Characterization of the fludioxonil and phenamacril dual resistant mutants of Fusarium graminearum.

Fusarium graminearum is an important fungal pathogen causing Fusarium head blight (FHB) in wheat and other cereal crops worldwide. Due to lack of resistant wheat cultivars, FHB control mainly relies on application of chemical fungicides. Both fludioxonil (a phenylpyrrole compound) and phenamacril (a cyanoacrylate fungicide) have been registered for controlling FHB in China, however, fludioxonil-resistant isolates of F. graminearum have been detected in field. To evaluate the potential risk of dual resistance of F. graminearum to both compounds, fludioxonil and phenamacril dual resistant (DR) mutants of F. graminearum were obtained via fungicide domestication in laboratory. Result showed that resistance of the DR mutants to both fludioxonil and phenamacril were genetically stable after sub-cultured for ten generations or stored at 4 °C for 30 days on fungicide-free PDA. Cross-resistance assay showed that the DR mutants remain sensitive to other groups of fungicides, including carbendazim, tebuconazole, pydiflumetofen, and fluazinam. In addition, the DR mutants exhibited defects in mycelia growth, conidiation, mycotoxin deoxynivalenol (DON) production, and virulence Moreover, the DR mutants displayed increased sensitivity to osmotic stress. Sequencing results showed that amino acid point mutations S217L/T in the myosin I protein is responsible for phenamacril resistance in the DR mutants. Our results indicate that mutations leading to fludioxonil and phenamacril dual resistance could result in fitness cost for F. graminearum. Our results also suggest that the potential risk of F. graminearum developing resistance to both fludioxonil and phenamacril in field could be rather low, which provides scientific guidance in controlling FHB with fludioxonil and phenamacril.

The jet-like chromatin structure defines active secondary metabolism in fungi.

Eukaryotic genomes are spatially organized within the nucleus in a nonrandom manner. However, fungal genome arrangement and its function in development and adaptation remain largely unexplored. Here, we show that the high-order chromosome structure of Fusarium graminearum is sculpted by both H3K27me3 modification and ancient genome rearrangements. Active secondary metabolic gene clusters form a structure resembling chromatin jets. We demonstrate that these jet-like domains, which can propagate symmetrically for 54 kb, are prevalent in the genome and correlate with active gene transcription and histone acetylation. Deletion of GCN5, which encodes a core and functionally conserved histone acetyltransferase, blocks the formation of the domains. Insertion of an exogenous gene within the jet-like domain significantly augments its transcription. These findings uncover an interesting link between alterations in chromatin structure and the activation of fungal secondary metabolism, which could be a general mechanism for fungi to rapidly respond to environmental cues, and highlight the utility of leveraging three-dimensional genome organization in improving gene transcription in eukaryotes.

Phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

Fusarium head blight caused by Fusarium asiaticum is an important cereal crop disease, and the trichothecene mycotoxins produced by F. asiaticum can contaminate wheat grain, which is very harmful to humans and animals. To effectively control FHB in large areas, the application of fungicides is the major strategy; however, the application of different types of fungicides has varying influences on the accumulation of trichothecene mycotoxins in F. asiaticum. In this study, phenamacril inhibited trichothecene mycotoxin accumulation in F. asiaticum; however, carbendazim (N-1H-benzimidazol-2-yl-carbamic acid, methyl ester) induced trichothecene mycotoxin accumulation. Additionally, phenamacril led to a lower level of reactive oxygen species (ROS) by inducing gene expression of the catalase and superoxide dismutase (SOD) pathways in F. asiaticum, whereas carbendazim stimulated ROS accumulation by inhibiting gene expression of the catalase and SOD pathways. Based on these results, we conclude that phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

SUMOylation regulates low-temperature survival and oxidative DNA damage tolerance in Botrytis cinerea.

SUMOylation as one of the protein post-translational modifications plays crucial roles in multiple biological processes of eukaryotic organisms. Botrytis cinerea is a devastating fungal pathogen and capable of infecting plant hosts at low temperature. However, the molecular mechanisms of low-temperature adaptation are largely unknown in fungi. Combining with biochemical methods and biological analyses, we report that SUMOylation regulates pathogen survival at low temperature and oxidative DNA damage response during infection in B. cinerea. The heat shock protein (Hsp70) BcSsb and E3 ubiquitin ligase BcRad18 were identified as substrates of SUMOylation; moreover, their SUMOylation both requires a single unique SUMO-interacting motif (SIM). SUMOylated BcSsb regulates ß-tubulin accumulation, thereby affecting the stability of microtubules and consequently mycelial growth at low temperature. On the contrary, SUMOylated BcRad18 modulates mono-ubiquitination of the sliding clamp protein proliferating cell nuclear antigen (PCNA), which is involved in response to oxidative DNA damage during infection. Our study uncovers the molecular mechanisms of SUMOylation-mediated low-temperature survival and oxidative DNA damage tolerance during infection in a devastating fungal pathogen, which provides novel insights into low-temperature adaptation and pathogenesis for postharvest pathogens as well as new targets for inhibitor invention in disease control.

Fungicide Resistance: Progress in Understanding Mechanism, Monitoring, and Management.

Fungicide treatments are often essential for maintaining healthy crops and to achieve reliable and high-quality yields. However, continued use of fungicides with the same modes of action can lead to development of fungicide resistance, which has emerged in various plant pathogens and is a serious threat to effective crop protection. Exploration of resistance mechanisms is critical for resistance monitoring and management. This brief review summarizes advances during the past five decades in understanding the molecular resistance mechanisms of plant pathogenic fungi and oomycetes to major classes of fungicides, including benzimidazoles, myosin inhibitors, sterol demethylation inhibitors, quinone outside inhibitors, succinate dehydrogenase inhibitors, anilinopyrimidines, carboxylic acid amides, and oxysterol-binding protein homolog inhibitors. Based on known resistance mechanisms, PCR- and loop-mediated isothermal amplification-based approaches have been developed to allow high-throughput monitoring and early/rapid detection of emerging resistance. Classical principles in fungicide resistance management are also summarized, including using different modes of action of fungicides, limiting the number of applications of the chemicals with site-specific modes of action, and avoidance of their eradicant use. Future studies on novel strategies of disease management, including development of epigenetics- and RNA-based fungicides, will provide valuable knowledge for management of fungicide resistance.

FaSmi1 Is Essential for the Vegetative Development, Asexual Reproduction, DON Production and Virulence of Fusarium asiaticum.

Smi1 is a protein required for cell cycle progression, morphogenesis, stress response and life span of Saccharomyces cerevisiae. FaSmi1 was identified as a Smi1 homolog in a wheat scab pathogenic fungus Fusarium asiaticum strain 2021. The deletion of FaSmi1 leads to defects in mycelial growth, asexual reproduction, and virulence. The FaSmi1 deletion mutant also exhibited increased sensitivity to osmotic stresses generated by NaCl and KCl, but increased tolerance to oxidative stresses and cell wall integrity inhibitors. All of these defects were restored by genetic complementation of the mutant with the whole parental FaSmi1 gene. Interestingly, the antioxidant system-associated genes exhibit a lower expression level and the mycotoxins' DON content was decreased in the FaSmi1 deletion mutant compared with the parental strain 2021. These results indicate that FaSmi1 plays a critical role in the vegetative development, asexual reproduction, DON production and virulence of F. asiaticum.

Biological and molecular characterizations of field fludioxonil-resistant isolates of Fusarium graminearum.

Fusarium head blight (FHB) predominately caused by F. graminearum, is an economical devastating disease for grain cereal crops especially on wheat. The phenylpyrrole fungicide fludioxonil exhibits excellent activity against F. graminearum and has been registered to control FHB in China. In this study, 6 fludioxonil-resistant (FludR) isolates of F. graminearum were identified from 2910 isolates collected from wheat cultivated field in Jiang Su, An Hui and Henan province of China in 2020. The sensitivity assay showed that resistance factor (RF) of FludR isolates ranges from 170.73 to >1000. In comparison with fludioxonil-sensitive (FludS) isolates, all of FludR isolates showed fitness defects in terms of mycelial growth, conidiation and virulence. Under fludioxonil treatment condition, the glycerol accumulation was obviously increased in FludS isolates, but was slightly increased in FludR isolates. Four FludR isolates exhibited increased sensitivity to osmotic stresses. Moreover, there is no positive cross-resistance between fludioxonil and other fungicides including phenamacril, carbendazim and tebuconazole. When treated with fludioxonil, the phosphorylation level of Hog1 was significantly decreased in the four FludR isolates, which was in contrast to the observation in the FludS and two FludR isolates where phosphorylation level of Hog1 was increased. Sequencing assay showed that the mutations were identified in different domains in FgOS1, FgOS2 or FgOS4 in FludR isolates. This was first reported that biological and molecular characterizations of field isolates of F. graminearum resistant to fludioxonil. The results can provide scientific directions for controlling FHB using fludioxonil.

Fusarium graminearum FgSdhC1 point mutation A78V confers resistance to the succinate dehydrogenase inhibitor pydiflumetofen.

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium graminearum complex (Fg) is a devastating disease of cereal crops worldwide. The succinate dehydrogenase inhibitor, pydiflumetofen, was registered for management of FHB in China in 2019. Previously, laboratory-induced pydiflumetofen-resistant (PyR) mutants of Fg have been characterized. However, resistance situation of Fg to pydiflumetofen in the field remains largely unknown. RESULTS: After screening 6468 isolates of Fg from various regions of China, six PyR isolates were identified. All six resistant isolates exhibited no fitness penalties based on mycelial growth, conidiation and virulence. However, no cross-resistance between pydiflumetofen and azoxystrobin, tebuconazole or fludioxonil in Fg was detected. Genome-sequencing revealed that all six PyR isolates contained a point mutation A78V in FgSdhC1 (FgSdhC1A78V ). Genetic replacement assay further confirmed that FgSdhC1A78V conferred resistance of Fg to pydiflumetofen. Based on this, a mismatch allele-specific polymerase chain reaction was developed for rapidly detecting the PyR isolates containing the FgSdhC1A78V mutation in Fg. CONCLUSION: This is the first time that resistance of Fg to pydiflumetofen in the field was reported and point mutation FgSdhC1A78V conferring resistance of Fg to pydiflumetofen was confirmed. This study provides critical information for monitoring and managing pydiflumetofen resistance in Fg.

The Importin FgPse1 Is Required for Vegetative Development, Virulence, and Deoxynivalenol Production by Interacting with the Nuclear Polyadenylated RNA-Binding Protein FgNab2 in Fusarium graminearum.

Karyopherins are involved in transport through nuclear pore complexes. Karyopherins are necessary for nuclear import and export pathways and bind to their cargos. Polyadenylation of messenger RNA (mRNA) is necessary for various biological processes, regulating gene expression in eukaryotes. Until now, the association of karyopherin with mRNA polyadenylation has been less understood in plant pathogenic fungi. In our study, we focused on the biological functions of the karyopherin FgPse1 in Fusarium graminearum. The results showed that FgPse1 is involved in mycelial growth, asexual reproduction, virulence, and deoxynivalenol (DON) production. Co-immunoprecipitation and bimolecular fluorescence complementation showed that FgPse1 interacts with the nuclear polyadenylated RNA-binding protein FgNab2. Moreover, a fluorescence localization assay indicated that FgPse1 is necessary for the nuclear import of FgNab2. The nuclear import of FgNab2 regulates the expression of FgTri4, FgTri5, and FgTri6, which are essential for DON production. Thus, ΔFgPse1 and ΔFgNab2 showed consistent defects in DON production. In summary, our data indicated that FgPse1 is necessary for mycelial growth, virulence, and DON production, interacting with FgNab2 in F. graminearum. These results contribute to our understanding of the functions of importins in phytopathogenic fungi.

Function of the Mitochondrial Transport Protein BcMtp1 in Regulating Vegetative Development, Asexual Reproduction, Stress Response, Fungicide Sensitivity, and Virulence of Botrytis cinerea.

In model fungi, mitochondrial transport proteins (MTPs), also known as "mitochondrial carriers" (MC), are known to facilitate the exchange of biochemical substances across the mitochondrial inner membrane. In this study, we characterized an MTP in Botrytis cinerea homologous to the known MTPs in Saccharomyces cerevisiae designated BcMtp1. The BcMtp1 deletion mutant phenotype was strikingly defective in vegetative development, conidiation, and sclerotia production. In addition, ΔBcMtp1 showed increased sensitivity to osmotic stress, oxidative stress, and cell wall biogenesis inhibitors. In the pathogenicity assay, ΔBcMtp1 displayed compromised virulence on various host-plant tissues. The BcMtp1 deletion mutant phenotype was rescued by transforming the wild-type BcMtp1 variant into the mutant. Together, these data indicate that BcMtp1 is critical for vegetative development, asexual reproduction, stress tolerance, and virulence of B. cinerea.

Plant volatile organic compound (E)-2-hexenal facilitates Botrytis cinerea infection of fruits by inducing sulfate assimilation.

Investigation into plant-fungal pathogen interactions is one of the most interesting fields in plant sciences. However, the roles of plant volatile organic compounds in the arms race are still largely unknown. Based on precise quantification of plant volatiles, we discovered that the plant volatile organic compound (E)-2-hexenal, at concentrations that were similar to or lower than those in tissues of strawberry and tomato fruits, upregulates sulfate assimilation in spores and hyphae of the phytopathogenic fungus Botrytis cinerea. This upregulation is independent of the types of sulfur sources in the plant and can be achieved in the presence of inorganic sulfate and organic sulfur sources. Using the fungal deletion mutants, we further found that sulfate assimilation is involved in the infection of tomato and strawberry fruits by B. cinerea, and that the severity of the disease is proportional to the sulfate content in the fruits. Both before and during the infection, (E)-2-hexenal induced utilisation of plant sulfate by B. cinerea facilitates its pathogenesis through enhancing its tolerance to oxidative stress. This work provides novel insights into the role of plant volatiles in plant-fungal pathogen interaction and highlights the importance of sulfur levels in the host in the prevention of grey mould disease.

Advances in Understanding Fungicide Resistance in Botrytis cinerea in China.

Gray mold, caused by Botrytis cinerea, is a devastating disease that causes significant yield losses in various economically important plants. Fungicide application is one of the main strategies for management of gray mold; however, B. cinerea has developed resistance to various groups of fungicide. In China, benzimidazole-, dicarboximide-, and quinone outside inhibitor-resistant populations of B. cinerea have become dominant. Substitute mutations in fungicide target genes are responsible for resistance in B. cinerea. Based on known resistance mechanisms, molecular methods including loop-mediated isothermal amplification have been developed for rapid detection of resistant isolates of B. cinerea. Because B. cinerea is able to quickly develop resistance to various fungicides, various integrated strategies have been implemented in the last decade, including biological and agricultural practices, to manage fungicide resistance in B. cinerea.

The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors.

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov), respectively, are among the most devastating diseases in cotton (Gossypium spp). To understand the cotton response to these soil-borne fungal pathogens, we performed a genome-wide in silico characterization and functional screen of diverse RLKs for their involvement in cotton wilt diseases. We identified Gossypium hirsutum GhWAK7A, a wall-associated kinase, that positively regulates cotton response to both Vd and Fov infections. Chitin, the major constituent of the fungal cell wall, is perceived by lysin-motif-containing RLKs (LYKs/CERK1), leading to the activation of plant defense against fungal pathogens. A conserved chitin sensing and signaling system is present in cotton, including chitin-induced GhLYK5-GhCERK1 dimerization and phosphorylation, and contributes to cotton defense against Vd and Fov Importantly, GhWAK7A directly interacts with both GhLYK5 and GhCERK1 and promotes chitin-induced GhLYK5-GhCERK1 dimerization. GhWAK7A phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation for its function. Consequently, GhWAK7A plays a crucial role in chitin-induced responses. Thus, our data reveal GhWAK7A as an important component in cotton response to fungal wilt pathogens by complexing with the chitin receptors.

