Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis. Fungal pathogens require virulence factors to be properly secreted and localized to guarantee complete infection. In common with many proteins, virulence factors must be post-translationally modified by glycosylation for normal localization, secretion and function. This is especially important for virulence factors, which are mainly comprised of cell wall and secreted proteins. Aberrant glycosylation leads to a loss of virulence in both animal and plant pathogenic fungi. We have previously demonstrated that glycosylation is important for virulence of the corn smut fungus, Ustilago maydis. However, the glycoproteins involved and their specific roles in the infection process have not yet been reported. Here, we describe a proteomic assay designed to identify glycoproteins involved in plant infection. Using this method, we define the role of Pdi1 protein disulfide isomerase in virulence. Interestingly, abolishing Pdi1 N-glycosylation mimics Δpdi1 defects observed during infection, suggesting that Pdi1 N-glycosylation is required for the normal secretion of virulence factors. We hypothesize that Pdi1 N-glycosylation is necessary for maintaining proper effector protein folding during the infection process, especially in the harsh conditions found inside the maize plant.

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs) play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis. Many pathogenic fungi need to undergo morphological changes in order to infect their hosts. Typically, pathogenic fungi switch from a non-pathogenic yeast-like form to a polarised pathogenic filament. This morphological switch is regulated genetically and is triggered by specific environmental conditions. Histone deacetylases (HDACs) are important regulators of chromatin structure and gene expression. In this study, we investigate the role of HDACs as targets of the signalling pathways that activate fungal virulence programs in response to specific external signals. We identify two specific HDACs, Hos2 and Clr3, that are required for the virulence of the corn smut fungus, Ustilago maydis. Our results reveal that Hos2 and Clr3 function in the cAMP-PKA cascade, a nutrient-sensing pathway conserved between all eukaryotes.

Plant pathogenic fungi must be able to degrade host cell walls in order to penetrate and invade plant tissues. Among the plant cell wall degrading enzymes (PCWDEs) produced, xylanases are of special interest since its degradation target, xylan, is one of the main structural polysaccharides in plant cell walls. In the biotrophic fungus Ustilago maydis, attempts to characterize PCWDEs required for virulence have been unsuccessful, most likely due to functional redundancy. In previous high-throughput screening, we found one xylanase to be important for U. maydis infection. Here, we characterize the entire U. maydis endo-xylanase family, comprising two enzymes from the glycoside hydrolase (GH) 10 family, Xyn1 and Xyn2, one from GH11, Xyn11A, and one from GH43, Xyn3. We show that all endo-xylanases except Xyn3 are secreted and involved in infection in a non-redundant manner, suggesting different roles for each xylanase in this process. Taking a closer look inside the plant during the pathogenic process, we observed that all secreted xylanases were necessary for fungal proliferation. Finally, we found that at least Xyn11A accumulated in the apoplast of the infected plant after three days, highlighting the role of these enzymes as important secreted proteins during fungal proliferation inside plant tissues.

CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates.