Search

Reactive oxygen/nitrogen species (ROS/RNS) play a fundamental role in plant–fungal interactions. How pathogenic fungi manipulate plant‐derived ROS/RNS is of importance to the outcomes of these interactions. In this study, we explored the individual and combined contributions of three transcription factors, VdAtf1, VdYap1, and VdSkn7, in the response to ROS/RNS, microsclerotia formation, and virulence in the plant wilt pathogen Verticillium dahliae. We showed that VdYap1 is essential for ROS response. Additionally, mutants lacking any combination of the three genes shared significant hypersensitivity to nitro‐oxidative stress like sodium nitroprusside dehydrate and double deletions lacking VdYap1 and VdAtf1 resulted in further increased sensitivity to ROS. Double deletion of VdAtf1 and VdSkn7 reduced melanin production and virulence while simultaneous lack of VdSkn7 and VdYap1 disrupted nitrogen metabolism and ROS resistance. Finally, comparison of transcriptional profiles of the respective single or double mutants in response to nitro‐oxidative stress revealed that the three transcription factors are involved in denitrification of nitrated alkanes and lipids to protect against nitro‐oxidative stress. Taken together, our results demonstrate convergent and distinctive functions of VdYap1, VdAtf1, and VdSkn7 in V. dahliae, and provide new data on their roles in response to ROS/RNS in fungi. [ABSTRACT FROM AUTHOR]

Summary: The fungus Verticillium dahliae causes vascular wilt disease on hundreds of plant species. Homologs of the bZIP transcription factor Atf1 are required for virulence in most pathogenic fungi, but the molecular basis for their involvement is largely unknown.We performed targeted gene deletion, expression analysis, biochemistry and pathogenicity assays to demonstrate that VdAtf1 governs pathogenesis via the regulation of nitrosative resistance and nitrogen metabolism in V. dahliae.VdAtf1 controls pathogenesis via the regulation of nitric oxide (NO) resistance and inorganic nitrogen metabolism rather than oxidative resistance and is important for penetration peg formation in V. dahliae. VdAtf1 affects ammonium and nitrate assimilation in response to various nitrogen sources. VdAtf1 may be involved in regulating the expression of VdNut1. VdAtf1 responds to NO stress by strengthening the fungal cell wall, and by causing over‐accumulation of methylglyoxal and glycerol, which in turn impacts NO detoxification. We also verified that the VdAtf1 ortholog in Fusarium graminearum mediates nitrogen metabolism, suggesting conservation of this function in related plant pathogenic fungi.Our findings revealed new functions of VdAtf1 in pathogenesis, response to nitrosative stress and nitrogen metabolism in V. dahliae. The results provide novel insights into the regulatory mechanisms of the transcription factor VdAtf1 in virulence. [ABSTRACT FROM AUTHOR]

Transcriptional regulation is critical for plant pathogen development and virulence. MADS-box transcription factors belong to a highly conserved transcriptional regulator family in eukaryotic organisms that are involved in various important biological processes. Only one predicted MADS-box gene, PsMAD1 , was identified in Phytophthora sojae , which was highly expressed during the sporangia and infection stages. To investigate its function, we generated PsMAD1 knockout mutants using the CRISPR/Cas9 system. Compared with the wild-type strain, the mutants showed no changes in vegetative growth, oospore production, or no differences in sensitivity to various abiotic stresses. Although sporangia production was normal, no zoospore release was detected in PsMAD1 mutants. Microscopy analyses revealed failure of cleavage of the cytoplasm into uninucleate zoospores in the mutants. In addition, the mutants showed reduced virulence in soybean. RNA-seq data indicated that PsMAD1 may regulate many zoospore development and infection associated genes. Thus, PsMAD1 may be a major regulator of P. sojae involved in zoosporogenesis and pathogenesis. [ABSTRACT FROM AUTHOR]

Yeast AP1 transcription factor is a regulator of oxidative stress response. Here, we report the identification and characterization of CgAP1, an ortholog of YAP1 in poplar anthracnose fungus Colletotrichum gloeosporioides . The expression of CgAP1 was highly induced by reactive oxygen species. CgAP1 deletion mutants displayed enhanced sensitivity to oxidative stress compared with the wild-type strain, and their poplar leaf virulence was obviously reduced. However, the mutants exhibited no obvious defects in aerial hyphal growth, conidia production, and appressoria formation. CgAP1::eGFP fusion protein localized to the nucleus after TBH ( tert -Butyl hydroperoxide) treatment, suggesting that CgAP1 functions as a redox sensor in C. gloeosporioides . In addition, CgAP1 prevented the accumulation of ROS during early stages of biotrophic growth. CgAP1 also acted as a positive regulator of several ROS-related genes (i.e., Glr1 , Hyr1 , and Cyt1 ) involved in the antioxidative response. These results highlight the key regulatory role of CgAP1 transcription factor in oxidative stress response and provide insights into the function of ROS detoxification in virulence of C. gloeosporioides . [ABSTRACT FROM AUTHOR]

