Two polyketide synthase genes VpPKS10 and VpPKS33 regulated by VpLaeA are essential to the virulence of Valsa pyri.

Valsa pyri, the causal agent of pear canker disease, typically induces cankers on the bark of infected trees and even leads to tree mortality. Secondary metabolites (SMs) produced by pathogenic fungi play a crucial role in the pathogenic process. In this study, secondary metabolic regulator VpLaeA was identified in V. pyri. VpLaeA was found to strongly affect the pathogenicity, fruiting body formation and toxicity of SMs of V. pyri. Additionally, VpLaeA was also found to be required for the response of V. pyri to some abiotic stresses. Transcriptome data analysis revealed that many of differentially expressed genes were involved in the secondary metabolite biosynthesis (SMB). Among them, about one third of SMB core genes were regulated by VpLaeA at different periods. Seven differentially expressed SMB core genes (VpPKS9, VpPKS10, VpPKS33, VpNRPS6, VpNRPS7, VpNRPS16, and VpNRPS17) were selected for knockout. Two modular polyketide synthase (PKS) genes (VpPKS10 and VpPKS33), which were closely related to the virulence of V. pyri from the above seven genes were identified. Notably, VpPKS10 and VpPKS33 also affected the production of fruiting body of V. pyri, but didn't participate in the resistance of V. pyri to abiotic stresses. Overall, this study demonstrates the multifaceted biological functions of VpLaeA in V. pyri, and identifies two toxicity-associated PKS genes in Valsa species fungi for the first time.

Drosophila hydei as a potential vector of Ceratocystis fimbriata, the causal agent of sweetpotato black rot, in storage facilities.

Ceratocystis fimbriata, the causal agent of sweetpotato black rot, is a pathogen capable of developing and spreading within postharvest settings. A survey of North Carolina sweetpotato storage facilities was conducted to determine the arthropods present and identify potential vectors of C. fimbriata. Sixteen taxonomic categories were recovered and the genus Drosophila (Diptera: Drosophilidae) accounted for 79% of individuals sampled with Drosophila hydei (Sturtevant) being the most abundant species. Behavioral assays were conducted to determine if D. hydei is attracted to C. fimbriata inoculated roots and if the pathogen could be recovered from external or internal surfaces of the insect. Flies were released in insect trapping pitchers containing either C. fimbriata inoculated or non-inoculated roots or Petri dishes. No significant differences in fly number were detected in sweetpotato-baited pitchers; however, significant differences were found in the pitcher baited with a mature C. fimbriata culture. Flies were subjected to washes to determine if viable C. fimbriata was present (internally or externally); washes were plated onto carrot agar plates and observed for the presence of C. fimbriata colonies. Both external and internal washes had viable C. fimbriata inoculum with no significant differences, and inoculated sweetpotatoes had a significantly higher number of flies carrying C. fimbriata. This study suggests that D. hydei can carry C. fimbriata from infected sweetpotatoes and move viable C. fimbriata inoculum both externally and internally, making this the first report of any Drosophila spp. serving as a potential vector for the Ceratocystis genus.

More than a Virulence Factor: Patulin Is a Non-Host-Specific Toxin that Inhibits Postharvest Phytopathogens and Requires Efflux for Penicillium Tolerance.

Mycotoxin contamination is a leading cause of food spoilage and waste on a global scale. Patulin, a mycotoxin produced by Penicillium spp. during postharvest pome fruit decay, causes acute and chronic effects in humans, withstands pasteurization, and is not eliminated by fermentation. While much is known about the impact of patulin on human health, there are significant knowledge gaps concerning the effect of patulin during postharvest fruit-pathogen interactions. Application of patulin on six apple cultivars reproduced some blue mold symptoms that were cultivar-independent and dose-dependent. Identical symptoms were also observed in pear and mandarin orange. Six Penicillium isolates exposed to exogenous patulin exhibited delayed germination after 24 h, yet all produced viable colonies in 7 days. However, four common postharvest phytopathogenic fungi were completely inhibited by patulin during conidial germination and growth, suggesting the toxin is important for Penicillium to dominate the postharvest niche. Using clorgyline, a broad-spectrum efflux pump inhibitor, we demonstrated that efflux plays a role in Penicillium auto-resistance to patulin during conidial germination. The work presented here contributes new knowledge of patulin auto-resistance, its mode of action, and inhibitory role in fungal-fungal interactions. Our findings provide a solid foundation to develop toxin and decay mitigation approaches.

