Detection of the gsh303 Gene Encoding a Candidate Protein with Effector Function during the Interaction between Ascochyta rabiei and Chickpea

In the context of pathogenicity in host plants, many fungal pathogens produce and secrete a range of small molecules, including proteins and secondary metabolites. Some of these molecules are known as pathogenic factors, and they play a specific role in manipulating interactions between the pathogen and host plants. Typically, these agents are referred to as effectors. Effector proteins are pivotal in the pathogenicity process of phytopathogenic fungi and their colonization of host plants. Identifying and analyzing the functions of effector proteins are essential steps in understanding pathogenicity mechanisms, symbiosis, and plant defense strategies.Ascochyta blight, caused by the fungus Ascochyta rabiei, stands as one of the primary limiting factors in chickpea cultivation, presenting a challenge wherever this plant is grown. The availability of genomic and transcriptomic data for A. rabiei, both in vitro and during its interaction with the plant, facilitates the identification of virulence and effector genes. In this study, our primary objective was to investigate the gsh303 gene as a candidate effector in different pathotypes (I, III, and VI) of A. rabiei.

Genomic data associated with A. rabiei were obtained from the NCBI database. We utilized SignalP v4.1, SecretomP v2.0, TMHM v2.0, and EffectorP v3.0 software for the screening of candidate effector proteins, ultimately identifying the gsh303 gene as a potential effector gene. We also conducted an investigation into the presence of homologous and orthologous sequences.Subsequently, specific primers were designed for the gsh303 gene, and we tracked its presence in different pathotypes of A. rabiei. This was achieved by performing polymerase chain reaction (PCR) both in vitro and during the interaction between the plant and pathogen, encompassing both the genome and transcriptome levels.

The findings of our study indicate that the nucleotide sequence of the gsh303 gene comprises a single exon without any introns. To detect the gsh303 gene, we designed PSh303F/R specific primers based on its upstream and downstream regions. A 599-base pair fragment was successfully amplified by PCR, confirming the presence of the gene in the genomes of all three pathotypes.While the expression of the gapdh housekeeping gene was verified at the transcriptome level in the culture medium, we were unable to confirm the detection of the gsh303 gene at the transcript level. To further investigate the expression of this gene during the plant-pathogen interaction, we inoculated two chickpea cultivars, one resistant (MCC133) and one sensitive (ILC1929), with three pathotypes of A. rabiei (I, III, and VI). After 96 hours of inoculation, we collected samples from the inoculated plants for DNA and RNA extraction. Our results from tracking the gsh303 gene during the interaction conditions at both the genome and transcript levels revealed the amplification of 599 bp and 591 bp fragments, respectively.

In our current study, we employed bioinformatics methods to predict the gsh303 effector gene and identified it as a candidate effector. Furthermore, we confirmed the presence of this gene in all three pathotypes of A. rabiei under in vitro conditions. Utilizing fungal genomic DNA as a template, a specific band of 599 base pairs was successfully amplified via PCR. Examining the expression pattern of the gsh303 gene in two cultivars infected with pathotypes I, III, and VI of A. rabiei at the transcriptome level revealed consistent expression 96 hours post-inoculation across all three pathotypes.The identification of effector genes holds promise for the development of resistance genes based on genomic data, paving the way for the production of more resilient cultivars resistant to disease.