Transformation of Alternaria dauci demonstrates the involvement of two polyketide synthase genes in aldaulactone production and fungal pathogenicity

Transformation of Alternaria dauci demonstrates the involvement of two polyketide synthase genes in aldaulactone production and fungal pathogenicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transformation of Alternaria dauci demonstrates the involvement of two polyketide synthase genes in aldaulactone production and fungal pathogenicity Jerome Monroe Bernardino, Elza Neau, Joséphine Kocuiba, Maïwenn Gadras, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6130137/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Chemical warfare between the host and the pathogen plays a crucial role in plant-necrotrophic pathogen interactions, but examples of its involvement in quantitative disease resistance in plants are poorly documented. In the Daucus carota-Alternaria dauci pathosystem, the novel toxin aldaulactone has been identified as a key factor in both fungal pathogenicity and the carrot’s partial resistance to the pathogen. Bioinformatic analyses have pinpointed a secondary metabolism gene cluster that harbors two polyketide synthase genes, AdPKS7 and AdPKS8 , that are likely responsible for the biosynthesis of aldaulactone. Here, we present the functional validation of AdPKS7 and AdPKS8 as genes responsible for aldaulactone production in A. dauci . We generated knock-out A. dauci mutants for AdPKS7 and AdPKS8 by replacing essential domains with a hygromycin resistance gene, marking the first reported case of genetic manipulation in A. dauci . Following transformation, the mutants were analyzed for toxin production via HPLC-UV and assessed for pathogenicity in planta . Aldaulactone production was abolished in all PKS mutants, which also exhibited significantly reduced pathogenicity on H1-susceptible carrot leaves. These findings confirm the roles of AdPKS7 and AdPKS8 in aldaulactone biosynthesis and their contribution to fungal pathogenicity. Biological sciences/Genetics/Microbial genetics/Fungal genetics Biological sciences/Microbiology/Fungi/Fungal pathogenesis Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Plant sciences Alternaria dauci transformation toxin biosynthesis pathogenicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The durable nature of partial or Quantitative Disease Resistance (QDR) in plants makes it a promising alternative to reduce the use of pesticides in agriculture. This is particularly true concerning pathogens with high evolutionary potential and necrotrophic pathogens 1 . Several genes control QDR and are associated with quantitative resistance loci (QRL) that each variably contribute to the phenotype 2 . Meanwhile, in qualitative disease resistance, R-genes are associated with total resistance to a disease. Their mechanism is generally based on the early detection of phytopathogen effectors. Specifically, R-genes facilitate a hypersensitivity response that causes the neighboring cells on the pathogen infection site to undergo programmed cell death 3 , 4 . The same response to necrotrophic pathogens would increase the plant’s susceptibility to the disease, as necrotrophic pathogens exploit cell death for their benefit. Hence, QDR appears to be a better means to mitigate the diseases caused by necrotrophic and hemi-biotrophic pathogens. However, in general, the levels of QDR obtained are insufficient to eliminate further phytosanitary measures such as the use of pesticides. Furthermore, breeding by exploiting QRLs is challenging as some of them can only be detected under certain environmental conditions or in specific genetic backgrounds 2 . As a result, observing QRL phenotypes and unveiling their underlying mechanisms have proven to be difficult. No comprehensive model of QDR has been realized so far. Moreover, very different mechanisms of QDR have been uncovered, suggesting that such a unifying model does not exist 1 , 5 . Alternatively, it can be envisioned that several mechanisms concur to yield a general QDR level. These mechanisms include (i) morphological and developmental variations, (ii) basal defense involvement, (iii) chemical warfare, (iv) defense signal transduction pathways, and (v) QRLs as a weak version of R-genes 1 . Recently, other mechanisms underlying QDR have been described, including vesicle trafficking, molecular chaperoning, and detoxification 5 . Phytotoxins are important determinants of plant diseases. Depending on their specificities, they can be classified as either non-host-specific toxins (NHSTs) or host-specific toxins (HSTs) 6 . Both NHSTs and HSTs fall under either of the two categories based on their chemical nature: ribosome synthesis-dependent peptides or secondary metabolites (SMs), which house most of the phytotoxins 7 . Several mechanisms of how phytotoxins are deployed have been described, including hindering lipid metabolism and disrupting plant membrane function. For instance, AAL toxins produced by the fungus Alternaria alternata are analogs of the plant’s ceramide synthase substrate. When exposed to this toxin, the plant cell undergoes rapid-fire production of sphingolipid precursors, leading to the loss of integrity of the plant’s plasma membrane 8 . An R-gene, Asc-1 , from tomato plants of the asc/asc genotype, confers total resistance to AAL-toxin-induced programmed cell death 9 . Meanwhile, fusicoccin, a toxin produced by the fungus Fusicoccum amygdali , renders the stomata unable to close and ultimately causes plant wilting. It does so by irreversibly activating the plant plasma membrane H + -ATPase 10 . While these examples pertain to the involvement of toxins in total resistance, some phytotoxins are also involved in QDR. Fungal tricothecenes play a role in QDR in wheat infected with Fusarium head blight. In this pathosystem, Fhb7 confers resistance to the disease by catalyzing the addition of glutathione (GSH) to a terminal epoxide of fungal tricothecenes 11 . A horizontally transferred glutathione S-transferase gene underlies this resistance mechanism conferred by Fhb7 . Meanwhile, the HC toxin produced by the fungus Cochliobolus carbonum race 1 takes part in QDR in maize. The maize Hm1 gene encodes an HC toxin reductase that inactivates the HC toxin 12 . Although