As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study. Fusarium head blight caused by Fusarium graminearum is one of the most important diseases on wheat and barley. Although protein kinases are known to play major regulatory roles in fungi, systematic characterization of fungal kinomes has not been reported in plant pathogens. In this study we generated deletion mutants for 96 protein kinase genes. All of the resulting knockout mutants were assayed for changes in 17 phenotypes, including growth, reproduction, stress responses, and plant infection. Overall, deletion of 64 kinase genes resulted in at least one of the phenotypes examined. In total, 42 kinase mutants were significantly reduced in virulence or non-pathogenic. A number of these protein kinase genes, including two that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores are the primary inoculum for wheat scab. We identified 26 mutants blocked in ascospore. We also used the in silico approach to predict the kinase-kinase interactions and verified some of them by yeast two-hybrid or co-IP assays. Overall, in this study we functionally characterize the kinome of F. graminearum. Protein kinase genes that are important for various aspects of growth, developmental, and plant infection processes were identified.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the ‘fosmid-to-fosmid’ strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.

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.