Mingshuang Wang

PdbrlA, PdabaA and PdwetA control distinct stages of conidiogenesis in Penicillium digitatum

Penicillium digitatum is one of the most important citrus postharvest pathogens worldwide. Reproduction of massive asexual spores is the primary factor contributing to the epidemic of citrus green mold. To understand the molecular mechanisms underlying conidiogenesis in P. digitatum, we functionally characterized the Aspergillus nidulans orthologs of brlA, abaA and wetA. We showed that deletion of PdbrlA completely blocked formation of conidiophores, whereas deletion of PdabaA led to the formation of aberrant and non-functional phialides. The PdwetA mutant showed various defective phenotypes, such as abnormal conidia with loose cell walls, delayed germination and reduced tolerance to osmotic, detergent, heat shock and menadione stresses, but elevated resistance to H2O2. PdbrlA-influenced genes were identified by comparing global gene expression profiles between the wild-type and the PdbrlA deletion mutant during conidiation. Gene ontology analysis of these differentially expressed genes (DEGs) revealed the diverse roles of PdbrlA in metabolism, transportation and cell structure. Moreover, out of 39 genes previously reported to be involved in conidiogenesis in Aspergillus, mRNA levels of 14 genes were changed in ΔPdbrlA. Our results confirm the roles of brlA, abaA and wetA in P. digitatum conidiogenesis and provide new insights into the genetics of conidiation in filamentous fungi.

Os2 MAP kinase-mediated osmostress tolerance in Penicillium digitatum is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis

High osmolarity glycerol (HOG) pathway is ubiquitously distributed among eukaryotic organisms and plays an important role in adaptation to changes in the environment. In this study, the Hog1 ortholog in Penicillium digitatum, designated Pdos2, was identified and characterized using a gene knock-out strategy. The ΔPdos2 mutant showed a considerably increased sensitivity to salt stress and cell wall-disturbing agents and a slightly increased resistance to fungicides iprodione and fludioxonil, indicating that Pdos2 is involved in response to hyperosmotic stress, regulation of cell wall integrity and sensitivity to fungicides iprodione and fludioxonil. Surprisingly, the mutant was not affected in response to oxidative stress caused by H2O2. The average lesion size in citrus fruits caused by ΔPdos2 mutant was smaller (approximately 25.0% reduction) than that caused by the wild-type strain of P. digitatum at 4 days post inoculation, which suggests that Pdos2 is needed for full virulence of P. digitatum. Interestingly, in the presence of 0.7 M NaCl, the glycerol content was remarkably increased and the ergosterol was decreased in mycelia of the wide-type P. digitatum, whereas the glycerol content was only slightly increased and the ergosterol content remained stable in the ΔPdos2 mutant, suggesting that Pdos2-mediated osmotic adaption is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

Glucosylceramides are required for mycelial growth and full virulence in Penicillium digitatum

Glucosylceramides (GlcCers) are important lipid components of the membrane systems of eukaryotes. Recent studies have suggested the roles for GlcCers in regulating fungal growth and pathogenesis. In this study, we report the identification and functional characterization of PdGcs1, a gene encoding GlcCer synthase (GCS) essential for the biosynthesis of GlcCers, in Penicilliumdigitatum genome. We demonstrated that the deletion of PdGcs1 in P. digitatum resulted in the complete loss of production of GlcCer (d18:1/18:0 h) and GlcCer (d18:2/18:0 h), a decrease in vegetation growth and sporulation, and a delay in spore germination. The virulence of the PdGcs1 deletion mutant on citrus fruits was also impaired, as evidenced by the delayed occurrence of water soaking lesion and the formation of smaller size of lesion. These results suggest that PdGcs1 is a bona fide GCS that plays an important role in regulating cell growth, differentiation, and virulence of P. digitatum by controlling the biosynthesis of GlcCers.

