Project description: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
Project description:Ascochyta blight of lentil is a prevalent disease in many lentil producing regions and can cause major yield and grain quality losses. The most environmentally acceptable and economically profitable method of control is to develop varieties with high levels of durable resistance. Genetic studies to date suggest that ascochyta blight resistance genes (R-gene) in lentil lines CDC Robin, ILL 7537, 964a-46, and ILL 1704 are non-allelic. To understand how different R-genes manifest resistance in these genotypes and an accession of Lens ervoides, L-01-827A, with high level of resistance to ascochyta blight, cellular and molecular defense responses were compared after inoculation with the causal pathogen Ascochyta lentis. Pathogenicity testing of the resistant lines to A. lentis inoculation revealed significantly lower disease severity on CDC Robin and ILL 7537 compared to ILL 1704 and 964a-46, and no symptoms of disease were observed on L-01-827A. Histological examinations indicated that cell death triggered by the pathogen might be disrupted as a mechanism of resistance in CDC Robin. In contrast, limiting colonization of epidermal cells by A. lentis is a suggested mechanism of resistance in 964a-46. A time-series comparison of the expressions of hallmark genes in salicylic acid (SA) and jasmonic acid (JA) signal transduction pathways between CDC Robin and 964a-46 was conducted. These partially resistant genotypes differed in the timing and the magnitude of SA and JA signaling pathway activation. The SA signaling pathway was only triggered in 964a-46, whereas the JA pathway was triggered in both partially resistant genotypes CDC Robin and 964a-46. The expression of JA-associated genes was lower in 964a-46 than CDC Robin. These observations corroborate the existence of diverse ascochyta blight resistance mechanisms in lentil genotypes carrying different R-genes.