Chloroplasts are important for photosynthesis and for plant immunity against microbial pathogens. Here we identify a haustorium-specific protein (Pst_12806) from the wheat stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), that is translocated into chloroplasts and affects chloroplast function. Transient expression of Pst_12806 inhibits BAX-induced cell death in tobacco plants and reduces Pseudomonas-induced hypersensitive response in wheat. It suppresses plant basal immunity by reducing callose deposition and the expression of defense-related genes. Pst_12806 is upregulated during infection, and its knockdown (by host-induced gene silencing) reduces Pst growth and development, likely due to increased ROS accumulation. Pst_12806 interacts with the C-terminal Rieske domain of the wheat TaISP protein (a putative component of the cytochrome b6-f complex). Expression of Pst_12806 in plants reduces electron transport rate, photosynthesis, and production of chloroplast-derived ROS. Silencing TaISP by virus-induced gene silencing in a susceptible wheat cultivar reduces fungal growth and uredinium development, suggesting an increase in resistance against Pst infection.

Summary: Small RNAs (sRNAs), an important type of pathogenicity factor, contribute to impairing host immune responses. However, little is known about sRNAs in Puccinia striiformis f. sp. tritici (Pst), one of the most destructive pathogens of wheat (Triticum aestivum L.). Here, we report a novel microRNA‐like RNA (milRNA) from Pst termed microRNA‐like RNA 1 (Pst‐milR1), which suppresses wheat defenses during wheat–Pst interactions. We identified Pst‐milR1 as a novel milRNA in Pst. Biological prediction and co‐transformation showed that Pst‐milR1 takes part in cross‐kingdom RNA interference (RNAi) events by binding the wheat pathogenesis‐related 2 (PR2) gene. Silencing of the Pst‐milR1 precursor resulted in increased wheat resistance to the virulent Pst isolate CYR31. PR2 knock‐down plants increased the susceptibility of wheat to the avirulent Pst isolate CYR23. This suggests that Pst‐milR1 represses the plant immune response by suppressing the expression of PR2. Taking our findings together, we postulate that Pst‐milR1 is an important pathogenicity factor in Pst, which acts as an effector to suppress host immunity. Our results provide significant new insights into the pathogenicity of the stripe rust pathogen.