AscoTransSeq · Identification of pathogenicity and virulence genes of the necrotrophic fungus Ascochyta spp. by genome-wide transcriptome analyses coupled to high-throughput next-generation sequencing

Identification of pathogenicity and virulence genes of the necrotrophic fungus Ascochyta spp. by genome-wide transcriptome analyses coupled to high-throughput next-generation sequencing.

Legumes are important crops for the European Union, providing a versatile and inexpensive protein source for animal feeding and human consumption. Furthermore, legumes are suitable candidates for a sustainable agriculture, being powerful natural soil fertilizers. However, legumes are affected by a number of foliar and root diseases. The most important foliar diseases in legumes worldwide are ascochyta blights. This group of diseases, incited by Ascochyta spp., causes severe yield losses in chickpea, pea, lentil, faba bean and vetchling. Health or disease is the result of the battle between the plants and their pathogens. Fungal pathogens use their pathogenicity factors to attack the plant. Recognition of the pathogen by the plant triggers defence responses. However, the pathogen has also efficient mechanisms to overcome these defence responses. Therefore, plant resistance or susceptibility depends on the genetic background of both pathogen and host. In the ascochyta-legume pathosystem most studies performed up to now have focused on the identification of resistance genes in the host, while very little is known about the pathogenicity factors of Ascochyta spp. Knowledge of Ascochyta pathogenic determinants will allow us to develop a better understanding of host-pathogen interactions to devise novel and more effective measures for managing the disease. The main objective of this project has been to identify the pathogenicity factors of ascochyta blight pathogens using Ascochyta rabiei as a model. Towards this objective the latest state-of-the-art genome-wide sequencing and transcriptomic technologies were used to sequence the genome and transcriptome of A. rabiei and to identify the genes involved in pathogenicity. In this project the genomes of four isolates of A. rabiei, each belonging to a different pathotype of A. rabiei, were sequenced using an Illumina HiSeq2500 sequencer. The resulting 122 million obtained reads were assembled into a draft 26.9 Mb genome. In addition to a consensus A. rabiei genome, the sequences of the four isolates have been compared and SNPs (Single Nucleotide Polymorphisms) been detected between the different isolates. In addition, de novo assembling, annotation and characterization of the transcriptome of A. rabiei was also carried out using RNAseq and MACE techniques. cDNA libraries were obtained from the fungus growing in PDB medium and from the fungus infecting chickpea leaves. A total of 152 million A. rabiei reads were processed to develop a transcriptome containing 22,725 different transcripts with an average length of 1,178 bp. This transcriptome will clearly enlarge the genomic resources available for A. rabiei and will be a useful tool to identify genes of interest in this species. Furthermore, this project has identified several putative pathogenic determinants and candidate effectors in A. rabiei. Using MACE, the transcriptome profile of A. rabiei growing in PDB medium was compared with that of the pathogen infecting chickpea leaves at different relevant steps of the infection process. This study identified 597 transcripts that were more expressed during infection than in the treatment without the host. A detailed analysis of these genes unravelled the pathogenicity factors that allow the pathogen to cause disease: A. rabiei produces a battery of cell wall-degrading enzymes, which are prerequisite for the pathogen to penetrate its host, secretes different types of toxins killing the host cells, and other enzymes needed to extract nutrients from chickpea dead cells. In addition, the pathogen expresses genes that detoxify the fungitoxic compounds produced by the plant as defense. While efforts to control ascochyta blight have mostly been based on the identification of resistance genes in the host, the present project explored a new approach through identifying the pathogen’s genes needed to cause the disease. The identification of these genes will open a wide range of alternatives to host resistance for the desirable control of ascochyta blight disease. In addition, knowledge of the fungal genes, that control virulence, and their functions is essential to better understand this host-pathogen system, including the breakdown of resistance. Results obtained through this project will also increase our knowledge on other necrotrophic fungi, which cause important losses in many crops. Controlling ascochyta blight, the main foliar disease in legumes, will promote and sustain legume cultivation in European agriculture. It should reduce the need for fertilizer and pesticides, thereby reducing production costs. This will have an important impact on farmers by increasing their competitiveness and improving their work conditions. Contact details: Prof. Dr. Günter Kahl Institute of Molecular Bioscience, Biocenter Johann Wolfgang Goethe-University, Frankfurt am Main, Germany e-mail: kahl@em.uni-frankfurt.de Dr. Sara Fondevilla Institute for Sustainable Agriculture Spanish National Research Council Córdoba, Spain e-mail: sfondevilla@ias.csic.es

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