Aggressiveness and Mycotoxin Production by Fusarium meridionale Compared with F. graminearum on Maize Ears and Stalks in the Field.

Fusarium meridionale and F. graminearum both cause Gibberella ear rot (GER) and Gibberella stalk rot (GSR) of maize in Brazil, but the former is much more common. Recent work with two isolates of each from maize suggested this dominance could be caused by greater aggressiveness and competitiveness of F. meridionale on maize. We evaluated pathogenicity and toxigenicity of 16 isolates of F. graminearum and 24 isolates of F. meridionale recovered from both wheat and maize. Strains were individually inoculated into ears of four maize hybrids in field trials. GER severity varied significantly between isolates within each species. Although ranges overlapped, the average GER severity induced by F. meridionale (25.2%) was two times as high overall as that induced by F. graminearum (12.8%) for isolates obtained from maize but was similar for those isolated from wheat (19.9 and 21.4%, respectively). In contrast, severity of GSR was slightly higher for F. graminearum (22.2%) than for F. meridionale (19.8%), with no effect of the host of origin. Deoxynivalenol and its acetylated form 15ADON were the main mycotoxins produced by F. graminearum (7/16 strains), and nivalenol toxin was produced by F. meridionale (17/24 strains). Six isolates of F. graminearum and three of F. meridionale also produced zearalenone. Results confirmed that F. meridionale from maize is, on average, more aggressive on maize but also suggested greater complexity related to diversity among the isolates within each species and their interactions with different hybrids. Further studies involving other components of the disease cycle are needed to more fully explain observed patterns of host dominance.

Brown Rot Disease in Stored Nectarines: Modeling the Combined Effects of Preharvest and Storage Conditions.

Brown rot in stored stone fruits, caused by Monilinia spp., may be due to preharvest and storage factors, but the combined effect of these factors has yet to be investigated. We set up two experiments to monitor the progression of brown rot during the storage of nectarines subjected to various preharvest and storage conditions. We assessed the effects of different agricultural practices (irrigation regimen × fruit load) and harvest dates on brown rot progress during storage in 2018 and the effect of different storage temperatures in 2019. We found that the cumulative incidence of brown rot during storage increased with individual fruit mass, which was influenced by agricultural practices, and for later harvest dates. It also increased with storage temperature. We observed that during storage no secondary infections developed in nectarines not in direct contact with fruits infected with Monilinia laxa. These findings led to the identification of candidate variables describing the brown rot risk on nectarines during storage, such as individual fruit mass, meteorological conditions before fruit harvest, prevalence of brown rot at harvest, and storage temperature. We used these variables to build a mathematical model for estimating the time-to-appearance of brown rot symptoms in stored nectarines. This model fitted the experimental data well, highlighting the need to pay greater attention to the interaction between preharvest and storage conditions. This model could be used to evaluate management strategies for reducing the impact of brown rot in nectarines during storage.

Occidiofungin Is the Key Metabolite for Antifungal Activity of the Endophytic Bacterium Burkholderia sp. MS455 Against Aspergillus flavus.

Aflatoxin is a secondary metabolite produced by Aspergillus fungi and presents a major food safety concern globally. Among the available methods for prevention and control of aflatoxin, the application of antifungal bacteria has gained favor in recent years. An endophytic bacterium MS455, isolated from soybean, exhibited broad-spectrum antifungal activity against economically important pathogens, including Aspergillus flavus. MS455 was identified as a strain of Burkholderia based on genomic analysis. Random and site-specific mutations were used in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA sequencing analysis demonstrated that ORF1, a homolog to the ambR1 LuxR-type regulatory gene, regulates occidiofungin biosynthesis in MS455. Additionally, 284 differentially expressed genes, including 138 upregulated and 146 downregulated genes, suggesting that, in addition to its role in occidiofungin production, ORF1 is involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate bioassays showed the growth of A. flavus was significantly inhibited by the wild-type strain MS455 as compared with the ORF1 mutant. Similarly, corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Collectively, the results demonstrated that production of occidiofungin is essential for antifungal activity of the endophytic bacterium MS455. This research has provided insights about antifungal mechanisms of MS455 and development of biological approaches to prevent aflatoxin contamination in plant production.

Unraveling the Host-Selective Toxic Interaction of Cassiicolin with Lipid Membranes and Its Cytotoxicity.

