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Host specialization is a key evolutionary process for the diversification and emergence of new pathogens. However, the molecular determinants of host range are poorly understood. Smut fungi are biotrophic pathogens that have distinct and narrow host ranges based on largely unknown genetic determinants. Hence, we aimed to expand comparative genomics analyses of smut fungi by including more species infecting different hosts and to define orphans and positively selected genes to gain further insights into the genetics basis of host specialization. We analyzed nine lineages of smut fungi isolated from eight crop and non-crop hosts: maize, barley, sugarcane, wheat, oats, Zizania latifolia (Manchurian rice), Echinochloa colona (a wild grass), and Persicaria sp. (a wild dicot plant). We assembled two new genomes: Ustilago hordei (strain Uhor01) isolated from oats and U. tritici (strain CBS 119.19) isolated from wheat. The smut genomes were of small sizes, ranging from 18.38 to 24.63 Mb. U. hordei species experienced genome expansions due to the proliferation of transposable elements and the amount of these elements varied among the two strains. Phylogenetic analysis confirmed that Ustilago is not a monophyletic genus and, furthermore, detected misclassification of the U. tritici specimen. The comparison between smut pathogens of crop and non-crop hosts did not reveal distinct signatures, suggesting that host domestication did not play a dominant role in shaping the evolution of smuts. We found that host specialization in smut fungi likely has a complex genetic basis: different functional categories were enriched in orphans and lineage-specific selected genes. The diversification and gain/loss of effector genes are probably the most important determinants of host specificity.

Sporisorium scitamineum is a biotrophic fungus causing sugarcane smut disease. In this study, we set up a pipeline and used genomic and dual transcriptomic data previously obtained by our group to identify candidate effectors of S. scitamineum and their expression profiles in infected smut-resistant and susceptible sugarcane plants. The expression profile of different genes after infection in contrasting sugarcane genotypes assessed by RT-qPCR depended on the plant genotypes and disease progression. Three candidate effector genes expressed earlier only in resistant plants, four expressed in both genotypes, and three later in susceptible plants. Ten genes were cloned and transiently expressed in N. benthamiana leaves to determine their subcellular location, while four localized in more than one compartment. Two candidates, g3890 having a nucleoplasmic and mitochondrial location and g5159 targeting the plant cell wall, were selected to obtain their possible corresponding host targets using co-immunoprecipitation (CoIP) experiments and mass spectrometry. Various potential interactors were identified, including subunits of the protein phosphatase 2A and an endochitinase. We investigated the presence of orthologs in sugarcane and using transcriptome data present their expression profiles. Orthologs of sugarcane shared around 70% similarity. Identifying a set of putative fungal effectors and their plant targets provides a valuable resource for functional characterization of the molecular events leading to smut resistance in sugarcane plants and uncovers further opportunities for investigation.