Occurrence and Characterization of Sclerotinia sclerotiorum Causing Fruit Rot on Sweet Cherry in Southern China.

Sweet cherry (Prunus avium L.) is widely planted in northern China due to its high economic value, and its cultivation has gradually spread south to warm regions. However, fruit rot, observed on the young fruits, poses a considerable threat to the development of sweet cherry. To determine the causal agent, morphological observation, molecular identification, and pathogenicity tests were performed on isolates obtained from diseased fruits. As a result, Sclerotinia sclerotiorum was identified as the pathogen. Pathogenicity tests on different sweet cherry cultivars indicated that 'Summit' was highly sensitive to S. sclerotiorum, whereas 'Hongmi' showed significant resistance. Besides sweet cherry, S. sclerotiorum could also infect other vegetable crops we tested, such as cowpea, soybean, tomato, and chili. Fungicide sensitivity and efficacy assays showed that both fludioxonil and pyraclostrobin can effectively inhibit the mycelial growth of S. sclerotiorum and decrease disease incidences on the young fruits of sweet cherry. Furthermore, genome sequencing resulted in a 37.8 Mb assembly of S. sclerotiorum strain ScSs1, showing abundant SNPs, InDels, and SVs with the genome of S. sclerotiorum reference strain 1980 UF-70. The above results provide an important basis for controlling the fruit rot of sweet cherry caused by S. sclerotiorum in China.

Transcriptome Analysis Reveals the Molecular Mechanism and Responsive Genes of Waterlogging Stress in Actinidia deliciosa Planch Kiwifruit Plants.

Waterlogging stress is one of the major natural issues resulting in stunted growth and loss of agricultural productivity. Cultivated kiwifruits are popular for their rich vitamin C content and unique flavor among consumers, while commonly sensitive to waterlogging stress. The wild kiwifruit plants are usually obliged to survive in harsh environments. Here, we carried out a transcriptome analysis by high-throughput RNA sequencing using the root tissues of Actinidia deliciosa (a wild resource with stress-tolerant phenotype) after waterlogging for 0 d, 3 d, and 7 d. Based on the RNA sequencing data, a high number of differentially expressed genes (DEGs) were identified in roots under waterlogging treatment, which were significantly enriched into four biological processes, including stress response, metabolic processes, molecular transport, and mitotic organization, by gene ontology (GO) simplify enrichment analysis. Among these DEGs, the hypoxia-related genes AdADH1 and AdADH2 were correlated well with the contents of acetaldehyde and ethanol, and three transcription factors Acc26216, Acc08443, and Acc16908 were highly correlated with both AdADH1/2 genes and contents of acetaldehyde and ethanol. In addition, we found that there might be an evident difference among the promoter sequences of ADH genes from A. deliciosa and A. chinensis. Taken together, our results provide additional information on the waterlogging response in wild kiwifruit plants.

Genome-wide identification of the Penicillium digitatum bZIP gene family and the roles of one key member, PdatfA.

Penicillium digitatum is the most common cause of postharvest decay in citrus fruits around the world. Previous studies revealed that the bZIP gene family plays crucial roles in development, stress adaptation, and pathogenicity in fungi. However, little is known about the bZIP genes in P. digitatum. In this study, we systematically identified the bZIP family in 23 Penicillium species and analyzed their evolutionary relationships. We found that gene loss and gene duplication shaped the evolution of the Penicillium bZIP family. P. digitatum experienced 3 bZIP gene loss events, but with no gene duplication. We subsequently characterized the biological functions of one important member, PdatfA in P. digitatum by constructing the deletion mutant. Results showed that ΔPdatfA exhibited a moderate growth defect, reduced pigmentation, and slightly increased resistance to fungicides iprodione and fludioxonil. However, ΔPdatfA displayed similar rot symptoms to that of the wild-type. The ΔPdatfA mycelia were not affected in response to oxidative stress while its conidia showed enhanced resistance due to the upregulation of catalases. Our results provide new insights into the evolution and functions of the bZIP gene family in Penicillium.

The completed genome sequence of the pathogenic ascomycete fungus Penicillium digitatum.

P. digitatum, the causative agent of green mold, is one of the most destructive pathogens in the citrus industry. To facilitate basal researches on this important plant pathogen, here we report a finished genome sequence for P. digitatum strain PDW03 using a combination of Illumina, PacBio, and Hi-C sequencing technologies. The assembly comprised 6 chromosomes from telomere to telomere and encodes approximately 9000 proteins. Genomic re-analyses identified 302 Carbohydrate-active enzymes, 420 secreted proteins, and 39 secondary metabolite (SM) gene clusters. Furthermore, we found 10 fragmentary SM clusters in the P. digitatum PDW03 genome. Pangenome analysis based on 5 P. digitatum genomes available showed that conserved orthogroups account for ~68% of the species pangenome. Taken together, this fully completed P. digitatum genome will provide an optimum resource for further researches to investigate the driving forces of fungal host switch and effectors functioning in plant-pathogen interaction.

Genome-wide identification and functional analysis of the horizontally transferred genes in Penicillium.

Horizontal gene transfer (HGT) is the transmission of genetic material between different evolutionary lineages and is believed to be an important source of genomic innovation in fungi. In this study, we searched for prokaryotic-derived HGTs in 23 fully sequenced genomes using a comprehensive phylogenomic pipeline followed by manual curation. We found strong support for 60 HGT events comprising 190 genes putatively acquired from bacteria. HGT affected all Penicillium species to various degrees. Gene duplication events happened to 3 HGT genes after the transmission. Most HGT events include genes encoding a variety of enzymes, which are associated with sugar, amino acid, and lipid metabolism. Transcriptome data from 6 Penicillium species revealed that 33 of 35 HGT genes showed expression under the conditions tested and 16 genes were differentially expressed. Our results suggest an important role for inter-domain gene transfers in shaping the genome of Penicillium fungi.

A Small Horizontally Transferred Gene Cluster Contributes to the Sporulation of Alternaria alternata.

Horizontal gene transfer (HGT) has been identified as an important source of genomic innovation in fungi. However, how HGT drove the evolution of Alternaria alternata, a necrotrophic fungus which can be ubiquitously isolated from soil and various plants and decaying plant materials is largely known. In this study, we identified 12 protein-encoding genes that are likely acquired from lineages outside Pezizomycotina. Phylogenetic trees and approximately unbiased comparative topology tests strongly supported the evolutionary origin of these genes. According to their predicted functions, these HGT candidates are involved in nitrogen and carbohydrate metabolism. Especially, five genes of them were likely transferred as a physically linked cluster from Tremellales (Basidiomycota). Functionally knocking out the five-gene cluster in an A. alternata isolate causing citrus brown spot resulted in an 80% decrease in asexual spore production in the deletion mutant. We further knocked out each of these five genes in this cluster and the resultant single-gene deletion mutants exhibited a various degree of reduction in spore production. Except for conidiation, functions of these genes associated with vegetative growth, stress tolerance, and virulence are very limited. Our results provide new evidence that HGT has played important roles over the course of the evolution of filamentous fungi.

Genomic features and evolution of the conditionally dispensable chromosome in the tangerine pathotype of Alternaria alternata.

The tangerine pathotype of the ascomycete fungus Alternaria alternata is the causal agent of citrus brown spot, which can result in significant losses of both yield and marketability for tangerines worldwide. A conditionally dispensable chromosome (CDC), which harbours the host-selective ACT toxin gene cluster, is required for tangerine pathogenicity of A. alternata. To understand the genetic makeup and evolution of the tangerine pathotype CDC, we isolated and sequenced the CDCs of the A. alternata Z7 strain and analysed the function and evolution of their genes. The A. alternata Z7 strain has two CDCs (~1.1 and ~0.8 Mb, respectively), and the longer Z7 CDC contains all but one contig of the shorter one. Z7 CDCs contain 254 predicted protein-coding genes, which are enriched in functional categories associated with 'metabolic process' (55 genes, P = 0.037). Relatively few of the CDC genes can be classified as carbohydrate-active enzymes (CAZymes) (4) and transporters (19) and none as kinases. Evolutionary analysis of the 254 CDC proteins showed that their evolutionary conservation tends to be restricted within the genus Alternaria and that the CDC genes evolve faster than genes in the essential chromosomes, likely due to fewer selective constraints. Interestingly, phylogenetic analysis suggested that four of the 25 genes responsible for the ACT toxin production were likely transferred from Colletotrichum (Sordariomycetes). Functional experiments showed that two of them are essential for the virulence of the tangerine pathotype of A. alternata. These results provide new insights into the function and evolution of CDC genes in Alternaria.

Functional diversification of sterol regulatory element binding proteins following gene duplication in a fungal species.

The sterol regulatory element binding proteins (SREBPs) are functionally well conserved and have been shown to regulate ergosterol synthesis in fungi. However, the distribution and evolution of the SREBPs in fungi, especially in the Pezizomycotina which comprised of a great many of animal and plant pathogens, are unexplored. In this study, we identified 641 SREBPs from 367 out of 530 fungi species. Reconstruction of their evolutionary history showed evidence of gene duplication and gene loss at multiple evolutionary scales. Especially, SREBPs undergo a gene duplication event in the common ancestor of Pezizomycotina, resulting in the formation of two clades of SREBPs. Besides, the conserved motifs in the bHLH domain of both clades within Eurotiomycetes are highly diverged. To better understand the evolutionary diversification of this biologically significant regulator, we performed a series of experiments using Penicillium digitatum, a member of the lineage of Eurotiomycetes, to investigate how the evolutionary process of gene duplication shaped its function. qRT-PCR analysis showed that although PdsreA and PdsreB can be induced by imazalil, they showed different expression pattern; the electrophoretic mobility shift assay showed that PdSreA but not PdSreB can directly bind to the PdMLE1 sequence, an element that leads to the increased resistance to demethylation inhibitors (DMI) fungicides in P. digitatum. These results demonstrated that functions of duplicated SREBPs have largely diverged in P. digitatum, which may be a major feature of the long-term adaptive evolution of a particular group of fungi.