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for Corynespora leaf fall disease in susceptible rubber trees. Currently, the molecular mechanisms of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more sensitive to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Mature Cas1 and Cas2 play an essential role in causing membrane disruption. Cytotoxicity tests on rubber leaves of Rubber Research Institute of Vietnam (RRIV) 1, RRIV 4, and Prang Besar (PB) 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryogenic scanning electron microscopy analyses of necrotic leaf tissues treated with Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and to disrupt specific membranes.

Sweetpotato Root Development Influences Susceptibility to Black Rot Caused by the Fungal Pathogen Ceratocystis fimbriata.

Black rot of sweetpotato, caused by Ceratocystis fimbriata, is an important reemerging disease threatening sweetpotato production in the United States. This study assessed disease susceptibility of the storage root surface, storage root cambium, and slips (vine cuttings) of 48 sweetpotato cultivars, advanced breeding lines, and wild relative accessions. We also characterized the effect of storage root development on susceptibility to C. fimbriata. None of the cultivars examined at the storage root level were resistant, with most cultivars exhibiting similar levels of susceptibility. In storage roots, Jewel and Covington were the least susceptible and significantly different from White Bonita, the most susceptible cultivar. In the slip, significant differences in disease incidence were observed for above- and below-ground plant structures among cultivars, advanced breeding lines, and wild relative accessions. Burgundy and Ipomoea littoralis displayed less below-ground disease incidence compared with NASPOT 8, Sunnyside, and LSU-417, the most susceptible cultivars. Correlation of black rot susceptibility between storage roots and slips was not significant, suggesting that slip assays are not useful to predict resistance in storage roots. Immature, early-developing storage roots were comparatively more susceptible than older, fully developed storage roots. The high significant correlation between the storage root cross-section area and the cross-sectional lesion ratio suggests the presence of an unfavorable environment for C. fimbriata as the storage root develops. Incorporating applications of effective fungicides at transplanting and during early-storage root development when sweetpotato tissues are most susceptible to black rot infection may improve disease management efforts.

Molecular and Alkaloid Characterization of Claviceps purpurea Sensu Lato From Grass Seed Production Areas of the U.S. Pacific Northwest.

Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), ß-tubulin and elongation factor-1α (EF-1α) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, ß-tubulin, and EF-1α sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.

Peeling the Onion: Towards a Better Understanding of Botrytis Diseases of Onion.

Onion is cultivated worldwide for its bulbs, but production is threatened by pathogens and pests. Three distinct diseases of onion are caused by species that belong to the fungal genus Botrytis. Leaf blight is a well-known foliar disease caused by B. squamosa that can cause serious yield losses. Neck rot is a postharvest disease that manifests in bulbs after storage and is associated with three species: B. aclada, B. allii, and B. byssoidea. The symptomless infection of onion plants in the field makes it difficult to predict the incidence of neck rot in storage, although progress on the detection of latent infection has been made. In onion cultivation for seed production, blighting of the inflorescence is caused by all four onion-specific Botrytis species plus the broad host range pathogen B. cinerea. Flower blight can reduce seed yield and contaminate seed. In this review, the long history of Botrytis diseases of onion is discussed, as well as recent and future approaches to acquire a better understanding of the biology and ecology of Botrytis spp. pathogenic on onion. New fundamental insights in the genetic, biochemical, and physiological aspects of Botrytis-onion interactions are essential to improve the breeding of Botrytis-resistant onion cultivars.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Regulation and Dynamics of Gene Expression During the Life Cycle of Fusarium graminearum.

Fungal pathogens survive harsh environments and overcome physical, temporal, and chemical barriers to colonize their hosts and reproduce. Fusarium graminearum was one of the first fungal plant pathogens for which transcriptomic tools were developed, making analysis of gene expression a cornerstone approach in studying its biology. The analysis of gene expression in diverse in vitro conditions and during infection of different cereal crops has revealed subsets of both unique and shared transcriptionally regulated genes. Together with genetic studies, these approaches have enhanced our understanding of the development and infection cycle of this economically important pathogen. Here, we will outline recent advances in transcriptional profiling during sporogenesis, spore germination, vegetative growth, and host infection. Several transcriptional regulators have been identified as essential components in these responses and the role of select transcription factors will be highlighted. Finally, we describe some of the gaps in our understanding of F. graminearum biology and how expression analysis could help to address these gaps.

Polyketide Synthase Gene Expression in Relation to Chloromonilicin and Melanin Production in Monilinia fructicola.

Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

Stability of Hybrid Maize Reaction to Gibberella Ear Rot and Deoxynivalenol Contamination of Grain.

Trials were conducted to quantify the stability (or lack of G × E interaction) of 15 maize hybrids to Gibberella ear rot (GER; caused by Fusarium graminearum) and deoxynivalenol (DON) contamination of grain across 30 Ohio environments (3 years × 10 locations). In each environment, one plot of each hybrid was planted and 10 ears per plot were inoculated via the silk channel. GER severity (proportion of ear area diseased) and DON contamination of grain (ppm) were quantified. Multiple rank-based methods, including Kendall's concordance coefficient (W) and Piepho's U, were used to quantify hybrid stability. The results found insufficient evidence to suggest crossover G × E interaction of ranks, with W greater than zero for GER (W = 0.28) and DON (W = 0.26), and U not statistically significant for either variable (P > 0.20). Linear mixed models (LMMs) were also used to quantify hybrid stability, accounting for crossover or noncrossover G × E interaction of transformed observed data. Based on information criteria and likelihood ratio tests for GER and DON response variables, the models with more complex variance-covariance structures-heterogeneous compound symmetry and factor-analytic-provided a better fit than the model with the simpler compound symmetry structure, indicating that one or more hybrids differed in stability. Overall, hybrids were stable based on rank-based methods, which indicated a lack of crossover G × E interaction, but the LMMs identified a few hybrids that were sensitive to environment. Resistant hybrids were generally more stable than susceptible hybrids.

Ceratocystis fimbriata Employs a Unique Infection Strategy Targeting Peltate Glandular Trichomes of Sweetpotato (Ipomoea batatas) Plants.

The infection processes of Ceratocystis fimbriata BMPZ13 (BMPZ13) was elucidated on vegetative tissues of sweetpotato plants employing light and scanning electron microscopy. Vegetative tissues infected with C. fimbriata BMPZ13 by either wounding or nonwounding inoculation methods developed typical disease symptoms, establishing black rot in stems and necrosis on buds, young leaves, and stems of sprouts, in addition to wilt on leaves and shoot cuttings, typical of vascular associated diseases. The runner hyphae of C. fimbriata BMPZ13 formed from germinated conidia were able to directly penetrate the epidermal cuticle for initial infection and invade sweetpotato peltate glandular trichomes, specialized secretory structures to store and secrete metabolites. A two-step biotrophic phase was observed with nonwounding inoculation on leaves and stems, featuring both intercellular and intracellular invasive hyphae, with the latter found within living cells of the leaf epidermis. Subsequent to the biotrophic phase was a necrotrophic phase displaying cell death in infected leaves and veins. Additionally, this cell death was an iron-associated ferroptosis, supporting the notion that iron is involved in the necrotrophic phase of C. fimbriata BMPZ13 infection. Significantly, we establish that C. fimbriata employs a unique infection strategy: the targeting of peltate glandular trichomes. Collectively, our findings show that C. fimbriata is a plant fungal pathogen with a hemibiotrophic infection style in sweetpotato vegetative tissues.

Differential roles, crosstalk and response to the Antifungal Protein AfpB in the three Mitogen-Activated Protein Kinases (MAPK) pathways of the citrus postharvest pathogen Penicillium digitatum.

Fungi have three mitogen-activated protein kinases (MAPKs): Kss1/Fus3 involved in the invasive growth and virulence of pathogens, Hog1 in response to osmotic stress, and Slt2/Mpk1 in response to cell wall (CW) stress. We conducted comparative analyses of these MAPKs in the phytopathogen Penicillium digitatum and studied their role in the mode of action of the novel self-antifungal protein AfpB. The sensitivity to different stresses of Δhog1 and the reduced growth of Δkss1 coincided with previous reports. However, Δslt2 showed a strong reduction of growth and conidiation, abnormal morphology, and sensitivity to CW stress and temperature. The complementation of Δslt2 validated this mutant. Immunodetection of P-Hog1 and P-Slt2 confirmed the loss and gain of MAPKs in the mutant and complemented strains. Mutants Δslt2 and Δkss1 showed a strong reduction in virulence, whereas Δhog1 was the least affected, and none sporulated during infection. We studied the MAPK signalling induction in response to different treatments. Our data revealed a complex crosstalk involving the three MAPKs, the differential responses of Hog1 and Slt2 to various stresses and their induction by AfpB or the fungicide fludioxonil (FD). Δhog1 resistance to FD confirmed that Hog1 mediates the activity of FD, whereas Δkss1 sensitivity is probably due to the basal activation of Hog1 in Δkss1. None of the three MAPK mutants showed increased sensitivity to AfpB, contrary to previous reports of other antifungal proteins, which indicates that the observed AfpB-mediated activation of Hog1 and Slt2 would not have a defensive role.

