Congyi Zhu

PdSNF1, a sucrose non-fermenting protein kinase gene, is required for Penicillium digitatum conidiation and virulence

The sucrose non-fermenting protein kinase 1 gene (SNF1) regulates the derepression of glucose-repressible genes in microorganisms. In this study, we cloned an ortholog of SNF1 from Penicillium digitatum and characterized its functions through a gene knock-out strategy. Growth of the PdSNF1 mutant (ΔPdSNF1) on the synthetic medium (SM) supplemented with pectin or polygalacturonic acid was severely disturbed. The appearance of disease symptoms on the ΔPdSNF1 mutant-inoculated citrus fruits was significantly delayed as well. The expression levels of the cell wall-degrading enzyme (CWDE) genes (e.g., XY1, PL1, PNL1, and EXPG2) after pectin induction were up-regulated in wild type, but unchanged or less up-regulated in the ΔPdSNF1 mutant. During infection in citrus fruit, the up-regulation of XY1 was delayed in the ΔPdSNF1 mutant. Disruption of PdSNF1 also resulted in impaired conidiation and caused malformation of the conidiophore structures. In addition, the expression of BrlA, a gene that regulates conidiophore development, was significantly impaired in the ΔPdSNF1 mutant. However, the expression of FadA, encoding the α-subunit of a heterotrimeric G protein, was up-regulated in this mutant. Collectively, our results demonstrate that the PdSNF1 plays a role in adapting P. digitatum to alternative carbon sources. Its involvements in the virulence of P. digitatum is probably via regulation of the expression of CWDE genes; and it is also involved in conidiation, probably through activation of the conidiation signaling pathway while inactivating the mycelial growth-signaling pathway.

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.

PdMLE1, a specific and active transposon acts as a promoter and confers Penicillium digitatum with DMI resistance

Previously, we found a 199 bp element which inserted into the promoter of PdCYP51B gene in Penicillium digitatum, was associated with the overexpression of this gene and DMI fungicides resistance. However, the mechanism how this 199 bp element upregulate the expression of downstream gene was completely unknown. In the current study, we confirmed that this 199 bp element was a MITE-like element, designated as PdMLE1. blast searching and Southern blot showed that this 199 bp element was unique to P. digitatum. Genome-wide localization of PdMLE1 showed that it preferentially inserted into A + T rich regions, and several copies localized at the coding or regulation regions of genes were found. Penicillium digitatum mutant harbouring the PdMLE1 fused GFP gene showed the strong green fluorescence, indicating the powerful promoter activity of PdMLE1. By promoter deletion method, we identified a 20 bp core sequence in PdMLE1 which was associated with its promoter activity. In addition, we also limited the core element of PdCYP51B promoter to a 368 bp region. Collectively, we proposed a model that PdMLE1 acted as a powerful promoter and most likely recruited the transcription factor(s), therefore led to the overexpression of PdCYP51B gene and conferred P. digitatum with DMI resistance. This is the first regulation model of transposon resulted fungicide resistance proved in plant pathogens.

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.

A Genomics Based Discovery of Secondary Metabolite Biosynthetic Gene Clusters in Aspergillus ustus

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum

Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation (ATMT), but the frequencies are often very low. In the present study, we replaced the Ku80 homolog (a gene of the non-homologous end-joining (NHEJ) pathway) with the hygromycin resistance cassette (hph) by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in Ku80-deficient strain (ΔPdKu80) of P. digitatum. However, using ΔPdKu80 as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous integration and the created ΔPdKu80 strain would be a good candidate for rapid gene function analysis in P. digitatum.

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.