Transcriptional profiling of Ascochyta blight resistance in Lentil

Using a cDNA microarray themed on Ascochyta-Pulse interaction resistance response was studied in two lentil varieties, specifically in response to A. lentis inoculation in a highly resistant (ILL7537) and highly susceptible (ILL6002) lentil variety. The experiments were conducted in reference design, where samples from mock-inoculated controls acted as references against post-inoculation samples and the plants were grown using a uniform and standardized experimental system that minimized environmental effects. Robust and high quality data was obtained through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and stringent selection criteria for differentially expressed genes including a fold change cutoff determined by self-self hybridizations, Students t-test and FDR (Fasle Discovary Rate) multiple testing correction (P<0.05). Microarray observations were validated by quantitative real time RT-PCR using the RNA from one of the bioassay used in the original microarray experiment. Ninety genes were differentially expressed in ILL7537 and 95 genes were differentially expressed in ILL6002. The expression profiles of the two varieties showed substantial difference in type and time of genes that were expressed in response to A. lentis. The resistant variety showed early up-regulation of PR proteins and other defence related genes. The susceptible genotype showed mainly down-regulation of defence related genes. The microarray experiment, the first in lentil, conducted with a small number of genes themed on Asochyta and Pulse interactions was able to identify different components of the defence mechanism by comparing the transcriptional profiles of the susceptible and resistant genotypes. This study will thus form the basis of future experiments to elaborate and corroborate the genomics of lentils defence to A. lentis. Keywords: time course, disease state analysis Total RNA was extracted from pooled stem and leaf samples for each genotype at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity, quality and integrity of the total RNA samples were assessed by OD260/OD280 ratios and by gel electrophoresis. Fifty μg of total RNA from each sample was reverse transcribed using Superscript II (Invitrogen) with incorporation of amino-allyl dUTP (Sigma) into the cDNA for post labeling with Cy3- or Cy5-NHS esters (Amersham). For each time point after inoculation, hybridisations were performed with six technical replicates (corresponding to the six complete grids on each microarray slide) and three biological replicates and included a dye-swapping (i.e. reciprocal labeling of Cy3 and Cy5) to eliminate any dye bias. In total 180 images were analysed from 30 slides, resulting in 18 data points for each time-point of each genotype. An Affymetrix® 428™ array scanner (Santa Clara, CA) was used to scan hybridized microarray slides at 660 nm for Cy5 (red laser) and 532 nm for Cy3 (green laser) at 10 µm resolution and the image was captured with Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Scanned image was analysed and quantified using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software. Transformation of quantified spot data with GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA) consisted of a local background correction (subtracting mean background intensity from mean signal intensity for each spot), omitting flagged spots (i.e. empty spots, negative spots (signal mean