Amino Acid Polymorphism in Succinate Dehydrogenase Subunit C Involved in Biological Fitness of Botrytis cinerea.

Succinate dehydrogenase (SDH) is an important respiratory enzyme which participates in the tricarboxylic acid cycle and oxidative phosphorylation. A previous study of the baseline sensitivity of Botrytis cinerea against SDH inhibitors (SDHIs) showed that intrinsic sensitivity of the small population against the SDHIs exhibited significant differences. In the sequencing assay, we found five kinds of amino acid polymorphism in SDH subunit C (SdhC) of B. cinerea isolates which were never exposed to the SDHIs. To validate that amino acid polymorphism in the SdhC of B. cinerea confers intrinsic sensitivity against the SDHIs, the replacement mutants containing each kind of amino acid polymorphism of SdhC exhibited phenotype differences in intrinsic sensitivity to SDHIs, mycelial growth, sporulation, virulence, oxidative stress response, and carbon source utilization. These results indicated that SdhC of B. cinerea experienced positive selection during evolution and resulted in amino acid polymorphism which is involved in intrinsic sensitivity to SDHIs and biological fitness.

The Receptor Kinases BAK1/SERK4 Regulate Ca2+ Channel-Mediated Cellular Homeostasis for Cell Death Containment.

Cell death is a vital and ubiquitous process that is tightly controlled in all organisms. However, the mechanisms underlying precise cell death control remain fragmented. As an important shared module in plant growth, development, and immunity, Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate plant cell death. By deploying an RNAi-based genetic screen for bak1/serk4 cell death suppressors, we revealed that cyclic nucleotide-gated channel 20 (CNGC20) functions as a hyperpolarization-activated Ca2+-permeable channel specifically regulating bak1/serk4 cell death. BAK1 directly interacts with and phosphorylates CNGC20 at specific sites in the C-terminal cytosolic domain, which in turn regulates CNGC20 stability. CNGC19, the closest homolog of CNGC20 with a low abundance compared with CNGC20, makes a quantitative genetic contribution to bak1/serk4 cell death only in the absence of CNGC20, supporting the biochemical data showing homo- and heteromeric assembly of the CNGC20 and CNGC19 channel complexes. Transcripts of CNGC20 and CNGC19 are elevated in bak1/serk4 compared with wild-type plants, further substantiating a critical role of homeostasis of CNGC20 and CNGC19 in cell death control. Our studies not only uncover a unique regulation of ion channel stability by cell-surface-resident receptor kinase-mediated phosphorylation but also provide evidence for fine-tuning Ca2+ channel functions in maintaining cellular homeostasis by the formation of homo- and heterotetrameric complexes.

The endocytic cargo adaptor complex is required for cell-wall integrity via interacting with the sensor FgWsc2B in Fusarium graminearum.

AP2 is a heterotetrameric clathrin adaptor complex that owns important roles in vesicle generation and cargo recognition. Cell-wall integrity (CWI) pathway is essential for fungal development, virulence, and adaptation to environment stresses. To date, the relationship between AP2 and CWI is largely unknown in phytopathogenic fungi. In this study, we identified the adaptor complex FgAP2 in Fusarium graminearum. The biological function analysis showed that FgAP2 complex contains FgAP2α, FgAP2ß, FgAP2σ, and FgAP2µ, and the subunit FgAP2µ, which is required for hyphal growth, conidiation, CWI, and virulence. Yeast two-hybrid showed that FgAP2µ interacts with the CWI sensor FgWsc2B. Consistently, western blotting analysis revealed that FgWsc2B positively regulates phosphorylation of FgMgv1, the MAP kinase of CWI. Moreover, the FgWsc2B deletion mutant exhibited defects in hyphal growth, virulence, and response to CWI damaging agents. Taken together, our data indicated that FgAP2µ is involved in CWI and virulence via interacting with FgWsc2B in F. graminearum.

Involvement of the two L-lactate dehydrogenase in development and pathogenicity in Fusarium graminearum.

Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD+. In this study, two L-type lactate dehydrogenase genes FgLDHL1 and FgLDHL2 were characterized in an ascomycete fungus Fusarium graminearum, a causal agent of wheat head blight. Both the single-gene deletion mutants of FgLDHL1 or FgLDHL2 exhibited phenotypic defects in vegetative growth, sporulation, spore germination, L-lactate biosynthesis and activity. Additionally, the two L-lactate dehydrogenases were involved in the utilization of carbon sources and maintenance of redox homeostasis during spore germination. Pathogenicity assays showed that ΔFgLDHL1 exhibits reduced virulence on wheat spikelets and on corn stigmas, suggesting that it was indirectly correlated with a reduced level of deoxynivalenol accumulation. These results indicate that FgLDHL1 and FgLDHL2 play multiple roles in the developmental processes and pathogenesis in F. graminearum, and help understand the functional diversity of D-/L-lactate dehydrogenase in phytopathogenic fungi.

Involvement of BcElp4 in vegetative development, various environmental stress response and virulence of Botrytis cinerea.

The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis and formation of modified wobble uridines in tRNA. In this study, we investigated the function of BcElp4 in Botrytis cinerea, which is homologous to S. cerevisiae Elp4. A bcelp4 deletion mutant was significantly impaired in vegetative growth, sclerotia formation and melanin biosynthesis. This mutant exhibited decreased sensitivity to osmotic and oxidative stresses as well as cell way-damaging agent. Pathogenicity assays revealed that BcElp4 is involved in the virulence of B. cinerea. In addition, the deletion of bcelp4 led to increased aerial mycelia development. All these defects were restored by genetic complementation of the bcelp4 deletion mutant with the wild-type bcelp4 gene. The results of this study indicated that BcElp4 is involved in regulation of vegetative development, various environmental stress response and virulence in B. cinerea.

The autophagy-related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea.

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that the expression of BcATG1 was up-regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re-introduction of an intact copy of the wild-type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.

The septins FaCdc3 and FaCdc12 are required for cytokinesis and affect asexual and sexual development, lipid metabolism and virulence in Fusarium asiaticum.

Septins are a highly conserved family of GTP-binding proteins that contribute to many cellular and metabolic functions, including cell polarity, cytokinesis, cell morphogenesis and pathogenesis. In this study, we characterized the septins FaCdc3 and FaCdc12 in the filamentous fungus Fusarium asiaticum. The functions of FaCdc3 and FaCdc12 were evaluated by constructing deletion mutants of FaCdc3 and FaCdc12, designated ΔFaCdc3-5 and ΔFaCdc12-71, respectively. The deletion mutants exhibited a reduced rate of mycelial growth, increased aerial hyphae formation, irregularly shaped hyphae, reduced conidiation and a lack of sexual reproduction in wheat kernels. Histochemical analysis revealed that the conidia and hyphae of ΔFaCdc3-5 and ΔFaCdc12-71 formed large lipid droplets (LDs). ΔFaCdc3-5 and ΔFaCdc12-71 also exhibited increased resistance to agents that induce osmotic stress and damage the cell membrane and cell wall. In addition, the hyphae and conidia of the two mutants formed fewer septa than those of the wild-type and exhibited aberrant nuclear distribution. Pathogenicity assays showed that ΔFaCdc3-5 and ΔFaCdc12-71 exhibited reduced virulence on wheat spikelets, which was indirectly correlated with a reduced level of deoxynivalenol accumulation. All of these defects were restored by genetic complementation of the two mutants with the parental FaCdc3 and FaCdc12. These results indicate that FaCdc3 and FaCdc12 play a critical role in various cellular processes in F. asiaticum.