Effector proteins secreted by necrotrophic fungi play key roles in pathogenesis. It has been considered essential to identify and understand their functions in necrotrophic fungi to develop effective defenses and breed resistant plant varieties. In recent years, bioinformatics-based analysis tools and artificial intelligence have increased the likelihood of identifying these effector genes by providing genomic and transcriptomic data on the stages of pathogen development within the host. In this study, a destructive agent of chickpea fields worldwide and a combination of in silico methods were used to identify the effectors of the fungus Ascochyta rabiei, focusing on the structural features of effector proteins, including secretion, short length, and disulfide-bond-forming ability. Also, the promoter sequence analysis of the gene encoding the effector protein and the presence of regulatory sites for transcription factors involved in the pathogen-host interaction were used to confirm the functional relationship of the candidate effector. For further investigation, the candidate gene was tracked at the genome and transcriptome levels in A. rabiei under growth conditions in culture medium and during interactions with resistant and susceptible chickpea genotypes. Based on the conducted studies, a 159-amino-acid protein, GSh302, was identified as a candidate effector, and its coding sequence (accession number OR875943) and protein sequence (accession number WPV74376) were registered in GenBank. Analysis of the upstream sequence of the gene confirmed the presence of regulatory elements, including transcription factor binding sites and the Sko1 (bZIP_1) transcription factor, which plays a key role in activating the expression of secretory genes during the fungus-plant interaction. The gene sequence was tracked in A. rabiei isolates grown in culture medium, but it was not detectable at the transcript level. This gene was discernible at the genome and transcriptome levels of the fungus in resistant and susceptible chickpea cultivars 96 hours after inoculation, and the expected specific bands were amplified. The combined method used in this study identified the GSh302 protein as a candidate effector in A. rabiei, expressed during the fungus-chickpea interaction. Conducting additional studies to investigate the expression pattern of this gene at different times after plant inoculation with this fungus can help better understand its expression. Moreover, identifying the target biomolecules of the GSh302 protein in the host plant can help determine its functions.

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