Heat shock protein 70s (Hsp70s) are a class of molecular chaperones that are highly conserved and ubiquitous in organisms ranging from microorganisms to plants and humans. Hsp70s play key roles in cellular development and protecting living organisms from environmental stresses such as heat, drought, salinity, acidity, and cold. However, their functions in pathogenic fungi are largely unknown. Here, a total of 14 FpHsp70 genes were identified in Fusarium pseudograminearum , including 3 in the mitochondria, 7 in the cytoplasm, 2 in the endoplasmic reticulum (ER), 1 in the nucleus, and 1 in the plastid. However, the exon–intron boundaries and protein motifs of the FpHsp70 have no consistency in the same subfamily. Expression analysis revealed that most FpHsp70 genes were up-regulated during infection, implying that FpHsp70 genes may play important roles in F . pseudograminearum pathogenicity. Furthermore, knockout of an ER lumenal Hsp70 homolog FpLhs1 gene reduced growth, conidiation, and pathogenicity in F . pseudograminearum . These mutants also showed a defect in secretion of some proteins. Together, FpHsp70s might play essential roles in F . pseudograminearum and FpLhs1 is likely to act on the development and virulence by regulating protein secretion.

The rice blast fungus Magnaporthe oryzae has been known to produce the phytohormone auxin/IAA from its hyphae and conidia, but the detailed biological function and biosynthesis pathway is largely unknown. By sequence homology, we identified a complete indole-3-pyruvic acid (IPA)-based IAA biosynthesis pathway in M. oryzae, consisting of the tryptophan aminotransferase (MoTam1) and the indole-3-pyruvate decarboxylase (MoIpd1). In comparison to the wild type, IAA production was significantly reduced in the motam1Δ mutant, and further reduced in the moipd1Δ mutant. Correspondingly, mycelial growth, conidiation, and pathogenicity were defective in the motam1Δ and the moipd1Δ mutants to various degrees. Targeted metabolomics analysis further confirmed the presence of a functional IPA pathway, catalyzed by MoIpd1, which contributes to IAA/auxin production in M. oryzae. Furthermore, the well-established IAA biosynthesis inhibitor, yucasin, suppressed mycelial growth, conidiation, and pathogenicity in M. oryzae. Overall, this study identified an IPA-dependent IAA synthesis pathway crucial for M. oryzae mycelial growth and pathogenic development.

Fusarium pseudograminearum is an important pathogen of Fusarium crown rot and Fusarium head blight, which is able to infect wheat and barley worldwide, causing great economic losses. Transcription factors (TFs) of the basic leucine zipper (bZIP) protein family control important processes in all eukaryotes. In this study, we identified a gene, designated FpAda1, encoding a bZIP TF in F. pseudograminearum. The homolog of FpAda1 is also known to affect hyphal growth in Neurospora crassa. Deletion of FpAda1 in F. pseudograminearum resulted in defects in hyphal growth, mycelial branching and conidia formation. Pathogenicity assays showed that virulence of the Δfpada1 mutant was dramatically decreased on wheat coleoptiles and barley leaves. However, wheat coleoptile inoculation assay showed that Δfpada1 could penetrate and proliferate in wheat cells. Moreover, the FpAda1 was required for abnormal nuclear morphology in conidia and transcription of FpCdc2 and FpCdc42. Taken together, these results indicate that FpAda1 is an important transcription factor involved in growth and development in F. 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. pseudograminearum. IMPORTANCE 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.

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