Hm1 confers major resistance, the partial loss-of-function of some Hm1 alleles confers QDR 13 . Two other phytotoxins have been involved in QDR: the SS toxin in the Stemphylium solani - Allium sativum pathosystem and, in our laboratory, aldaulactone from Alternaria dauci , the causal agent of Alternaria Leaf Blight (ALB) on carrots ( Daucus carota ) 14 – 16 . The SS toxin causes plant cell death by inhibiting the H + -ATPase activity, NADH oxidation rate, and Fe(CN) 6 3− reduction rate in a dose-dependent manner 14 . Thus, the SS toxin targets the standard redox system and the plasma membrane H + -ATPase, the latter mechanism being similar to that of fusicoccin. These findings bolster the long-standing notion that the plasma membrane is one of the primary sites of action of phytotoxins 17 . Meanwhile, aldaulactone is produced by the necrotrophic fungus Alternaria dauci , causing ALB, the most prevalent and damaging foliar disease in carrots 18 – 21 . This disease is characterized by necrotic lesions surrounded by a chlorotic halo on carrot leaves. Out of the over seventy phytotoxins known to be produced by the phytopathogenic species of Alternaria , six are known to be produced by A. dauci : zinniol, alternariol, alternariol monomethyl ether, α-acetylorcinol, p -hydroxybenzoic acid, and aldaulactone 16 , 22 – 26 . Moreover, the existence in its genome of 19 secondary metabolite gene clusters indicates that this list is most likely not exhaustive 27 . This quite diverse toxin weaponry perhaps helps A. dauci infect a wide range of dicotyledonous plants, mainly inside but also outside of the Apiaceae family, with the carrot being its main host 28 . There are various strains of A. dauci , each characterized by different levels of aggressiveness. Similarly, various genotypes of carrots exhibit different levels of resistance to A. dauci , broadening the disease resistance levels to a continuum 29 . In recent years, we took advantage of the diversity of both carrot resistance level and A. dauci aggressiveness level to decipher the molecular basis of carrot QDR to the fungus. First, a correlation between plant partial resistance to A. dauci with in vitro cultured carrot cell resistance to fungal exudates was observed, indicating toxin resistance as a QDR mechanism 15 . Then, aldaulactone was isolated and characterized, dubbing it as an original A. dauci -produced phytotoxin. It likely plays an important role in the carrot- A. dauci interaction, as the production of aldaulactone was found to correlate with fungal pathogenicity levels 16 . In addition, in vitro assays showed that I2- partially resistant carrot cells were less susceptible to aldaulactone when compared to H1-susceptible carrot cells 16 . These results prompted us to better understand the structure of aldaulactone and its biosynthetic pathway. Aldaulactone is a benzenediol lactone of a polyketide (PK) nature 16 . Generally, PK benzenediol lactones are comprised of two molecule families bearing a 1,3-benzenediol moiety connected to a macrolactone: either a dihydroxyphenylacetic acid lactone (DAL) or a resorcyclic acid lactone (RAL). Through bidimensional NMR analyses, aldaulactone was identified as a PK containing a DAL 16 . The biosynthesis of PKs entails polyketide synthase (PKS) genes that are usually organized in the same cluster. Tailoring enzymes that catalyze functional group transfer or redox reactions are also encoded by other genes found within the same cluster 30 . In fungi, two types of PKSs are known: type I and type III PKSs. While Type III fungal PKSs are less characterized, Type I fungal PKSs are widely studied since most fungal PKSs fall under this category 31 , 32 . Type I PKSs iteratively catalyze the head-to-tail Claisen condensation of acetyl-CoA, leading to the eventual formation of a polyketide 30 , 33 , 34 . These multifunctional enzymes can be grouped into three types, according to the resulting PK’s degree of reduction: non-reducing (NR)-, partially reducing (PR)-, and highly reducing (HR)-PKSs 35 , 36 . Moreover, PKSs are multidomain enzymes that harbor a minimal core module of three domains: ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) 37 . The biosynthesis of some RALs such as zearalenone and 10,11-dehydrocurvularin have been described, owing to their genetic and molecular underpinnings 38 – 41 . In these pathways, the molecular backbones of the toxins are synthesized by the joint effort of Type I HR-PKS and NR-PKS. The same collaborative effort between the two Type I PKSs is also observed in the synthesis of DALs 42 . The regioselectivity of the cyclization spells out the difference between the synthesis of an RAL from that of a DAL: a C2-C7 aldol condensation produces an RAL, while a C3-C8 aldol condensation yields a DAL 42 . The construction of the biosynthetic pathway of aldaulactone requires probing into the genetic and molecular foundations of the PKSs involved. Bioinformatic analyses on the A. dauci genome revealed a single cluster (cluster 8) harboring two PKSs— AdPKS7 and AdPKS8 —thought to be involved in aldaulactone biosynthesis 27 . The AdPKS7 encodes an HR-PKS, while the AdPKS8 encodes an NR-PKS. Based on the organization of both PKS genes in the cluster and the structure of aldaulactone, a biosynthetic pathway of this toxin was proposed 27 . The expression patterns of AdPKS7 and AdPKS8 correlated with aldaulactone production under different experimental conditions 27 . These findings support the hypothesis that both AdPKS7 and AdPKS8 are involved in aldaulactone biosynthesis. Here, we present experiments designed to prove the implication of the two PKSs in aldaulactone biosynthesis and the involvement of aldaulactone in A. dauci pathogenicity. To our knowledge, no A. dauci transformation experiment has been published until now. The first aim of this study was thus to generate knock-out mutants of AdPKS7 and AdPKS8 in the FRA001 strain of A. dauci. Protoplast production, double-joint PCR-produced cassette uptake, and homologous recombination permitted the transformation of A. dauci. Two domains were targeted in each of the two AdPKS genes: the AT and the KS domains, which are minimally required to synthesize the aldaulactone backbone. In the mutants, the target domains were replaced by the Hygromycin Phosphotransferase gene ( Hph ), which confers resistance to hygromycin B. A non-coding mutant ( i.e ., the target domain was outside of the coding region of both AdPKS genes) was also constructed. In total, five mutants were generated: AdPKS7∆AT, AdPKS7∆KS, AdPKS8∆AT, AdPKS8∆KS, and ∆NC. The second aim of our study was to characterize the A. dauci mutants based on their ability to produce aldaulactone and their pathogenicity on carrot leaves. We analyzed the organic exudates from the five fungal mutants and the wild-type FRA001 strain through HPLC-UV. In a controlled environment, we also infected carrot leaves of both the H1-susceptible genotype and the I2-partially resistant genotype using conidial suspensions from all mutants and the wild-type. Our results indicate that the transformation method employed on A. dauci is efficient in carrying out functional validation experiments. Subsequently, through analyzing the mutants, we provided definitive proof of the function of AdPKS7 and AdPKS8 in aldaulactone production and the important role of this toxin in A. dauci pathogenicity on carrots. Results Transformation of A. dauci and molecular characterization of the transformed strains The transformation of A. dauci was realized in several steps, which include the generation of the hygromycin resistance cassette, protoplast production through enzymatic digestion, cassette DNA uptake in protoplasts, and selection of transformed fungal cells. The enzymatic cocktail Driselase/Kitalase allowed the A. dauci FRA001 strain to produce a high yield of protoplasts after four to five hours of digestion. The protoplast transformation protocol originally developed for A. brassicicola was adaptable to A. dauci , although differences in hygromycin sensitivity necessitated some modifications in selection concentration. The initial transformation to create A. dauci KO mutants was done using an agar layer containing 12 μg·mL -1 of hygromycin B, following the protocol for A. brassicicola 43,44 . However, no A. dauci transformants were recovered under this condition. Subsequent hygromycin sensitivity assays with conidia from the A. dauci FRA001 and A. brassicicola Abra43 strains revealed that A. dauci is more sensitive to hygromycin B than A. brassicicola is, with complete inhibition of conidial germination at concentrations above 12 μg/ml. A second transformation attempt was thus conducted with a reduced hygromycin concentration of 2 μg·mL -1 to select KO mutants. This second transformation revealed that 2 μg·mL -1 hygromycin B was insufficient for effective selection, as both transformants and non-transformants traversed the hygromycin B-supplemented agar overlay. The transformants were then transplanted anew to PDA with either 5 or 8 μg·mL -1 hygromycin B. This process determined that an agar overlay supplemented with 8 μg·mL -1 of hygromycin B was effective for initial selection, with subsequent transfer to PDA supplemented with 5 μg·mL -1 of hygromycin B to support sustained fungal colony growth without excessive inhibition. The molecular characterization of the wild-type A. dauci FRA001 strain, along with all the mutants, was performed by PCR verification. Specifically, sequences corresponding to the internal transcribed spacer ( ITS ), hygromycin B phosphotransferase ( Hph ) gene, the acyltransferase (AT) and ketosynthase (KS) domains of AdPKS7 and AdPKS8, and the non-coding region targeted in the ΔNC strain were amplified via standard PCR and analyzed using gel electrophoresis. All A. dauci strains exhibited amplification of the ITS , whereas only the mutant strains displayed amplification of the Hph (Figure 1). In the wild-type strain, the AT, KS, and non-coding regions were amplified. The targeted domains showed no amplification in the respective mutants. To verify the correct insertion of the Hph gene in place of the target domains, PCR amplification was performed across the region spanning the upstream sequence of the respective AdPKS gene ( AdPKS7 or AdPKS8 ) and the downstream sequence of Hph . Similarly, amplification was carried out for the downstream region of the AdPKS gene and the upstream region of Hph . The presence of PCR products corresponding to both regions in the gel confirmed the correct insertion of Hph into the genomes of the A. dauci mutants. These results corroborate the successful knockout of the PKS genes, replaced by Hph in the PKS mutants, as well as the substitution of the non-coding region with Hph in the ΔNC mutant. Morphological profiling of A. dauci mutants To our knowledge, this study presents the first reported case of genetic transformation in A. dauci . Consequently, we documented the morphological characteristics of the resulting mutants, focusing specifically on their radial mycelial growth on V8 agar medium and their conidial morphology and length. The mycelial growth rates of all five A. dauci strains were evaluated by computing the Area Under the growth Curve (AUC) values. Figure 2a presents a statistical comparison of the mean AUC values across nine replicates per strain, analyzed using Tukey’s post-hoc test. The mycelial growth rates of all five mutant strains were slightly yet significantly faster than that of the wild-type. Moreover, the ΔNC mutant displayed a growth rate that was statistically similar to those of the PKS mutants. A visual comparison of mycelial growths on V8 agar medium at seven days post-transplant is shown in Figure 2b. While differences in growth rate between the wild-type and mutant strains were subtle, a marked contrast in mycelial pigmentation was evident: the wild-type strain displayed a lighter hue compared to the black pigmentation observed in all mutant strains. Conidial phenotyping of the mutants was also conducted to evaluate further pleiotropic effects of the transformation. Micrographs of conidia per strain were captured using a light microscope, and conidial length was manually measured. Figure 3a shows a statistical comparison of mean conidial lengths for all A. dauci strains, analyzed using Tukey’s post-hoc test. All mutants exhibited significantly longer average conidial lengths than the wild-type. Figure 3b visually compares representative conidia from each A. dauci strain, highlighting the noticeably shorter conidium of the wild-type in contrast