Utility of a Multilevel Modeling Approach to Investigate Differences in Isolation Frequency of Fusarium culmorum in Agricultural Soil Across the Inland Pacific Northwest.

The plant pathogen Fusarium culmorum represents an inoculum source capable of contaminating grains with deoxynivalenol in the Inland Northwest region of the United States. A multilevel modeling approach utilizing varying intercepts for different sampling quadrats, fields, and iterations in the dataset was performed to characterize differences in isolation frequency of F. culmorum collected during a 2-year soil survey. Differences in the isolation frequency of F. culmorum varied the most by sampled field followed by quadrat and iteration, respectively. Higher relative elevation within the sampling region of a field limited the amount of F. culmorum recovered. The effect of annual climate variables was investigated using combinations of single-variable and multivariable model equations with linear and polynomial terms. The same data analysis approach was applied to an external dataset of F. culmorum isolation frequencies in grains from fields across eastern Australia. These results represent a case study for investigating variability within datasets containing overdispersed fungal counts and incorporating climate summaries as predictor variables.

Modeling Epidemics in Seed Systems and Landscapes To Guide Management Strategies: The Case of Sweet Potato in Northern Uganda.

Seed systems are critical for deployment of improved varieties but also can serve as major conduits for the spread of seedborne pathogens. As in many other epidemic systems, epidemic risk in seed systems often depends on the structure of networks of trade, social interactions, and landscape connectivity. In a case study, we evaluated the structure of an informal sweet potato seed system in the Gulu region of northern Uganda for its vulnerability to the spread of emerging epidemics and its utility for disseminating improved varieties. Seed transaction data were collected by surveying vine sellers weekly during the 2014 growing season. We combined data from these observed seed transactions with estimated dispersal risk based on village-to-village proximity to create a multilayer network or "supranetwork." Both the inverse power law function and negative exponential function, common models for dispersal kernels, were evaluated in a sensitivity analysis/uncertainty quantification across a range of parameters chosen to represent spread based on proximity in the landscape. In a set of simulation experiments, we modeled the introduction of a novel pathogen and evaluated the influence of spread parameters on the selection of villages for surveillance and management. We found that the starting position in the network was critical for epidemic progress and final epidemic outcomes, largely driven by node out-degree. The efficacy of node centrality measures was evaluated for utility in identifying villages in the network to manage and limit disease spread. Node degree often performed as well as other, more complicated centrality measures for the networks where village-to-village spread was modeled by the inverse power law, whereas betweenness centrality was often more effective for negative exponential dispersal. This analysis framework can be applied to provide recommendations for a wide variety of seed systems.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.

Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum.

Antifungal proteins (AFPs) of fungal origin have been described in filamentous fungi. AFPs are small, highly stable, cationic cysteine-rich proteins (CRPs) that are usually secreted in high amounts and show potent antifungal activity against non-self fungi. The role of AFPs in the biology of the producer fungus remains unclear. AFPs have been proposed as promising lead compounds for the development of new antifungals. The analyses of available antifungal CRP sequences from fungal origin and their phylogenetic reconstruction led us to propose a new classification of AFPs in three distinct classes: A, B and C. We initiate for the first time the characterization of an AFP in a fungal pathogen, by analysing the functional role of the unique afpB gene in the citrus fruit pathogen Penicillium digitatum. Null ΔafpB mutants revealed that this gene is dispensable for vegetative growth and fruit infection. However, strains that artificially express afpB in a constitutive way (afpB (C)) showed a phenotype of restricted growth, distortion of hyphal morphology and strong reduction in virulence to citrus fruits. These characteristics support an antifungal role for AfpB. Surprisingly, we did not detect the AfpB protein in any of the P. digitatum strains and growth conditions that were analysed in this study, regardless of high gene expression. The afpB (C) phenotype is not stable and occasionally reverts to a wild type-like phenotype but molecular changes were not detected with this reversion. The reduced virulence of afpB (C) strains correlated with localized fruit necrosis and altered timing of expression of fruit defence genes.

The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.

Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit.