Transcriptome profiling of lentil (Lens culinaris) through the first 24 hours of Ascochyta lentis infection reveals key defence response genes

Abstract

Background: Ascochyta blight, caused by the fungus Ascochyta lentis, is one of the most destructive lentil diseases worldwide, resulting in over $16 million AUD annual loss in Australia alone. The use of resistant cultivars is currently considered the most effective and environmentally sustainable strategy to control this disease. However, little is known about the genes and molecular mechanisms underlying lentil resistance against A. lentis.

Results: To uncover the genetic basis of lentil resistance to A. lentis, differentially expressed genes were profiled in lentil plants during the early stages of A. lentis infection. The resistant 'ILL7537' and susceptible 'ILL6002' lentil genotypes were examined at 2, 6, and 24 h post inoculation utilising high throughput RNA-Sequencing. Genotype and time-dependent differential expression analysis identified genes which play key roles in several functions of the defence response: fungal elicitors recognition and early signalling; structural response; biochemical response; transcription regulators; hypersensitive reaction and cell death; and systemic acquired resistance. Overall, the resistant genotype displayed an earlier and faster detection and signalling response to the A. lentis infection and demonstrated higher expression levels of structural defence-related genes.

Conclusions: This study presents a first-time defence-related transcriptome of lentil to A. lentis, including a comprehensive characterisation of the molecular mechanism through which defence against A. lentis is induced in the resistant lentil genotype.

Keywords: Ascochyta lentis; De novo assembly; Defence response; Fabaceae; Lens culinaris; Lentil; RNA sequencing and transcriptome analysis.

Fig. 1

Experimental design of the Ascochyta lentis resistant (ILL7537) and susceptible (ILL6002) lentil genotypes used for RNA extraction

Fig. 2

Bioinformatics flowchart of tools and methods used to process and analyse the RNA-Sequencing data and produce the transcriptome

Fig. 3

Expression-dependant N50 (ExN50), as calculated against a fraction of the total expressed data (Ex). ExN50 at the point of assembly saturation (96%) and traditional N50 are highlighted

Fig. 4

Taxonomy distribution of significant* Blast matches. Annotation was considered as significant with a BitScore >100, pie slices are calculated in logarithmic scale to assist in visualisation

Fig. 5

Principal component analysis of the variance-stabilized estimated raw counts. Samples are categorized by Genotype (as marker shapes) and Hours post inoculation (HPI, marker colour)

Fig. 6

The number of unique and common differentially expressed genes between the subgroups of inoculated samples (time post inoculation and genotype). Comparison of inoculated resistant vs. susceptible genotypes at each time point (2 hpi, 6 hpi and 24 hpi, a); and within the inoculated resistant (ILL7537) genotype samples between the different time points (b). Circle area is plotted to scale (Euler diagram) when geometrically possible

Fig. 7

GO and KEGG pathway enrichment analysis, based on over-expressed DE genes at each time point (2, 6, and 24 hpi) in the resistant lentil genotype ILL7537. GO pathway enrichment at 2 and 6 hpi (a) and 6 and 24 hpi (b); KEGG pathway enrichment at 2 and 6 hpi (c) and 6 and 24 hpi (d)

Fig. 8

Expression levels of selected genes with exceptional DE trends in the earlier stages of the defence response to A. lentis in ILL7537 and ILL6002 over 2, 6, and 24 hpi. Expression levels of the following genes are presented: CDPK, ERF and LRR-RK, with PP2A and MYB49 as examples of stable reference genes (a); Delta (12)-FAD, EXO70A1 and XTH (b); PR genes and UPL-BOI (c); ARP, PGIP and PMEI (d). A full line represents the expression level in the resistant genotype ILL7537 and the dashed line represents the expression level of the gene in the susceptible genotype ILL6002. Y-axis is in logarithmic scale, error bars represent standard error values between replicates

Fig. 9

Expression levels of selected genes with exceptional DE trends in the earlier stages of the defence response to A. lentis in ILL7537 and ILL6002 over 2, 6, and 24 hpi. Expression levels of the following genes are presented: R-S/T-K1, RING/U-box and SAG (a); R-S/T-K1, RING/U-box and SAG (b); DELLA, GID1, NB-ARC and UPL-SHPRH (c). A full line represents the expression level in the resistant genotype ILL7537 and the dashed line represents the expression level of the gene in the susceptible genotype ILL6002. Y-axis is in logarithmic scale, error bars represent standard error values between replicates

Fig. 10

Expression ratios between resistant (ILL7537) and susceptible (ILL6002) lentil genotypes, measured by RT-qPCR. Expression ratios of selected defence-related genes at 2, 6, and 24 hpi (a, b, c, respectively). Asterisks denote statistic significance (DE ≠ 1), with the following p-values: * <0.05, ** <0.01, *** <0.005

Fig. 11

Defence-related molecules involved in response of lentil to A. lentis during the first 24 h