Comment[ArrayExpressAccession]	E-GEOD-43282
MAGE-TAB Version	1.1					
Public Release Date	2013-01-07
Investigation Title	Assessing global transcriptomic changes in response to South African cassava mosaic virus [ZA-99] infection in susceptible Arabidopsis thaliana					
Comment[Submitted Name]	Assessing global transcriptomic changes in response to South African cassava mosaic virus [ZA-99] infection in susceptible Arabidopsis thaliana
Experiment 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.					
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl				
Person Last Name	Rey	Pierce	Rey			
Person First Name	Chrissie	Erica	M			
Person Mid Initials			E			
Person Email	chrissie.rey@wits.ac.za					
Person Affiliation	University of the Witswatersrand					
Person Phone	27117176324					
Person Fax	27117176351					
Person Address	School of Molecular and Cell Biology, University of the Witswatersrand, 1 Jan Smuts Avenue, Braamfontein, Johannesburg, Gauteng, South Africa					
Person Roles	submitter					
Protocol Name	P-GSE43282-1	P-GSE43282-5	P-GSE43282-4	P-GSE43282-2	P-GSE43282-3	P-GSE43282-6
Protocol Description	ID_REF =  VALUE = Normalized Log2 (infection/control) ratios	Genepix 4000B scanner using Genepix Pro 6 software	Microarray hybridization was carried out according to manufacturerM-bM-^@M-^Ys instructions (Agilent). 100 pmol of each cyanine dye, linearly amplified cRNA was added to a hybridization mix containing 10 X blocking agent and 25 X fragmentation buffer were incubated for 30 mins at 60M-0C to fragment the RNA. Fifty five microliters of 2 X GE buffer was then added to the solution, spun gently and placed on ice, ready for hybridization. One hundred and ten microliters of solution was added onto three Arabidopsis Agilent 4 X 44 slides (Version 3), and placed in a rotating hybridization chamber (Agilent) set at 65M-0C for 18 h. Slides were then washed using AgilentM-bM-^@M-^Xs Gene Expression Wash Buffers 1 and 2.	Total RNA was extracted from pooled SACMV-[ZA;99] - infected and healthy Arabidopsis plants at 14, 24, and 36 dpi using a QIAzol lysis reagent method	Total RNA (1 ug) was amplified using the Amino Allyl MessageAmpM-bM-^DM-"II aRNA Amplification Kit (Ambion) following manufacturerM-bM-^@M-^Ys instructions. During aRNA:Dye coupling, 4 ug of aRNA was vacuum dried at 45M-0C and resuspended in 5 ul of 0.2M NAHCO3 (pH 9.0) at RT for 20 min. Two microlitres of each dye (Cy5 or Cy3) was added to the sample.	Fluorescence data obtained from the microarray was imported into Limma in the R computing environment, where the data was normalized (M-bM-^@M-^Xwithin-arrayM-bM-^@M-^Y global loess normalization and M-bM-^@M-^Xbetween-arrayM-bM-^@M-^Y quantile normalization), and linear models were fitted in order to contrast SACMV-[ZA:99] - infected expression values with those of mock-inoculated controls. Dye swap experiments were carried out for each replicate. Normalized Log2 fold change ratios showing direct comparison of data representing infection/control are shown below.
Protocol Type	bioassay_data_transformation	image_aquisition	hybridization	nucleic_acid_extraction	labeling	feature_extraction
Experimental Factor Name	TIME	INFECTION				
Experimental Factor Type	time	infection				
Comment[SecondaryAccession]	GSE43282					
Comment[GEOReleaseDate]	2013-01-07					
Comment[ArrayExpressSubmissionDate]	2013-01-04					
Comment[GEOLastUpdateDate]	2013-01-07					
Comment[AEExperimentType]	transcription profiling by array					
SDRF File	E-GEOD-43282.sdrf.txt