The citrus postharvest pathogen Penicillium digitatum depends on the PdMpkB kinase for developmental and virulence functions

The postharvest pathogen Penicillium digitatum causes green mold decay on citrus fruit, resulting in severe economic losses. To explore possible factors involved in fungal pathogenesis, phenotypic characterization of the budding yeast Fus3/Kiss1 mitogen-activated protein (MAP) kinase homolog was carried out. The P. digitatum MAP kinase B coding gene, designated PdMpkB, was functionally inactivated via homologous recombination. The fungal strain (∆PdMpkB) carrying a PdMpkBdeletion demonstrated altered gene expression profiles, reduced growth and conidiogenesis, elevated resistance to osmotic stress, and failed to induce green mold decay on citrus fruit. ∆PdMpkB was more resistant to CaCl2, NaCl and sorbitol than its progenitor strain, indicating a negative regulatory function of PdMpkB in osmotic stress adaptation. Fungal infection assays on citrus fruit revealed that ∆PdMpkB proliferated poorly within host tissues, induced water-soaking lesions, failed to break through host cuticle layers and thus, failed to produce aerial hyphae and conidia. Introduction of a functional copy of PdMpkB into a null mutant restored all defective phenotypes. Transcriptome analysis revealed that inactivation of PdMpkB impacted expression of the genes associated with cell wall-degrading enzyme activities, carbohydrate and amino acid metabolisms, conidial formation, and numerous metabolic processes. Our results define pivotal roles of the PdMpkB-mediated signaling pathway in developmental and pathological functions in the citrus postharvest pathogen P. digitatum.

Genomic and transcriptomic analyses of the tangerine pathotype of Alternaria alternata in response to oxidative stress.

The tangerine pathotype of Alternaria alternata produces the A. citri toxin (ACT) and is the causal agent of citrus brown spot that results in significant yield losses worldwide. Both the production of ACT and the ability to detoxify reactive oxygen species (ROS) are required for A. alternata pathogenicity in citrus. In this study, we report the 34.41 Mb genome sequence of strain Z7 of the tangerine pathotype of A. alternata. The host selective ACT gene cluster in strain Z7 was identified, which included 25 genes with 19 of them not reported previously. Of these, 10 genes were present only in the tangerine pathotype, representing the most likely candidate genes for this pathotype specialization. A transcriptome analysis of the global effects of H2O2 on gene expression revealed 1108 up-regulated and 498 down-regulated genes. Expressions of those genes encoding catalase, peroxiredoxin, thioredoxin and glutathione were highly induced. Genes encoding several protein families including kinases, transcription factors, transporters, cytochrome P450, ubiquitin and heat shock proteins were found associated with adaptation to oxidative stress. Our data not only revealed the molecular basis of ACT biosynthesis but also provided new insights into the potential pathways that the phytopathogen A. alternata copes with oxidative stress.

Functional analysis of two sterol regulatory element binding proteins in Penicillium digitatum

The sterol regulatory element binding proteins (SREBPs) are key regulators for sterol homeostasis in most fungi. In the citrus postharvest pathogen Penicillium digitatum, the SREBP homolog is required for fungicide resistance and regulation of CYP51 expression. In this study, we identified another SREBP transcription factor PdSreB in P. digitatum, and the biological functions of both SREBPs were characterized and compared. Inactivation of PdsreA, PdsreB or both genes in P. digitatum reduced ergosterol contents and increased sensitivities to sterol 14-α-demethylation inhibitors (DMIs) and cobalt chloride. Fungal strains impaired at PdsreA but not PdsreB increased sensitivity to tridemorph and an iron chelator 2,2’-dipyridyl. Virulence assays on citrus fruit revealed that fungal strains impaired at PdsreA, PdsreB or both induce maceration lesions similar to those induced by wild-type. However, ΔPdsreA, ΔPdsreB or the double mutant strain rarely produce aerial mycelia on infected citrus fruit peels. RNA-Seq analysis showed the broad regulatory functions of both SREBPs in biosynthesis, transmembrane transportation and stress responses. Our results provide new insights into the conserved and differentiated regulatory functions of SREBP homologs in plant pathogenic fungi.

