.

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