Assessing Global Transcriptome Changes in Response to South African Cassava Mosaic Virus [ZA-99] Infection in Susceptible Arabidopsis thaliana

Abstract

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.

Figure 1. Infectivity assay of SACMV-agroinoculated Arabidopsis.

A: Mock-inoculated Arabidopsis plants displaying no symptoms (healthy). B: SACMV – infected leaves displaying leaf curl and deformation. C: SACMV copy number (copies/200 ng TNA) over time. Large error bars indicate variability in virus copy number due to biological differences between replicates. D and E: AGL1 detection in 200 ng of TNA from healthy and SACMV – infected leaf tissue across time points 14, 24, and 36 dpi.

Figure 2. Venn diagram depicting the distribution of 13,934 differentially expressed genes (p<0.05) in SACMV - infected leaf tissue at three time points post infection.

Figure 3. MIPS functional distribution categories of 2-fold differentially expressed transcripts in SACMV - infected Arabidopsis leaf tissues at 14, 24 and 36 dpi.

Figure 4. Gene tree heat map showing hierarchical clustering of 37 out of 41 transcripts expressed continuously across time points 14, 24, and 36 dpi (4 unknowns were not displayed).

Red bars indicated induction (>2.0) and green bars, repression (<−2.0). Abbreviations: FC (Fold Change).

Figure 5. Validation of microarray expression data by relative quantitative real-time RT-PCR (qRT-PCR).

Expression changes of 10 selected transcripts depicting similarities in expression patterns between the two technologies are shown. Signal intensities for each transcript were normalized with CBP20 for 14 dpi and Actin2 for 24 dpi. The x-axis represents validated genes at time points 14 and 24 dpi. The y-axis represents normalized fold-change expression values for each transcript. The error bars show standard deviation from 3 biological replicates.

Figure 6. Gene tree heat map of differentially expressed core-cyclin genes in response to SACMV infection.

All listed Arabidopsis accession numbers refer to cyclin-related genes.

Figure 7. Map of potential links between hormonal signals and cell cycle regulators.

Abbreviations: CK, cytokinin; E2F/DP, transcription factors; RBR, retinoblastoma-related protein; P, phospho-protein; CYC, cyclin; CDK, cyclindependent kinase; PP2A, phosphatase; SCR, SCARECROW; SHR, SHORT ROOT; SCF, SKP1+ CULLIN+F-box (SKP2); EBP1, plant homologue of epidermal growth factor-binding protein; SKP2, F-box protein; STM, SHOOT MERISTEMLESS; KRP, CDK inhibitor; CaM, calmodulin; CPK, calmodulin-like domain protein kinase; ABAP1, armadillo BTB Arabidopsis protein 1; TCP24, transcription factor; CDT1, DNA replication-licensing factor; ABP1, auxin binding protein 1; ANT, aintegumenta; ARGOS, auxin-regulated gene in organ size; AXR1, RUB1-activating enzyme; ABA, abscisic acid; GL2, GLABRA (root hair); GEM, GL2 expression regulator; ACS5, 1-aminocyclo-propane-1-carboxil acid synthase . Stars depict SACMV-[ZA:99] involvement in hormone signals and cell cycle regulators. Red stars show up-regulation, while blue stars show down-regulation.