Thioredoxin and Glutaredoxin Systems Required for Oxidative Stress Resistance, Fungicide Sensitivity, and Virulence of Alternaria alternata

ABSTRACT This study determined the function of thioredoxin and glutaredoxin systems in the phytopathogenic fungus Alternaria alternata via analyzing mutants obtained from the targeted deletion of genes encoding thioredoxin peroxidase (Tsa1), thioredoxin reductase (Trr1), and glutathione reductase (Glr1). Trr1 and Glr1, but not Tsa1, are required for growth and conidiation. The reduced growth and conidiation seen in the Trr1 or Glr1 deletion mutant can be restored by glutathione. Deletion mutants showing growth inhibition by oxidants are defective for H2O2 detoxification and induce smaller lesions on citrus leaves. Trr1 and Glr1, but not Tsa1, also contribute to NaCl resistance. Glr1 is required for sorbitol resistance and is responsible for resistance to mancozeb and boscalid but not chlorothalonil fungicides, a novel phenotype that has not been reported in fungi. Trr1 is required for resistance to boscalid and chlorothalonil fungicides but confers susceptibility to mancozeb. The Tsa1 deletion mutant displays wild-type sensitivity to the tested fungicides. The expression of Tsa1 and Trr1 is regulated by the oxidative stress responsive regulators Yap1, Hog1, and Skn7. The expression of Tsa1, but not Trr1, is also regulated indirectly by the NADPH oxidase. The results indicate that the capability to resist oxidative stress is required for virulence of A. alternata. IMPORTANCE The thioredoxin and glutaredoxin systems are important thiol antioxidant systems in cells, and knowledge of these two systems in the plant-pathogenic fungus A. alternata is useful for finding new strategies to reduce the virulence of this pathogen. In this study, we demonstrated that thiol antioxidant system-related genes (Tsa1, Trr1, and Glr1) are required for H2O2 detoxification and virulence in A. alternata. Moreover, deletion of Trr1 results in hypersensitivity to the fungicides chlorothalonil and boscalid, and Glr1 deletion mutants are highly sensitive to mancozeb, which is the fungicide mostly used in citrus fields. Therefore, our findings demonstrate that the ability to detoxify reactive oxygen species (ROS) plays a critical role in pathogenesis on citrus and provide novel insights into the physiological functions of thiol-containing systems in fungicide sensitivity for A. alternata.

Adenylyl cyclase is required for cAMP production, growth, conidial germination, and virulence in the citrus green mold pathogen Penicillium digitatum

Penicillium digitatum is the causative agent of green mold decay on citrus fruit. The cAMP-mediated signaling pathway plays an important role in the transduction of extracellular signals and has been shown to regulate a wide range of developmental processes and pathogenicity in fungal pathogens. We cloned and characterized a Pdac1 gene of P. digitatum, which encodes a polypeptide similar to fungal adenylyl cyclases. Using a loss-of-function mutation in the Pdac1 gene we demonstrated a critical requirement for hyphal growth and conidial germination. Deletion of Pdac1 resulted in decreased accumulation of cAMP and down-regulation of genes encoding a G protein α subunit, both catalytic and regulatory subunits of PKA, and two transcriptional regulators StuA and Som1. Fungal mutants lacking Pdac1 produced abundant conidia, which failed to germinate effectively and displayed an elevated sensitivity to heat treatment. Pdac1 mutant failed to utilize carbohydrates effectively and thus displayed severe growth retardation on rich and synthetic media. Slow growth seen in the Pdac1 mutants could be due to a defect in nutrient sensing and acquisition. Quantitative RT-PCR analysis revealed that Pdac1 was primarily expressed at the early stage of infection. Fungal pathogenicity assayed on citrus fruit revealed that P. digitatum strains impaired for Pdac1 delayed lesion formation. Our results highlight important regulatory roles of adenylyl cyclase-mediated cAMP production in P. digitatum and provide insights into the critical role of cAMP in fungal growth, development and virulence.