to the mutants. Under typical conditions, A. dauci conidia display an ellipsoidal body with multiple transverse septa and one or more longitudinal septa, tapering into a long, slender filiform beak (Figure 3b, FRA001). While most conidia from the transformed strains retained this characteristic morphology, some displayed abnormal forms, such as bent bodies often with a clump of cells, as seen in the micrograph of the AdPKS7ΔKS conidium. HPLC-UV analysis for aldaulactone detection The organic exudates (OEs) of the various A. dauci strains were analyzed using HPLC-UV to assess their ability to produce aldaulactone. For each fungal strain, three biological replicates were included. Since aldaulactone has a maximum absorbance at 305 nm, all chromatograms were examined at this wavelength 16 . Aldaulactone exhibits a retention time of 18.5 minutes, as seen in the chromatogram of the aldaulactone standard ( i.e., pure aldaulactone; Figure 4). A peak corresponding to aldaulactone was detected in the wild-type and ΔNC strains. However, this peak was absent in all PKS mutants. To confirm the identity of the purported aldaulactone detected in the wild-type and ΔNC strains, the UV profiles of the compounds from the 18.5-min peak in each chromatogram were compared with that of aldaulactone. The profiles were consistent across three biological replicates, showing characteristic absorbance peaks at 196 nm and 302 nm (Supplementary Figure 1). These results confirm that the peak with a retention time of 18.5 min observed in both the wild-type and ΔNC strains corresponds to aldaulactone. Carrot in planta pathogenicity test To assess the potential pathogenicity of the mutant strains in inducing symptoms characteristic of Alternaria Leaf Blight (ALB), we inoculated carrot leaves with conidial suspensions from both the wild-type and mutant A. dauci strains. The degree of pathogenicity of the A. dauci strains on carrot leaves was then compared by analyzing the log-transformed Area Under the Disease Progression Curve (LogAUDPC) values. An illustration of symptom severity on carrot leaves showing different LogAUDPC values is provided in Figure 5a. A Waller-Duncan post-hoc analysis was conducted to statistically compare between symptom severity of each A. dauci strain. Given that the ΔNC strain shares closer genetic similarity to the PKS mutants due to the insertion of the Hph gene, and exhibits similar morphological characteristics, we deemed it more appropriate to compare the pathogenicity of the PKS mutants to the ΔNC strain rather than the wild-type FRA001 strain. In the H1-susceptible carrot genotype, the ΔNC strain exhibited significantly higher pathogenicity compared to the PKS mutants, though no significant difference was observed relative to the wild-type strain (p < 0.05; Figure 5b). For the wild-type FRA001 strain, a significant difference in disease severity was noted between the H1-susceptible and I2-partially resistant carrot genotypes, with the former showing a markedly higher level of symptom severity. A similar pattern was observed for the ΔNC strain, where the H1-susceptible genotype displayed significantly higher disease severity than the I2-partially resistant genotype. In contrast, no significant difference was seen between the H1-susceptible and I2-partially resistant leaves infected with any of the PKS mutants. The identity of the A. dauci used for inoculation was confirmed by microscopic viewing of the conidia still attached to the carrot leaves 48h following leaf detachment (supplementary figure 2). Discussion As a necrotrophic pathogen, A. dauci is expected to rely on phytotoxins to establish pathogenicity in its carrot host. To date, six secondary metabolites (SMs) with a phytotoxic activity have been identified as being produced by A. dauci 16 , 22 – 26 . While the role of zinniol as a phytotoxin has been questioned, aldaulactone accounts for most—but not all—of the in vitro toxicity of A. dauci exudates to carrot cells 16 , 45 . In addition, aldaulactone production is strongly correlated with A. dauci pathogenicity. Finally, a notable correlation was observed between carrot cell resistance to A. dauci organic extracts and carrot plant resistance to the fungus, which led us to think that toxin resistance is a major resistance mechanism in the carrot- A. dauci interaction 15 , 16 . To obtain a snapshot of the genetic determinants of secondary metabolism in A. dauci , our lab has provided the first transcriptome of this species, along with the first appraisal of SM core gene diversity within Alternaria genomes 27 . These investigations pinpointed AdPKS7 and AdPKS8 , both located in gene cluster 8, as candidate genes responsible for aldaulactone biosynthesis 27 . To confirm this, we conducted a functional validation of these genes that prompted us to produce and analyze KO mutants. For this to be realized, we adapted a transformation method originally developed for A. brassicicola Abra43. The method was successfully applied to the A. dauci FRA001 strain with a couple of modifications. First was the use of 8 µg·mL − 1 hygromycin in the agar overlay, compared to 12 µg·mL − 1 used for the transformation of A. brassicicola Abra43. This adjustment of the hygromycin concentration suggests that the A. dauci FRA001 cells exhibit lower resistance to hygromycin B than the A. brassicicola Abra43 cells. Another modification involved extending the enzymatic digestion time for A. dauci FRA001. While A. brassicicola Abra43 cells underwent digestion for four hours, those of A. dauci FRA001 were treated for six hours. To our knowledge, this is the first reported case of targeted genetic manipulation in A. dauci . Morphological profiling of the transformed A. dauci strains revealed phenotypic effects associated with its genetic transformation. The significantly faster mycelial growth and longer conidia observed in all mutant strains including the ΔNC strain, compared to the wild-type, suggests that the transformation process itself impacted their physiology. Form variations in A. dauci FRA001 mutant conidia were also observed alongside changes in conidial length: the conidia of these transformed strains occasionally exhibited clumps of cells in the conidial body, a feature absent in the wild-type FRA001 strain. These effects