Transcriptome Analysis and Functional Validation Identify a Putative bZIP Transcription Factor, Fpkapc, that Regulates Development, Stress Responses, and Virulence in Fusarium pseudograminearum.

Fusarium pseudograminearum is a soilborne, hemibiotrophic phytopathogenic fungus that causes Fusarium crown rot and Fusarium head blight in wheat. The basic leucine zipper proteins (bZIPs) are evolutionarily conserved transcription factors that play crucial roles in a range of growth and developmental processes and the responses to biotic and abiotic stresses. However, the roles of bZIP transcription factors remains unknown in F. pseudograminearum. In this study, a bZIP transcription factor Fpkapc was identified to localize to the nucleus in F. pseudograminearum. A mutant strain (Δfpkapc) was constructed to determine the role of Fpkapc in growth and pathogenicity of F. pseudograminearum. Transcriptomic analyses revealed that many genes involved in basic metabolism and oxidation-reduction processes were downregulated, whereas many genes involved in metal iron binding were upregulated in the Δfpkapc strain, compared with the wild type (WT). Correspondingly, the mutant had severe growth defects and displayed abnormal hyphal tips. Conidiation in the Fpkapc mutant was reduced, with more conidia in smaller size and fewer septa than in the WT. Also, relative to WT, the Δfpkapc strain showed greater tolerance to ion stress, but decreased tolerance to H2O2. The mutant caused smaller disease lesions on wheat and barley plants, but significantly increased TRI gene expression, compared with the WT. In summary, Fpkapc plays multiple roles in governing growth, development, stress responses, and virulence in F. pseudograminearum.

Alternative splicing of the conserved drug-resistant orthologue FpNcb2 is associated with its nuclear accumulation of products and full virulence of Fusarium pseudograminearum.

BACKGROUND: Negative cofactor 2 NC2ß (Ncb2 or Dr1) is the beta subunit of a conserved heterodimeric regulator of transcription negative cofactor 2 (NC2) complex that has been identified as key regulator of drug resistance in model fungi. However, its role in plant pathogens is still unclear. RESULTS: We identified an NC2ß orthologue, FpNcb2, in Fusarium pseudograminearum, which is not only a significant regulatory function in drug resistance, but also essential for growth, conidiation and penetration. Moreover, FpNcb2 undergoes alternative splicing which creates two mRNA isoforms. As a putative CCAAT binding protein, FpNcb2 concentrates in the nuclei, contributing to the expression of two spliced mRNA of FpNcb2 in hypha, conidiophores and conidia, with exception of FpNcb2ISOA in germlings. Expression of each spliced mRNA of FpNcb2 in Δfpncb2 mutant could full complement the defects on growth, conidiation and fungicides sensitivity to that of wild type. However, FpNcb2ISOA and FpNcb2ISOB have different effects on virulence. FpNcb2 acts as a regulator for the transcription of some genes encoding drug efflux and hydrolases. CONCLUSION: Our analysis showed the existence of alternative mRNA splicing in the NC2ß orthologue, which is associated with protein subcellular localization and fungal virulence. The further elucidation of the target genes of NC2ß will provide insights into the potential regulation mechanisms in the antifungal resistance and pathogenesis of F. pseudograminearum. © 2024 Society of Chemical Industry.

Analysis of Apoptosis-Related Genes Reveals that Apoptosis Functions in Conidiation and Pathogenesis of Fusarium pseudograminearum.

Apoptosis, a type of programmed cell death, plays crucial roles in various physiological processes, from development to adaptive responses. Key features of apoptosis have been verified in various fungal microbes but not yet in Fusarium species. Here, we identified 19 apoptosis-related genes in Fusarium pseudograminearum using a genome-wide survey. Expression profile analysis revealed that several apoptosis-related genes were significantly increased during conidiation and infection stages. Among these is FpBIR1, with two BIR (baculovirus inhibitor-of-apoptosis protein repeat) domains at the N-terminal end of the protein, a homolog of Saccharomyces cerevisiae BIR1, which is a unique apoptosis inhibitor. FpNUC1 is the ortholog of S. cerevisiae NUC1, which triggers AIF1- or YCA1-independent apoptosis. The functions of these two proteins were assessed by creating Δfpbir1 and Δfpnuc1 mutants via targeted gene deletion. The Δfpbir1 mutant had more cells with nuclear fragmentation and exhibited reduced conidiation, conidial formation, and infectivity. Correspondingly, the Δfpnuc1 mutant contained multiple nuclei, produced thicker and more branched hyphae, was reduced in conidiation, and exhibited faster conidial formation and higher infection rates. Taken together, our results indicate that the apoptosis-related genes FpBIR1 and FpNUC1 function in conidiation, conidial germination, and infection by F. pseudograminearumIMPORTANCE The plant-pathogenic fungus F. pseudograminearum is the causal agent of Fusarium crown rot (FCR) in wheat and barley, resulting in substantial yield losses worldwide. Particularly, in the Huanghuai wheat-growing region of China, F. pseudograminearum was reported as the dominant Fusarium species in FCR infections. Apoptosis is an evolutionarily conserved mechanism in eukaryotes, playing crucial roles in development and cell responses to biotic and abiotic stresses. However, few reports on apoptosis in plant fungal pathogens have been published. In this study, we identified 19 conserved apoptosis-related genes in F. pseudograminearum, several of which were significantly increased during conidiation and infection stages. Potential apoptosis functions were assessed by deletion of the putative apoptosis inhibitor gene FpBIR1 and apoptosis trigger gene FpNUC1 in F. pseudograminearum The FpBIR1 deletion mutant exhibited defects in conidial germination and pathogenicity, whereas the FpNUC1 deletion mutant experienced faster conidial formation and higher infection rates. Apoptosis appears to negatively regulate the conidial germination and pathogenicity of F. pseudograminearum To our knowledge, this study is the first report of apoptosis contributing to infection-related morphogenesis and pathogenesis in F. pseudograminearum.

Heat Stress Tolerance Gene FpHsp104 Affects Conidiation and Pathogenicity of Fusarium pseudograminearum.

Heat shock protein Hsp104, a homolog of the bacterial chaperone ClpB and plant Hsp100, plays an essential part in the response to heat and various chemical agents in Saccharomyces cerevisiae. However, their functions remain largely unknown in plant fungal pathogens. Here, we report the identification and functional characterization of a plausible ortholog of yeast Hsp104 in Fusarium pseudograminearum, which we termed FpHsp104. Deletion mutant of FpHsp104 displayed severe defects in the resistance of heat shock during F. pseudograminearum mycelia and conidia when exposed to extreme heat. We also found that the protein showed dynamic localization to small particles under high temperature. However, no significant differences were detected in osmotic, oxidative, or cell wall stress responses between the wild-type and Δfphsp104 strains. Quantitative real-time PCR analysis showed that FpHsp104 was upregulated in the conidia, and disruption of FpHsp104 gene resulted in defects in conidia production, morphology, and germination. The transcript levels of conidiation-related genes of FpFluG, FpVosA, FpWetA, and FpAbaA were reduced in the Δfphsp104 mutant vs. the wild-type strain, but heat-shocked mRNA splicing repair was not affected in Δfphsp104. Moreover, Δfphsp104 mutant also showed attenuated virulence, but its DON synthesis was normal. These data from the first study of Hsp104 in F. pseudograminearum strongly suggest that FpHsp104 gene is an important element in the heat tolerance, development, and pathogenicity processes of F. pseudograminearum.