Expression of a Structural Protein of the Mycovirus FgV-ch9 Negatively Affects the Transcript Level of a Novel Symptom Alleviation Factor and Causes Virus Infection-Like Symptoms in Fusarium graminearum

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus- like mycovirus, debilitates Fusarium graminearum, the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1. This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence. IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics

Complete Functional Verification

The dissertation describes a practically proven, particularly efficient approach for the verification of digital circuit designs. The approach outperforms simulation based verification wrt. final circuit quality as well as wrt. required verification effort. In the dissertation, the paradigm of transaction based verification is ported from simulation to formal verification. One consequence is a particular format of formal properties, called operation properties. Circuit descriptions are verified by proof of operation properties with Interval Property Checking (IPC), a particularly strong SAT based formal verification algorithm. Furtheron, a completeness checker is presented that identifies all verification gaps in sets of operation properties. This completeness checker can handle the large operation properties that arise, if this approach is applied to realistic circuits. The methodology of operation properties, Interval Property Checking, and the completeness checker form a symbiosis that is of particular benefit to the verification of digital circuit designs. On top of this symbiosis an approach to completely verify the interaction of completely verified modules has been developed by adaptation of the modelling theories of digital systems. The approach presented in the dissertation has proven in multiple commercial application projects that it indeed completely verifies modules. After reaching a termination criterion that is well defined by completeness checking, no further bugs were found in the verified modules. The approach is marketed by OneSpin Solutions GmbH, Munich, under the names "Operation Based Verification" and "Gap Free Verification"

Unbalanced roles of fungal aggressiveness and host cultivars in the establishment of the Fusarium head blight in bread wheat

International audienceFusarium head blight (FHB), caused mainly by Fusarium graminearum, is the foremost destructive disease of cereals worldwide. Effector-like molecules produced by F. graminearum play key roles in the infection process and are assumed to be one of the essential components of the pathogen's aggressiveness. However, their nature and role in the disease are still largely misunderstood. As a mean to provide relevant information about the molecular determinism of F. graminearum aggressiveness, we surveyed three F. graminearum strains on three wheat cultivars contrasted by their susceptibility to FHB. F. graminearum strains revealed large differences in aggressiveness which were mostly unchanged when facing hosts of contrasted susceptibility, suggesting that their behavior rely on intrinsic determinants. Surveying the fungal mass progress and the mycotoxin production rate in the spikes did not evidence any simple relationship with aggressiveness differences, while clues were found through a qualitative and quantitative characterization of the three strain proteomes established in planta especially with regards to early synthesized putative effectors. Independently of the wheat cultivar, the three F. graminearum strains produced systematically the same protein set during the infection but substantial differences in their abundance enabled the categorization of fungal aggressiveness. Overall, our findings show that the contrasts in F. graminearum aggressiveness were not based on the existence of strain-specific molecules but rather on the ability of the strain to ensure their sufficient accumulation. Protein abundance variance was mostly driven by the strain genetics and part was also influenced by the host cultivar but strain by cultivar interactions were marginally detected, depicting that strain-specific protein accumulations did not depend on the host cultivar. All these data provide new knowledge on fungal aggressiveness determinants and provide a resourceful repertoire of candidate effector proteins to guide further research

Comparative genomics of two Fusarium graminearum strains of contrasting aggressiveness in bread wheat

By targeting host cellular processes, fungal effectors promote growth and spreading of pathogenic fungi in plant tissues. During the wheat/Fusarium graminearum (Fg) interaction causing the Fusarium head blight (FHB) disease, the nature and the function of these fungal molecular components which control plant susceptibility factors, remain largely unknown. Two Fg strains (Fg1 and Fu10008) contrasting for their aggressiveness in field trials were sequenced using Sequel technology. Fg1 and Fu10008 displayed 11,171 CDS (37,2Mb) and 10,629 CDS (36,1Mb), respectively. The comparative analysis of the predicted proteomes revealed 1,320 specific proteins in Fg1 and 693 in Fu10008, comprising 205 and 81 candidate putative effectors, respectively. A thorough pathotyping of the two Fg strains on three wheat genotypes of different susceptibility to FHB was assessed to test their mycotoxin production and their infection dynamics, including symptoms development and fungal biomass progress in point-inoculated spikelets (PI) and in the uninoculated peripheral ones (Up or Dn). Fg1 strain induced systematically the most severe symptoms in the PI, Up and Dn parts of each wheat genotypes. Whatever the infected genotype, Fg1 initiated symptom development 24 h earlier than Fu10008. Spreading of both strains in Up and Dn spikelets appeared preferentially towards the top of the spike as early as 5 days post-inoculation, displaying symptoms very close to those of the PI parts suggesting that they are related to fungal migration in healthy plant tissues. qRT-PCR and mycotoxin analyzes (DON and ZEA) of the different spike zones are currently performed to refine the early stages of the infection process of both strains. This will allow for connecting these Fg data to recent work investigating the wheat susceptibility factors (Chetouhi et al., 2016) and will contribute to shape an integrated picture of the molecular events piloting FHB