could be due to the insertion of foreign DNA, such as the Hph gene, which may have activated a general stress response pathway, potentially leading to physiological reprogramming. A study on the development of spontaneous hygromycin B resistance in the necrotrophic pathogen Monilinia fructicola demonstrated that hygromycin B-resistant mutant colonies exhibited slower growth on the PDA medium when compared to the wild-type 46 . Although this study did not employ site-specific mutagenesis, it suggests that the insertion of the Hph gene might lead to developmental changes in fungi. Interestingly, similar conidial form variations have also been observed in other A. dauci strains, such as FRA017, a wild strain resistant to the fungicide, iprodione. Also, similar morphological variations were observed in A. dauci conidia exposed to environmental stress, such as fungicide 47 . These findings suggest that the observed morphological changes can correspond to the expression of stress resistance-associated mechanisms. The shared morphological features observed among the ΔPKS mutants and the ΔNC strain, coupled with the insertion of Hph into their genomes, led us to use the ΔNC strain rather than the wild-type as the basis of comparison in experiments assessing the effects of AdPKS gene knockouts. Organic exudates from the A. dauci strains were analyzed by HPLC equipped with a photodiode array detector. Chromatograms were extracted at 305 nm, the wavelength where aldaulactone shows maximum absorbance. The ΔPKS mutants did not show the characteristic aldaulactone peak at 18.5 min. In contrast, both the wild-type and the ΔNC strains exhibited the aldaulactone peak, which is consistent across all three biological replicates. Moreover, the UV profile of the compound detected at 18.5 min in these two strains closely matches that of aldaulactone (Supplementary document 2). Minor peaks were likewise observed beyond the 18.5 min retention time across all fungal strains, suggesting that other metabolic pathways involved in the production of the compounds in these peaks were not affected by the transformation process. However, minor peaks detected before 18.5 min were present in the wild-type FRA001 strain but not in the transformed strains. Other metabolic pathways involved in the production of the compounds detected in these peaks may have been affected by the transformation process and not the knock-out of either of the AdPKS genes, as the ΔNC strain did not show similar peaks. All in all, these results provide strong evidence that AdPKS7 and AdPKS8 are responsible for aldaulactone biosynthesis. In addition, they provide us with a set of isogenic strains that only differs in their production of aldaulactone. To evaluate the role of aldaulactone production in fungal pathogenicity, we conducted an in planta pathogenicity test on six-week-old carrot leaves. In the H1-susceptible carrot genotype, a significant difference was observed between the ΔNC strain and all ΔPKS mutants, but not with the wild-type. This suggests that aldaulactone plays an essential role in the pathogenicity of A. dauci . In contrast, no significant difference was observed across all fungal strains when inoculated on the I2-partially resistant carrot genotype. Aldaulactone production is the only component of pathogenicity that differs between the ΔNC and the ΔPKS mutants. In a previous study, we saw that the I2 cultivar resists aldaulactone toxicity 16 . A possible explanation for the in planta observation could be that since the I2 genotype resists aldaulactone, it is solely the other components of A. dauci pathogenicity that are responsible for the symptoms observed in this genotype. As a result, the absence or presence of aldaulactone alone does not affect the outcome of the confrontation with A. dauci strains unable to produce aldaulactone, suggesting that the other components of I2 resistance are not strong enough to be detected in our setup when compared with that of the H1 genotype. Our study demonstrates that the knockout of AdPKS7 or AdPKS8 , resulting in the complete loss of aldaulactone production, significantly reduces disease symptom severity in the H1-susceptible carrot genotype. This finding strongly indicates that aldaulactone is the primary phytotoxin responsible for causing ALB symptoms in H1-susceptible carrots. These results are in contrast with a recent study on the functional redundancy of pathogenicity factors in Botrytis cinerea . In that study, multiple knockout mutants, lacking up to 12 cell-death-inducing proteins (CDIPs) and metabolites, exhibited a progressive decrease in virulence 48 . Interestingly, the reduction in virulence correlated with the increasing number of gene knockouts, and was not significant when less than four genes were deleted, suggesting functional redundancy among the pathogenicity factors. Despite the substantial reduction in known phytotoxins, significant phytotoxic activity persisted in the mutants' secretomes, pointing to the presence of yet-unidentified CDIPs in B. cinerea . In stark contrast, our findings show that the knockout of AdPKS7 or AdPKS8 leads to a clear reduction in ALB symptoms in carrots. Unlike B. cinerea , A. dauci does not exhibit compensatory mechanisms to recover its pathogenicity when the aldaulactone biosynthesis pathway is disrupted. This underscores the central role of aldaulactone in A. dauci pathogenicity on carrots. Our previous findings have established that aldaulactone is toxic to carrot cells and also to tobacco leaves when directly injected into them, suggesting that aldaulactone may be classified as a non-host specific toxin (NHST) 27 . This finding contrasts aldaulactone with the HC toxin, a host-specific toxin (HST) produced by Cochliobolus carbonum race 1. While the HC toxin targets specific maize genotypes owing to the absence of the HM1 gene in their genomes, aldaulactone exhibits toxicity across distant plant species, such as D. carota and N. benthamiana 12 , 16 , 27 . This broader host range suggests that aldaulactone potentially targets conserved cellular processes, a hallmark of NHSTs. The resistance mechanisms against NHSTs, such as aldaulactone, are often associated with QDR rather than race-specific total resistance typical of HSTs. In this context, the partial resistance observed in the carrot I2 genotype likely demonstrates an array of possible defense responses, such as detoxification mechanisms and cellular defense mechanisms, among others. This is in contrast with the HC toxin resistance mechanism in maize, where the single dominant HM1 gene encodes a specific HM toxin reductase that inactivates the toxin. The NHST classification of aldaulactone thus corresponds to the general notion of this group of toxins in that they are not the sole determinant of pathogenicity 49 . In turn, the ALB symptoms observed on carrot leaves infected with the ΔPKS mutants are attributed to the other components of A. dauci pathogenicity and not aldaulactone. Both the ingenuity and downfall of toxin resistance mechanisms in plants are reflected in the plants’ evolutionary race against necrotrophic pathogens. For instance, the Pc2 gene renders some plants susceptible to victorin, a toxin produced by the necrotrophic fungus, Cochliobolus victoriae 50 . Various Tsn genes, on the other hand, recognize effectors encoded by Tox genes in Parastagonospora nodorum 51 . Upon recognition of the effectors, the Tsn genes activate programmed cell death, a means for necrotrophic fungi such as P. nodorum to undermine plant defense responses and promote infection. These are examples of the inverse gene-for-gene hypothesis, where a susceptible gene, instead of an R-gene, is compatible with the gene of a pathogen encoding an avirulence factor. Another example is the Asc1 gene in tomatoes that confers susceptibility to AAL-toxin produced by Alternaria alternata f. sp. lycopersici 52 . The mutation in Asc1 in some tomato cultivars is responsible for the susceptibility to the toxin. These cases show that plants resist fungal-derived phytotoxins in various ways, whether by detoxifying the toxins or by lacking susceptibility genes. However, how carrots resist aldaulactone is yet to be determined. As mentioned, the NSHT nature of aldaulactone suggests that resistance against it may not depend on a single R-gene, but rather on general cellular defense mechanisms or perhaps, on the inverse gene-for-gene hypothesis. All in all, we provided an efficient site-directed transformation method for A. dauci , offering a valuable tool for genetic manipulation in this species. Through the analysis of the transformed strains, definitive proof of the roles of AdPKS7 and AdPKS8 in aldaulactone production was provided. Furthermore, fungal transformation gave us isogenic lines that helped us isolate the role of aldaulactone in both fungal pathogenicity and carrot plant resistance. This study represents the first demonstration of the role of both aldaulactone itself and aldaulactone resistance in the carrot- A. dauci interaction. Further research is needed to elucidate the toxin mode of action and the resistance mechanisms involved in this pathosystem. That is, studies streamlined on identifying the molecular target of aldaulactone and the key defense pathways it activates could reveal whether the resistance against it fits into known mechanisms or follows an entirely different one. Beyond the recessive resistance to necrotrophic effectors such as those encoded by P. nodorum Tox genes, there are only three examples where pathogen resistance genes were shown to encode toxin resistance factors 11 , 12 , 52 , 53 . Lengthening this short list would invite insights into the diversity of plant toxin resistance mechanisms, a crucial part of plant resistance mechanisms against necrotrophic pathogens. Methods Fungal material and fungal growth conditions Alternaria dauci was grown on Petri dishes containing V8 agar and incubated at 24°C in darkness for 14 d. Only one strain of A. dauci was used in this paper: the FRA001 strain, which is moderately aggressive on carrot and was also used in other studies 28 , 16,27 . It was collected as described in 28,54 and is freely available from the COMIC collection (COMIC-SFR Quasav, 42 rue Georges Morel, 49070 Beaucouzé cedex, France). For liquid cultures, all fungal strains were grown in Difco TM Potato Dextrose Broth (PDB) as in 16 . Ten colonized agar plugs (about 5 mm in diameter) were taken from solid V8 culture and ground for 1 min in 40 mL of PDB. Sixty mL of PDB was then used to rinse any adhering mycelia on the sterile metal grinder (DuPont Instruments, Sorvall ® Omni-Mixer), then poured into the flask containing the earlier collected 40 mL suspension. The 100-mL suspension was then sealed and incubated in the dark at 24°C for 72 h with an agitation of 125 rpm (MINITRON ® , Infors HT). HPLC-UV analysis FRA001 PDB culture suspensions for HPLC-UV analysis were grown for 72 h. Then, the raw exudates were recovered by serial filtration using 200 µm, 50 µm, and 1 µm sterile nylon membranes from Sefar Nitex (Sefar AG, Heiden, Switzerland) as in 15 . The mycelia were weighed and stored at -80°C for future use, while the filtered raw exudates were neutralized to pH 7 and underwent liquid-liquid extraction through the addition of an equal volume of ethyl acetate. This was done three times to ensure that the organic exudate (OE) was maximally extracted. The OEs were then dried over sodium sulphate and evaporated under reduced pressure, and stored as described in 15 . The OEs were resuspended in HPLC-grade methanol (5 mg·mL -1 ) and centrifuged at 11,000g for 10 min. The supernatants were analyzed using a Shimadzu Prominence-I LC-2030 3D coupled with PDA detector and assisted by LabSolutions software. The mobile phase (flow rate: 0.7 mL·min -1 ) was composed of water and HPLC-grade methanol with the following gradient: 90% water and 10% methanol at 0 min, then 100% methanol after 25 min, maintained for 5 min. All analyses were performed at 25°C on an UPTISPHERE C18-ODB column (150 x 4.6 mm; 3 µm). Genomic DNA extraction and standard PCR The genomic DNA of A. dauci was extracted using the microwave miniprep method as described by 55 . The molecular characterization of fungal strains through standard PCR (Thermocycler T100, Bio-rad) was performed on various genomic regions, with the internal transcribed spacer (ITS) region as a positive control. The PCR mixtures contained 1x Green GoTaq® Flexi Buffer (Promega), 1.25u of GoTaq® Hot Start DNA polymerase (Promega), 0.1 µM of each primer, 1.5 mM of MgCl 2 solution, 0.2 mM of dNTP, 1 µL of the DNA suspension, and diluted to a total volume of 25 µL using nuclease-free water. The primers used in this study are listed and detailed in Supplementary document 4 . The PCR programs used vary depending on the regions amplified. Detailed programs are specified in Supplementary document 5 . Gene replacement cassette production and fungal transformation The gene replacement cassettes were created by the double-joint PCR method as detailed by 56 . They bore the 5’ and 3’ flanking regions of each target gene fused with the Hph gene (LT726869) conferring resistance to hygromycin B. For each amplification, the Phusion Hot Start II High-Fidelity DNA polymerase pack (Thermo Fisher Scientific) and the appropriate primers were used. The final volume for each reaction was 50 µL, which was comprised of Phusion Hot Start DNA polymerase (0.02 u·µL -1 ), 3% v/v DMSO, 1x Buffer HF, dNTP (0.2 mM each), primers (20 µmol each), and 1 µL of the DNA. The first round of PCR amplified the 5’ and 3’ flanking regions of the target gene, as well as the Hph gene (1699 bp) from the plasmid pCB1636 57 . The second round of PCR ligated the products from the first PCR round to form the gene replacement cassette. The third round of PCR amplified the entire cassette from the second PCR round. Products of the first and third rounds of PCR were purified using the NucleoSpin ® Gel and PCR Clean-up kit (Macherey Nagel) according to the manufacturer’s instructions. Protoplast production was initiated by culturing A. dauci conidia (minimum of 80 cfu·mL -1 ) in 100 mL of PDB for 15 h at 24°C with an agitation of 175 rpm. The mycelia were then collected by centrifugation (10 min, 1700 g), washed in 0.7 M NaCl, and then centrifuged again using the same conditions. This washing step was performed twice before adding the mycelia to a 20-mL digestion solution containing Driselase (20 mg·mL -1 ; Sigma D9515-5G) and Kitalase (10 mg·mL -1 ; Wako 1W114-0037) prepared in 0.7 M NaCl. The resulting mixture was carefully mixed by inversion every 30 min for 6 h. The protoplasts were collected by centrifugation (7 min, 1700 g) and then delicately resuspended in 10 mL STC buffer (1.2 M Sorbitol, 10 mM Tris pH 7.5, 50 mM CaCl 2 ). Centrifugation was repeated and the protoplasts were resuspended in 1 mL STC at a concentration between 10 6 -10 8 protoplasts·mL -1 . For each mutant, 10-20 ng of the gene replacement cassette was added to 500 µL of protoplast suspension, which was then incubated on ice for 20 min. PEG [6 % w/v Polyethylene glycol MW 3350 (Sigma-Aldrich P3640), 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2 ] was added three times, with the suspension warmed in the hand in between each addition. The suspension was gently mixed, incubated on ice for 5 min, and added with 1 mL of STC. Then, 250 µL of the protoplast suspension was added to 20 mL of the regeneration medium (1 M sucrose, 0.1% w/v yeast extract, 0.1% w/v casein hydrolase, and 1.6% w/v bacteriological agar), and the resulting mixture was poured into a Petri dish. After 24 h, a 10-mL agar overlay supplemented with hygromycin B (8 µg·mL -1 ) was poured. The plates were stored at 24°C in the dark. The hygromycin B-resistant mutants were identified based on their ability to cross the agar overlay 5 to 7 days after. They were then sub-cultured on PDA media supplemented with hygromycin B (5 µg·mL -1 ). The purity of the mutants was checked by standard PCR as described above. In cases where the mutants were not pure ( i.e ., when a band corresponding to the target gene appeared on the agarose gel), monosporing on PDA media supplemented with hygromycin B (5 µg·mL -1 ) was conducted. The mutant strains were conserved in cryotubes with 1 mL 30% glycerol and stored at -80°C. In total, five A. dauci mutants were constructed from the A. dauci FRA001 strain: AdPKS7∆AT, AdPKS7∆KS, AdPKS8∆AT, AdPKS8∆KS, and ∆NC. The ∆NC strain was generated by knocking out a non-coding region 946 bp downstream the AdPKS7 gene through its replacement with Hph . Fungal phenotyping The mycelial radial growth assay was carried out according to 58 . Briefly, agar disks were sourced from the margin of a 7d-old colony grown on V8 agar media and were transferred to another V8 agar media. The radius of the mycelial colony was measured in cm at 3-, 4-, 5-, 6-, and 7 days post-transfer. The area under the growth curve was then calculated as in 59 and photos of the obverse view of the Petri dishes bearing the mycelia were captured on the final day of measurement. Conidial phenotyping was performed by first scraping the surface of 14-day-old fungal colonies from V8 agar medium with 100 mL 0.05% Tween 20 solution. Three µL of the conidial suspension was mounted on a slide and then viewed under a light microscope at 40x magnification (ZEISS Axio Imager 2). The length from the tip of the conidial body to the end of the filiform beak was measured, with at least 35 measurements of different conidia for each strain. The ImageJ software was used to measure conidial lengths. Plant material and in planta pathogenicity test The Daucus carota genotypes used in this study were H1 (susceptible) and I2 (partially resistant). The H1 and I2 seeds were obtained as described in 15 . Plant cultivation was performed according to 21 . Briefly, plants were grown in pots containing peat moss/sand mixture in greenhouse conditions (16h of day, 22°C day/19°C night, 60% humidity) for six weeks. Plant inoculation procedures have been described in detail in 19 . Sporulation of A. dauci strains was realized by individually growing the fungi on V8 agar and incubating them at 24°C in darkness for 14 d. The conidial suspensions were adjusted to 200 conidia mL -1 in 0.05% Tween 20. The third leaf of six-week-old carrot plants was inoculated with the conidial suspension. For this, leaves still-attached to the plant were placed in incubation chambers made of a Petri dish anchored to the substrate. In an incubation chamber, a moist filter paper was placed at the bottom, allowing the leaf to rest on top of the paper and be held in place by paper clips and 6 mm nuts. Forty drops of 5 µL of the conidial suspension were applied on the adaxial side of the leaf using a micropipette. Symptom intensity was evaluated at 7-, 11-, and 13-days