Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley

Barley leaf stripe is a significant agricultural disease caused by Pyrenophora graminea, which severely affects the yield and quality of barley. During the process of infecting its host, P. graminea secretes a variety of effector proteins to modulate the plant's defense system, thereby enhancing its pathogenicity. The objective of this study is to clarify the function and mechanism of action of the P. graminea effector protein Pg00778, and to provide a theoretical reference for the study of P. graminea.

Based on the previously sequenced genome data of the highly virulent P. graminea strain QWC from our research group, combined with transcriptomic analysis of the P. graminea-highly susceptible barley interaction, a candidate effector protein, Pg00778, was identified. Using the P. graminea wild-type strain QWC as the experimental material, the Pg00778 was cloned, sequence characteristics were analyzed, and a phylogenetic tree was constructed using MEGA11.0 with homologous proteins from P. graminea and other pathogenic fungi. The virulence function of Pg00778 was validated through transient expression in Nicotiana benthamiana leaf cells, while its subcellular localization was determined using tobacco leaf localization assays. Both RNAi mutants and overexpression transformants of Pg00778 were generated via CaCl₂-PEG4000-mediated protoplast transformation. Subsequent assessments of mycelial growth and pathogenicity were conducted to elucidate the function of Pg00778 during P. graminea infection in barley.

Pg00778 lacks signal peptides, transmembrane domains, and known functional domains, categorizing it as a non-classical effector protein. Phylogenetic analysis indicated that Pg00778 has the closest evolutionary relationship with the P. tritici-repentis (XM_066107220.1). The transient expression of Pg00778 in N. benthamian leaves was ineffective in inducing cell death and inhibiting cell death caused by BAX. Pg00778 is mainly localized in the nucleus, cell membrane and cytoplasm. Compared with the wild-type strain QWC, the relative gene expression levels of the Pg00778 in RNAi mutants were reduced by 41.6% and 54.0%, respectively, while the relative gene expression levels of the Pg00778 in OE mutants increased by 290.0%, 397.4%, and 263.7%, respectively. A significant reduction was detected in the growth rate of the RNAi mutants by 21.4% to 30.1%, and an increase in the relative content of chlorophyll in barley leaf by 34.1% to 39.1% after infection. The colony growth rate of the Pg00778-OE mutants was significantly faster than that of the wild-type strain by 7.3% to 12.6%, and the relative content of chlorophyll in barley leaf infected by the OE mutants was significantly lower than that of the wild-type strain by 20.0% to 23.0%. There was no significant difference in the microscopic morphology of mycelium. Pathogenicity test revealed that the incidence of Pg00778-RNAi mutants decreased by 51.5% and 49.0% compared to the QWC, while the Pg00778-OE mutants increased by 19.0%, 20.3%, and 21.2%. Compared with ‘Alexis’ infected by wild-type strain QWC, ‘Alexis’ infected by Pg00778-OE mutants showed more severe stripe disease symptoms, deeper and bluer trypan blue staining, and significantly increased accumulation of reactive oxygen species in the leaves; the stripe invasion symptoms of leaf infected by Pg00778-RNAi mutants were significantly reduced, the cell membrane was more intact, and the accumulation of reactive oxygen species was significantly reduced.

Effector protein Pg00778 positively regulates the pathogenicity of P. graminea and serves as a crucial effector protein influencing its infection to the host.