Genomic Sequencing of Phyllosticta citriasiana Provides Insight Into Its Conservation and Diversification With Two Closely Related Phyllosticta Species Associated With Citrus.

Phyllosticta capitalensis, Phyllosticta citricarpa, and Phyllosticta citriasiana are three very important Phyllosticta species associated with citrus. P. capitalensis is an endophyte fungus of citrus while P. citricarpa can cause black spot of citrus (e.g., oranges and mandarins). P. citriasiana was identified recently which is the causal agent of the pomelo tan spot. Here, we present the ∼34 Mb genome of P. citriasiana. The genome is organized in 92 contigs, encompassing 9202 predicted genes. Comparative genomic analyses with two other Phyllosticta species (P. citricarpa and P. capitalensis) associated with citrus was conducted to understand their evolutionary conservation and diversification. Pair-wise genome alignments revealed that these species are highly syntenic. All species encode similar numbers of CAZymes and secreted proteins. However, the molecular functions of the secretome showed that each species contains some enzymes with distinct activities. The three Phyllosticta species investigated shared a core set of 7261 protein families. P. capitalensis had the largest set of orphan genes (1991), in complete contrast to that of P. citriasiana (364) and P. citricarpa (262). Most of the orphan genes are functionally unknown, but they contain a certain number of species-specific secreted proteins. A total of 23 secondary metabolites biosynthesis clusters were identified in the three Phyllosticta species, 21 of them being highly conserved among these species while the remaining two showed whole cluster gain and loss polymorphisms or gene content polymorphisms. Taken together, our study reveals insights into the genetic mechanisms of host adaptation of three species of Phyllosticta associated with citrus and paves the way to identify effectors that function in infection of citrus plants.

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

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. alternataIMPORTANCE 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.

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.

Functional characterization of the Dsc E3 ligase complex in the citrus postharvest pathogen Penicillium digitatum.

Sterol regulatory element binding proteins (SREBPs) are required for sterol homeostasis in eukaryotes. Activation of SREBPs is regulated by the Dsc E3 ligase complex in Schizosaccharomyces pombe and Aspergillus spp. Previous studies indicated that an SREBP-coding gene PdsreA is required for fungicide resistance and ergosterol biosynthesis in the citrus postharvest pathogen Penicillium digitatum. In this study, five genes, designated PddscA, PddscB, PddscC, PddscD, and PddscE encoding the Dsc E3 ligase complex were characterized to be required for fungicide resistance, ergosterol biosynthesis and CoCl2 tolerance in P. digitatum. Each of the dsc genes was inactivated by target gene disruption and the resulted phenotypes were analyzed and compared. Genetic analysis reveals that, of five Dsc complex components, PddscB is the core subunit gene in P. digitatum. Although the resultant dsc mutants were able to infect citrus fruit and induce maceration lesions as the wild-type, the mutants rarely produced aerial mycelia on affected citrus fruit peels. P. digitatum Dsc proteins regulated not only the expression of genes involved in ergosterol biosynthesis but also that of PdsreA. Yeast two-hybrid assays revealed a direct interaction between the PdSreA protein and the Dsc proteins. Ectopic expression of the PdSreA N-terminus restored fungicide resistance in the dsc mutants. Our results provide important evidence to understand the mechanisms underlying SREBP activation and regulation of ergosterol biosynthesis in plant pathogenic fungi.

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.

Deletion of PdMit1, a homolog of yeast Csg1, affects growth and Ca(2+) 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.

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.

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.

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.

Genomewide investigation into DNA elements and ABC transporters involved in imazalil resistance in Penicillium digitatum.

Penicillium digitatum, causing citrus green mold, is one of the most devastating pathogenic fungi for postharvest fruits. The disease control is becoming less efficient because of the dispersal of fungicide-resistant strains. However, genome-scale analyses of its resistance mechanism are scarce. In this work, we sequenced the whole genome of the R1 genotype strain Pd01-ZJU and investigated the genes and DNA elements highly associated with drug resistance. Variation in DNA elements related to drug resistance between P. digitatum strains was revealed in both copy number and chromosomal location, indicating that their recent and frequent translocation might have contributed to environmental adaptation. In addition, ABC transporter proteins in Pd01-ZJU were characterized, and the roles of typical subfamilies (ABCG, ABCC, and ABCB) in imazalil resistance were explored using real-time PCR. Seven ABC proteins, including the previously characterized PMR1 and PMR5, were induced by imazalil, which suggests a role in drug resistance. In summary, this work presents genome information of the R1 genotype P. digitatum and systematically investigates DNA elements and ABC proteins associated with imazalil resistance for the first time, which would be indicative for studying resistant mechanisms in other pathogenic fungi.

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.