Cloning and functional characterization of candidate virulence/avirulence factors in parasitic plant Striga gesnerioides

The root parasitic weed Striga gesnerioides significantly impedes cowpea (Vigna unguiculata L.) cultivation in sub-Saharan Africa (SSA), where cowpea is a crucial and nutrient-rich legume, fundamental as both a dietary staple and an agricultural asset. Cowpea cultivars such as B301, which are resistant (incompatible), exhibit a hypersensitive response (HR) at the site of parasite attachment, leading to the death of the invading S. gesnerioides. In contrast, susceptible (compatible) varieties like Blackeye allow the parasite to establish a xylem-xylem connection, resulting in successful parasitization.
Previous comparative transcriptomic study identified multiple virulence/avirulence genes that may shed light on the molecular mechanisms underlying Striga parasitism. Through the alignment of contig sequences from the comparative transcriptomic analysis with the S gesnerioides race 3 (SG3) genome assembly, I successfully identified and cloned the full-length coding sequence (CDS) of five genes. These genes are: SGall_094658.3 (SGCHE) encoding a carbohydrate esterase, SGall_013151.2 (SGRE) encoding a steroid 5-α reductase domain-containing protein, SGall_037195.1 (SGCYP) encoding a cytochrome P450 superfamily protein, SGall_060151.1 (SGFAD) encoding a flavin adenine dinucleotide (FAD)-binding berberine family protein, and SGall_073984.4 (SGPOX), encoding a peroxidase. In addition, I isolated three expansin genes from S. gesnerioides. The successful cloning of these genes lays the groundwork for further exploration of their roles in Striga parasitism.
Next, I conducted functional characterization of candidate factors SGCHE and SGFAD. Full-length SGCHE and SGFAD genes and corresponding truncated versions lacking the N-terminal apoplastic signal peptide sequences, SGCHE∆SP and SGFAD∆SP were transformed into cowpea B301 to create composite plants. I found that transgenic B301 roots overexpressing SGCHE, SGCHE∆SP, SGFAD, and SGFAD∆SP demonstrated enhanced host immunity to S gesnerioides race 4z (SG4z) attack as indicated by increased HR and reduced parasite cotyledon expansion (CE). Furthermore, RT-qPCR analysis showed that B301 roots overexpressing SGCHE, SGCHE∆SP, SGFAD, and SGFAD∆SP exhibited significantly upregulated levels of cowpea resistance genes RSG3-301, VuPOB1, GRF, Narbonin, NIP1, Lipoxygenase, Heat shock protein, Endochitinase, COI1, PR1 compared to non-transgenic roots when challenged by SG4z. These results suggest that SGCHE and SGFAD are important avirulence factors capable of activating host immune responses against S. gesnerioides.
Finally, I also prove that papain-like cysteine protease from S. gesnerioides (SGCP) plays a role as a potential avirulence factor that can elicit host immunity against S. gesnerioides.
In summary, this dissertation offers fresh perspectives on the mechanisms through which avirulence factors in parasitic plants trigger host defense responses during infestation. It advances the understanding of molecular interactions between S. gesnerioides and host cowpea, providing a foundation for developing effective strategies to combat this parasitic weed in crop cultivation.