Calcium signaling plays crucial roles in ion stress tolerance, sporulation and pathogenicity in fungi. Although the signaling pathway mediated by calcineurin and the calcineurin-responsive zinc finger transcription factor Crz1 is well characterized in other fungi, this pathway is not well characterized in the phytopathogenic fungus, Verticillium dahliae . To better understand the role of this calcineurin-dependent transcription factor in V. dahliae , an ortholog of CRZ1 , VdCrz1 , was identified and characterized functionally. Transcriptional analysis of VdCrz1 and GFP expression driven by the VdCrz1 promoter indicated that VdCrz1 was involved in microsclerotia development. After targeted deletion of VdCrz1 , microsclerotia formation and melanin accumulation were impaired. Furthermore, the Δ VdCrz1 mutants were hypersensitive to high concentrations of Ca 2+ and cell wall-perturbing agents, such as sodium dodecyl sulfate. The addition of Mg 2+ to the medium restores the microsclerotia formation in Δ VdCrz1 mutants. The Δ VdCrz1 mutants exhibited delayed Verticillium wilt symptoms on smoke tree. These results suggest that VdCrz1 plays important roles in Ca 2+ signaling, cell wall integrity, microsclerotia development and full virulence in V. dahliae . [ABSTRACT FROM AUTHOR]

Abstract: The C2H2 zinc finger proteins form one of the largest families of transcriptional regulators in eukaryotes. We identified a Phytophthora sojae C2H2 zinc finger (PsCZF1), that is highly conserved in sequenced oomycete pathogens. In transformants of P. sojae containing the PsCZF1 promoter fused to the β-glucuronidase (GUS) reporter gene, GUS activity was highly induced in the P. sojae oospore stage and upregulated after infection. To elucidate the function of PsCZF1, its expression was silenced by introducing anti-sense constructs into P sojae. PsCZF1-silenced transformants did not exhibit altered cell size or morphology of sporangia and hyphae; however, hyphal growth rate was reduced by around 50% in the mutants. PsCZF1-deficient mutants were also impaired in production of oospores, swimming zoospores and germinating cysts, indicating that the gene is involved in various stages of the life cycle. Furthermore, we found that PsCZF1-deficient mutants lost virulence on host soybean cultivars. Our results suggest that this oomycete-specific C2H2-type zinc finger protein plays an important role in growth, development, and pathogenesis; therefore, PsCZF1 might be an attractive oomycete-specific target for chemical fungicide screening. [Copyright &y& Elsevier]

Verticillium dahliae is a notorious pathogen that causes vascular wilt disease in numerous plant species worldwide. The fungus produces melanized microsclerotia, which helps it survive adverse environmental conditions that it may encounter within its hosts and in the soil. Previously, we determined that the high osmolarity glycerol (HOG) pathway is involved in the environmental stress response of V. dahliae . In this study, we investigated the function of VdMsn2, a homologue of the yeast C 2 H 2 transcription factor Msn2, which is predicted to function as a downstream player in the HOG pathway. Disruption of VdMsn2 has a discernible effect on hyphal growth and septation, but not on diverse stresses including hyperosmotic stresses and cell wall inhibitory agents. Furthermore, we show that VdMsn2 deletion mutants produce significantly more microsclerotia than the wild-type and exhibit attenuated virulence to smoke trees because of poor penetration. Taken together, our findings suggest that VdMsn2 controls hyphal growth, microsclerotia formation, and virulence but does not significantly contribute to stress responses in V. dahliae . [ABSTRACT FROM AUTHOR]

bZIP transcription factors play various biological roles in stress responses, conidiation, and pathogenicity in pathogenic fungi. Here, we report two bZIP transcription factors (VDAG_08640 and VDAG_08676) of Verticillium dahliae , which were differentially expressed during microsclerotia development and induced by hydrogen peroxide as well. We find that deletion of either gene does not affect microsclerotia formation and the sensitivity to hydrogen peroxide; however, the mutants manifest decreased activity of extracellular peroxidase and laccase. Other phenotypic characterization reveals that VDAG_08676 disruption results in significant reduction of conidial production and virulence, while VDAG_08640 disruption does not lead to observable phenotypic variances compared with the wild-type strain. To elucidate whether they exhibit functional redundancy, double deletion mutants were generated. The double deletion mutants show remarkably increased sensitivity to hydrogen peroxide stress, whereas the two genes are not involved in microsclerotia formation. Taken together, our data demonstrate that a bZIP transcription factor gene VDAG_08676 is involved in the conidial production, oxidative stress response and virulence which may lay a foundation for further analysis of other bZIP transcription factors in V. dahliae . [ABSTRACT FROM AUTHOR]