CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in Sporisorium reilianum.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum. Key features • First CRISPR/Cas9 application in S. reilianum. • Generation of S. reilianum mutants without genomic integration of resistance marker. • Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.

A transcriptional activator effector of Ustilago maydis regulates hyperplasia in maize during pathogen-induced tumor formation.

Ustilago maydis causes common smut in maize, which is characterized by tumor formation in aerial parts of maize. Tumors result from the de novo cell division of highly developed bundle sheath and subsequent cell enlargement. However, the molecular mechanisms underlying tumorigenesis are still largely unknown. Here, we characterize the U. maydis effector Sts2 (Small tumor on seedlings 2), which promotes the division of hyperplasia tumor cells. Upon infection, Sts2 is translocated into the maize cell nucleus, where it acts as a transcriptional activator, and the transactivation activity is crucial for its virulence function. Sts2 interacts with ZmNECAP1, a yet undescribed plant transcriptional activator, and it activates the expression of several leaf developmental regulators to potentiate tumor formation. On the contrary, fusion of a suppressive SRDX-motif to Sts2 causes dominant negative inhibition of tumor formation, underpinning the central role of Sts2 for tumorigenesis. Our results not only disclose the virulence mechanism of a tumorigenic effector, but also reveal the essential role of leaf developmental regulators in pathogen-induced tumor formation.

Predicting Task-Driven Attention via Integrating Bottom-Up Stimulus and Top-Down Guidance.

Task-free attention has gained intensive interest in the computer vision community while relatively few works focus on task-driven attention (TDAttention). Thus this paper handles the problem of TDAttention prediction in daily scenarios where a human is doing a task. Motivated by the cognition mechanism that human attention allocation is jointly controlled by the top-down guidance and bottom-up stimulus, this paper proposes a cognitively-explanatory deep neural network model to predict TDAttention. Given an image sequence, bottom-up features, such as human pose and motion, are firstly extracted. At the same time, the coarse-grained task information and fine-grained task information are embedded as a top-down feature. The bottom-up features are then fused with the top-down feature to guide the model to predict TDAttention. Two public datasets are re-annotated to make them qualified for TDAttention prediction, and our model is widely compared with other models on the two datasets. In addition, some ablation studies are conducted to evaluate the individual modules in our model. Experiment results demonstrate the effectiveness of our model.

Cross-species analysis between the maize smut fungi Ustilago maydis and Sporisorium reilianum highlights the role of transcriptional change of effector orthologs for virulence and disease.

The constitution and regulation of effector repertoires shape host-microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies. We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross-species RNA-sequencing analyses. We identified 207 of 335 one-to-one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies. Using CRISPR-Cas9-mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species-specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization. Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins.

Triple Multi-scale Adversarial Learning with Self-attention and Quality Loss for Unpaired Fundus Fluorescein Angiography Synthesis.

Clinically, the Fundus Fluorescein Angiography (FA) is a more common mean for Diabetic Retinopathy (DR) detection since the DR appears in FA much more contrasty than in Color Fundus Image (CF). However, acquiring FA has a risk of death due to the fluorescent allergy. Thus, in this paper, we explore a novel unpaired CycleGAN-based model for the FA synthesis from CF, where some strict structure similarity constraints are employed to guarantee the perfectly mapping from one domain to another one. First, a triple multi-scale network architecture with multi-scale inputs, multi-scale discriminators and multi-scale cycle consistency losses is proposed to enhance the similarity between two retinal modalities from different scales. Second, the self-attention mechanism is introduced to improve the adaptive domain mapping ability of the model. Third, to further improve strict constraints in the feather level, quality loss is employed between each process of generation and reconstruction. Qualitative examples, as well as quantitative evaluation, are provided to support the robustness and the accuracy of our proposed method.

Cas9HF1 enhanced specificity in Ustilago maydis.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.

Effectors with Different Gears: Divergence of Ustilago maydis Effector Genes Is Associated with Their Temporal Expression Pattern during Plant Infection.

Plant pathogens secrete a variety of effector proteins that enable host colonization but are also typical pathogen detection targets for the host immune system. Consequently, effector genes encounter high selection pressures, which typically makes them fast evolving. The corn smut pathogen Ustilago maydis has an effector gene repertoire with a dynamic expression across the different disease stages. We determined the amino acid divergence of U. maydis effector candidates with Sporisorium reilianum orthologs, a close relative of U. maydis. Intriguingly, there are two distinct groups of effector candidates, ones with a respective conserved and diverged protein evolution. Conservatively evolving effector genes especially have their peak expression during the (pre-)penetration stages of the disease cycle. In contrast, expression of divergently evolving effector genes generally peaks during fungal proliferation within the host. To test if this interspecific effector diversity corresponds to intraspecific diversity, we sampled and sequenced a diverse collection of U. maydis strains from the most important maize breeding and production regions in China. Effector candidates with a diverged interspecific evolution had more intraspecific amino acid variation than candidates with a conserved evolution. In conclusion, we highlight diversity in evolution within the U. maydis effector repertoire with dynamically and conservatively evolving members.

Molecular Interactions Between Smut Fungi and Their Host Plants.

Smut fungi are a large group of biotrophic plant pathogens that infect mostly monocot species, including economically relevant cereal crops. For years, Ustilago maydis has stood out as the model system to study the genetics and cell biology of smut fungi as well as the pathogenic development of biotrophic plant pathogens. The identification and functional characterization of secreted effectors and their role in virulence have particularly been driven forward using the U. maydis-maize pathosystem. Today, advancing tools for additional smut fungi such as Ustilago hordei and Sporisorium reilianum, as well as an increasing number of available genome sequences, provide excellent opportunities to investigate in parallel the effector function and evolution associated with different lifestyles and host specificities. In addition, genome analyses revealed similarities in the genomic signature between pathogenic smuts and epiphytic Pseudozyma species. This review elaborates on how knowledge about fungal lifestyles, genome biology, and functional effector biology has helped in understanding the biology of this important group of fungal pathogens. We highlight the contribution of the U. maydis model system but also discuss the differences from other smut fungi, which raises the importance of comparative genomic and genetic analyses in future research.

Cytological and Molecular Characterization of ZmWAK-Mediated Head-Smut Resistance in Maize.

Head smut, caused by the fungal pathogen Sporisorium reilianum, poses a threat to maize production worldwide. ZmWAK, a cell wall-associated receptor kinase, confers quantitative resistance to head smut disease. Here, two near-isogenic lines (NILs), susceptible line Huangzao4 and its ZmWAK-converted resistant line Huangzao4R, were used to decipher the role of ZmWAK in head smut resistance. Cytological and molecular characterization in response to S. reilianum infection was compared between two NILs. Upon S. reilianum infection, the growth of pathogen hyphae was severely arrested in the ZmWAK-converted resistant line Huangzao4R, relative to its susceptible parental line Huangzao4. Infected cells exhibited apoptosis-like features in Huangzao4R and hyphae were sequestered within dead cells, whereas pathogen invasion caused autophagy in Huangzao4, which failed to prevent hyphal spreading. Integrated transcriptomic and metabolomic analysis indicated that ZmWAK functions as a hub in the trade-off between growth and defense, whereby ZmWAK promotes cell growth in the absence of the pathogen and switches to a defense response upon S. reilianum attack. These findings shed light on an elegant regulatory mechanism governed by ZmWAK in the trade-off between growth and head smut defense.

An Atypical Thioredoxin Imparts Early Resistance to Sugarcane Mosaic Virus in Maize.

Sugarcane mosaic virus (SCMV) causes substantial losses of grain yield and forage biomass in susceptible maize worldwide. A major quantitative trait locus, Scmv1, has been identified to impart strong resistance to SCMV at the early infection stage. Here, we demonstrate that ZmTrxh, encoding an atypical h-type thioredoxin, is the causal gene at Scmv1, and that its transcript abundance correlated strongly with maize resistance to SCMV. ZmTrxh alleles, whether they are resistant or susceptible, share the identical coding/proximal promoter regions, but vary in the upstream regulatory regions. ZmTrxh lacks two canonical cysteines in the thioredoxin active-site motif and exists uniquely in the maize genome. Because of this, ZmTrxh is unable to reduce disulfide bridges but possesses a strong molecular chaperone-like activity. ZmTrxh is dispersed in maize cytoplasm to suppress SCMV viral RNA accumulation. Moreover, ZmTrxh-mediated maize resistance to SCMV showed no obvious correlation with the salicylic acid- and jasmonic acid-related defense signaling pathways. Taken together, our results indicate that ZmTrxh exhibits a distinct defense profile in maize resistance to SCMV, differing from previously characterized dominant or recessive potyvirus resistance genes.

A maize wall-associated kinase confers quantitative resistance to head smut.

Head smut is a systemic disease in maize caused by the soil-borne fungus Sporisorium reilianum that poses a grave threat to maize production worldwide. A major head smut quantitative resistance locus, qHSR1, has been detected on maize chromosome bin2.09. Here we report the map-based cloning of qHSR1 and the molecular mechanism of qHSR1-mediated resistance. Sequential fine mapping and transgenic complementation demonstrated that ZmWAK is the gene within qHSR1 conferring quantitative resistance to maize head smut. ZmWAK spans the plasma membrane, potentially serving as a receptor-like kinase to perceive and transduce extracellular signals. ZmWAK was highly expressed in the mesocotyl of seedlings where it arrested biotrophic growth of the endophytic S. reilianum. Impaired expression in the mesocotyl compromised ZmWAK-mediated resistance. Deletion of the ZmWAK locus appears to have occurred after domestication and spread among maize germplasm, and the ZmWAK kinase domain underwent functional constraints during maize evolution.