Deletion of PdMit1, a homolog of yeast Csg1, affects growth and Ca2+ sensitivity of the fungus Penicillium digitatum, but does not alter virulence

GDP-mannose:inositol-phosphorylceramide (MIPC) and its derivatives are important for Ca(2+) sensitization of Saccharomyces cerevisiae and for the virulence of Candida albicans, but its role in the virulence of plant fungal pathogens remains unclear. In this study, we report the identification and functional characterization of PdMit1, the gene encoding MIPC synthase in Penicillium digitatum, one of the most important pathogens of postharvest citrus fruits. To understand the function of PdMit1, a PdMit1 deletion mutant was generated. Compared to its wild-type control, the PdMit1 deletion mutant exhibited slow radial growth, decreased conidia production and delayed conidial germination, suggesting that PdMit1 is important for the growth of mycelium, sporulation and conidial germination. The PdMit1 deletion mutant also showed hypersensitivity to Ca(2+). Treatment with 250 mmol/l Ca(2+) induced vacuole fusion in the wild-type strain, but not in the PdMit1 deletion mutant. Treatment with 250mmol/lCaCl2 upregulated three Ca(2+)-ATPase genes in the wild-type strain, and this was significantly inhibited in the PdMit1 deletion mutant. These results suggest that PdMit1 may have a role in regulating vacuole fusion and expression of Ca(2+)-ATPase genes by controlling biosynthesis of MIPC, and thereby imparts P. digitatum Ca(2+) tolerance. However, we found that PdMit1 is dispensable for virulence of P. digitatum.

Csn5 Is Required for the Conidiogenesis and Pathogenesis of the Alternaria alternata Tangerine Pathotype

The COP9 signalosome (CSN) is a highly conserved protein complex involved in the ubiquitin-proteasome system. Its metalloisopeptidase activity resides in subunit 5 (CSN5). Functions of csn5 in phytopathogenic fungi are poorly understood. Here, we knocked out the csn5 ortholog (Aacsn5) in the tangerine pathotype of Alternaria alternata. The ΔAacsn5 mutant showed a moderately reduced growth rate compared to the wildtype strain and was unable to produce conidia. The growth of ΔAacsn5 mutant was not affected in response to oxidative and osmotic stresses. Virulence assays revealed that ΔAacsn5 induced no or significantly reduced necrotic lesions on detached citrus leaves. The defects in hyphal growth, conidial sporulation, and pathogenicity of ΔAacsn5 were restored by genetic complementation of the mutant with wildtype Aacsn5. To explore the molecular mechanisms of conidiation and pathogenesis underlying Aacsn5 regulation, we systematically examined the transcriptomes of both ΔAacsn5 and the wildtype. Generally, 881 genes were overexpressed and 777 were underexpressed in the ΔAacsn5 mutant during conidiation while 694 overexpressed and 993 underexpressed during infection. During asexual development, genes related to the transport processes and nitrogen metabolism were significantly downregulated; the expression of csn1–4 and csn7 in ΔAacsn5 was significantly elevated; secondary metabolism gene clusters were broadly affected; especially, the transcript level of the whole of cluster 28 and 30 was strongly induced. During infection, the expression of the host-specific ACT toxin gene cluster which controls the biosynthesis of the citrus specific toxin was significantly repressed; many other SM clusters with unknown products were also regulated; 86 out of 373 carbohydrate-active enzymes responsible for breaking down the plant dead tissues showed uniquely decreased expression. Taken together, our results expand our understanding of the roles of csn5 on conidiation and pathogenicity in plant pathogenic fungi and provide a foundation for future investigations.