Comparative Transcriptomic Analysis of Virulence Factors in Leptosphaeria maculans during Compatible and Incompatible Interactions with Canola

Abstract

Leptosphaeria maculans is a hemibiotrophic fungus that causes blackleg of canola (Brassica napus), one of the most devastating diseases of this crop. In the present study, transcriptome profiling of L. maculans was performed in an effort to understand and define the pathogenicity genes that govern both the biotrophic and the necrotrophic phase of the fungus, as well as those that separate a compatible from an incompatible interaction. For this purpose, comparative RNA-seq analyses were performed on L. maculans isolate D5 at four different time points following inoculation on susceptible cultivar Topas-DH16516 or resistant introgression line Topas-Rlm2. Analysis of 1.6 billion Illumina reads readily identified differentially expressed genes that were over represented by candidate secretory effector proteins, CAZymes, and other pathogenicity genes. Comparisons between the compatible and incompatible interactions led to the identification of 28 effector proteins whose chronology and level of expression suggested a role in the establishment and maintenance of biotrophy with the plant. These included all known Avr genes of isolate D5 along with eight newly characterized effectors. In addition, another 15 effector proteins were found to be exclusively expressed during the necrotrophic phase of the fungus, which supports the concept that L. maculans has a separate and distinct arsenal contributing to each phase. As for CAZymes, they were often highly expressed at 3 dpi but with no difference in expression between the compatible and incompatible interactions, indicating that other factors were necessary to determine the outcome of the interaction. However, their significantly higher expression at 11 dpi in the compatible interaction confirmed that they contributed to the necrotrophic phase of the fungus. A notable exception was LysM genes whose high expression was singularly observed on the susceptible host at 7 dpi. In the case of TFs, their higher expression at 7 and 11 dpi on susceptible Topas support an important role in regulating the genes involved in the different pathogenic phases of L. maculans. In conclusion, comparison of the transcriptome of L. maculans during compatible and incompatible interactions has led to the identification of key pathogenicity genes that regulate not only the fate of the interaction but also lifestyle transitions of the fungus.

Non-parent of Origin Expression of Numerous Effector Genes Indicates a Role of Gene Regulation in Host Adaption of the Hybrid Triticale Powdery Mildew Pathogen

“…triticale genome shows a hybrid architecture with segments originating from the wheat mildew donor and others from the rye mildew donor, with a very variable origin of the different segments in different isolates (Menardo et al, 2016 ). Many published RNAseq studies compared gene expression levels of the same fungal genotype under different conditions, at different times after infection, or between compatible and incompatible interactions (Cantu et al, 2013 ; Hacquard et al, 2013 ; Kellner et al, 2014 ; Palma-Guerrero et al, 2016 ; Sonah et al, 2016 ). In our work, we used a reference genome-based approach allowing both intra- and inter- forma specialis comparisons.…”

“…Several transcriptomic studies in biotrophic fungal pathogens such as Puccinia striiformis , as well as hemibiotrophs such as Magnaporthe grisea, Zymoseptoria tritici , and Leptosphaeria maculans all found strong upregulation of putative effector genes during host infection, thus substantiating their role in host-pathogen interactions (Kawahara et al, 2012 ; Garnica et al, 2013 ; Hacquard et al, 2013 ; Sonah et al, 2016 ). It was also shown that effector gene expression in plant pathogenic fungi occurs in different waves during host invasion, suggesting stage-specific regulation of different subsets of the effector repertoire (Cantu et al, 2013 ; Hacquard et al, 2013 ; Rudd et al, 2015 ; Palma-Guerrero et al, 2016 ; Sonah et al, 2016 ). In Leptosphaeria maculans , stage-specific expression of different effector waves was identified in transcriptomic studies comparing cotyledon and stem colonization of oilseed rape by the pathogen (Sonah et al, 2016 ).…”

“…It was also shown that effector gene expression in plant pathogenic fungi occurs in different waves during host invasion, suggesting stage-specific regulation of different subsets of the effector repertoire (Cantu et al, 2013 ; Hacquard et al, 2013 ; Rudd et al, 2015 ; Palma-Guerrero et al, 2016 ; Sonah et al, 2016 ). In Leptosphaeria maculans , stage-specific expression of different effector waves was identified in transcriptomic studies comparing cotyledon and stem colonization of oilseed rape by the pathogen (Sonah et al, 2016 ). Finally, studies of quantitative differences in gene expression between pathogen races and lineages revealed the existence of both isolate and lineage-specific patterns of gene expression (Duplessis et al, 2011 ; Palma-Guerrero et al, 2017 ).…”

Understanding Aquaporin Transport System in Eelgrass (Zostera marina L.), an Aquatic Plant Species

“…A heat map for expression of AQPs was constructed using TIGR Multi Experiment Viewer (MeV 4 ). Hierarchical clustering with average linkage method was performed to cluster the genes ( Sonah et al, 2016 ).…”

Advances in Omics Approaches for Abiotic Stress Tolerance in Tomato

“…QTL regions are quite large, and harboring too many genes makes it difficult to identify the candidate gene, but the combination of QTL and GWAS approaches has been successfully used to identify candidate genes in soybean [107]. Furthermore, the integration of RNA sequencing profiles with gene expression profiles can provide vital clues for the identification of functions of unknown genes [108,109]. Therefore, combining QTL and GWAS with transcriptome profiling will be helpful to identify differentially expressed candidate genes [110,111].…”