Identification and characterization of genes involved in the defense response of Arabidopsis thaliana towards the soil-borne pathogen Rhizoctonia solani.

Plants are permanently subjected to external biotic and abiotic factors that hamper an optimal yield. In agriculture, crop losses due to plant pathogens remain an important yield limiting factor despite agricultural measures such as selective breeding and the use of phytopharmaceuticals. Especially plant diseases caused by soil-borne pathogens and pests remain difficult to treat due to their below-ground interactions with the roots. Because of this additional complexity, research on plant defense mechanisms so far mainly focused on air-borne leaf diseases.In this study we challenged the model plant Arabidopsis thaliana with Rhizoctonia solani AG 2-2IIIB in order to get insight in the plant defense response towards this economically important soil-borne fungus. In first instance we conducted a leaf transcriptome analysis on plants grown on R. solani inoculated soil and revealed 417 differentially expressed (DE) genes at 48 hours post inoculation (hpi). By clustering the DE genes that function in the same biological processes, we identified processes that were differentially regulated in leaf tissue upon infection by R. solani. This analysis elucidated the up-regulation of biological processes involved in defense signaling events mediated by the plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (Et). To evaluate the biological significance of these defense signaling cascades we monitored the susceptibility of A. thaliana mutant lines affected in these defense signaling pathways, to R. solani infection using the soil inoculation assay. In this way we could demonstrate that all three signaling pathways contributed to the plant resistance level against R. solani.In a second research part we developed a R. solani in vitro assay, which in comparison to the earlier used soil assay allows easy and sufficient collection of R. solani inoculated root tissue and in addition shows less variability. Using this in vitro assay we subsequently conducted a transcriptome analysis on roots at 24 hpi with R. solani and identified 1353 DE genes. Using clustering analysis we could show the involvement of SA, JA and Et defense signaling events in the induced root defense response. Hence a comparison between the data obtained for both leaves and roots suggests a very similar regulation of induced defense responses. Nevertheless, when analyzing specific gene expression profiles we show that marker genes commonly used in leaf defense research can exert different expression profiles in root tissue, and as such need re-evaluation before they can be applied to unravel root defense responses.In a last part we used our generated transcriptome data to select 4 specific genes or gene families for an in-depth evaluation of their potential defense-related role. First, we obtained experimental support for a defense-related function of AtPDR12, a gene coding for ABC-transporter reported to mediate abscisic acid (ABA) transport. The involvement of this phytohormone further extends our earlier defined complexity of the regulation of the induced defense response towards this pathogen. Secondly, we investigated the relevance of a putative ß-1,3-endoglucanase (At4g16260) for the plant defense towards R. solani. We demonstrated elevated endoglucanase activity in leaf extracts of plant lines overexpressing At4g16260, which is the first experimental support for an in planta activity of this gene. Although none of the transgenic lines with a modulated At4g16260 expression (up- or down-regulated) showed an altered susceptibility towards R. solani, we were able to demonstrate that At4g16260 overexpression lines display an elevated resistance towards the necrotrophic leaf pathogen Botrytis cinerea. Thirdly, evaluating the leaf transcriptome analysis we observed that 4 members of the plant defensin (AtPDF) family 1, coding for the same or very similar peptides, were all highly up-regulated during the induced leaf defense response towards R. solani. To evaluate the relevance of these AtPDFs, we generated silencing lines that simultaneously down-regulated all 4 genes to circumvent possible redundancy effects. Although we were unable to observe an altered susceptibility towards R. solani and B. cinerea, these transgenic lines will be valuable to further elucidate the biological function of these plant defensins. Finally, we initiated research investigating the defense-related role of genes that were specifically up-regulated in our root transcriptome analysis but not or marginally during the leaf defense response upon infection with the soil-borne pathogen R. solani. Our first series of disease assays on A. thaliana plants with altered expression levels for these genes suggested their potential defense-related role against R. solani. Moreover, using quantitative and visual analysis of their expression, we generated additional experimental data supporting a root-specific induction for two of them. Since genes displaying such an expression profile are rarely studied, they are a potential source of valuable resistance genes towards soil-borne pathogens such as R. solani, Fusarium oxysporum and Verticillium longisporum, and therefore deserve special attention.nrpages: 147status: publishe