post-inoculation and is expressed as the number of symptoms per conidia. A value of 1 was assigned to a necrotic lesion smaller than the drop deposited, while a value of 10 was assigned to the lesion larger than the drop. The ratio of the number of lesions per viable conidia was calculated at each scoring date, and the area under the disease progression curve (AUDPC) was determined according to 19 . In each condition ( i.e ., carrot genotype inoculated with the fungal strain), three technical replicates were made. This was done in four repetitions. Statistical analyses All statistical analyses were performed using the R v3.2.4 software in R Studio v1.0.136 60 . Normality and homoscedasticity of residues were checked using a Shapiro test and graphical assessments, respectively. To determine significant differences between fungal strains in the morphological characterization experiments, one-way analysis of variance (ANOVA) alongside Tukey’s post-hoc test was used. Meanwhile, the in planta pathogenicity test, the AUDPC values were log-transformed (LogAUDPC), then underwent the same ANOVA, but coupled with the Waller-Duncan multiple comparisons test. In all three analyses, significant differences were noted at the p < 0.05. Declarations Additional information The authors declare no competing interests. Author Contribution R.B. and P.P. designed and managed the project. J.M.B., R.B., and P.P. wrote the article. M.G., E.N., N.B.S., and J.Colou performed fungal transformation. J.M.B., J.C., E.N., M.G., J.Colou, and F.B. contributed to the molecular analysis and characterization of fungal strains. J.M.B. and A.R. performed the morphological profiling of fungal strains. B.H. and F.B. managed the fungal collection. J.M.B., E.N., J.K., A.R., J.J.H., and D.B. produced, extracted, and analyzed the fungal extracts. J.M.B., E.N., J.K., A.R., and S.A. conducted the in planta pathogenicity test. J.M.B. and R.B. performed the statistical analyses. All authors reviewed the manuscript and approved the final version. Acknowledgement The authors would alike to thank Jérôme Collemare of the Westerdijk Fungal Biodiversity Institute, Julie Chong of Université de Haute-Alsace, and Sandrine Giraud of Université d’Angers for their valuable insights in the fulfillment of this scientific work. They would also like to thank the following people from the Institut de Recherche en Horticulture et Semences (IRHS): Thomas Guillemette of the FungiSem team for his insights on fungal transformation, Mathilde Briard of the QuaRVeg team for the provision of carrot seeds, Aurelia Rolland and Fabienne Simonneau of the IMAC team for the realization of the conidial phenotyping, and Kaat Hellyn and Daniel Sochard of the PHENOTIC team for participating in the in planta experiments. Data Availability All data generated or analyzed during this study are available upon request to the corresponding author. References Poland, J. A., Balint-Kurti, P. J., Wisser, R. J., Pratt, R. C. & Nelson, R. J. Shades of gray: the world of quantitative disease resistance. Trends in Plant Science 14 , 21–29 (2009). Pilet-Nayel, M.-L. et al. Quantitative Resistance to Plant Pathogens in Pyramiding Strategies for Durable Crop Protection. Front. Plant Sci. 8 , 1838 (2017). Jones, J. D. G. & Dangl, J. L. 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QUASAV","correspondingAuthor":false,"prefix":"","firstName":"Dimitri","middleName":"","lastName":"Bréard","suffix":""},{"id":425507985,"identity":"72f2cf08-c543-4a6b-85ec-2ee68d96e38b","order_by":8,"name":"Sophie Aligon","email":"","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Aligon","suffix":""},{"id":425507986,"identity":"124bee01-267e-4a07-b6d4-7a83f0a32835","order_by":9,"name":"Franck Bastide","email":"","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":false,"prefix":"","firstName":"Franck","middleName":"","lastName":"Bastide","suffix":""},{"id":425507987,"identity":"da605c2c-73bc-4256-b7d0-f6093abb3936","order_by":10,"name":"Bruno Hamon","email":"","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"","lastName":"Hamon","suffix":""},{"id":425507988,"identity":"e358341f-0e3b-45f6-b341-df0c97b3bc51","order_by":11,"name":"Justine Colou","email":"","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":false,"prefix":"","firstName":"Justine","middleName":"","lastName":"Colou","suffix":""},{"id":425507989,"identity":"05c2ea72-bf81-443e-8419-68de26292857","order_by":12,"name":"Pascal Poupard","email":"","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":false,"prefix":"","firstName":"Pascal","middleName":"","lastName":"Poupard","suffix":""},{"id":425507990,"identity":"e8094e4c-9b25-4dc0-aadb-ac631794a61e","order_by":13,"name":"Nelly Bataillé-Simoneau","email":"","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":false,"prefix":"","firstName":"Nelly","middleName":"","lastName":"Bataillé-Simoneau","suffix":""},{"id":425507991,"identity":"ad056e54-20a7-410b-b2d4-b92448774fd7","order_by":14,"name":"Romain Berruyer","email":"data:image/png;base64,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","orcid":"","institution":"Université d’Angers, Institut Agro, INRAE, UMR IRHS 1345","correspondingAuthor":true,"prefix":"","firstName":"Romain","middleName":"","lastName":"Berruyer","suffix":""}],"badges":[],"createdAt":"2025-02-28 16:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6130137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6130137/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-17441-z","type":"published","date":"2025-10-02T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78258056,"identity":"21692d38-f992-42c0-b37a-31043e4f4e91","added_by":"auto","created_at":"2025-03-11 11:08:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":814120,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular characterization of \u003cem\u003eA. dauci \u003c/em\u003estrains through PCR verification, visualized using 1.2% agarose gels. The individual gel photos are numbered 1 to 17 from top to bottom. Gel 1, amplification of \u003cem\u003eITS\u003c/em\u003e; gel 2, \u003cem\u003eHph\u003c/em\u003e; gel 3, AT domain of \u003cem\u003eAdPKS7\u003c/em\u003e; gel 4, KS domain of \u003cem\u003eAdPKS7\u003c/em\u003e; gel 5, AT domain of \u003cem\u003eAdPKS8\u003c/em\u003e; gel 6, KS domain of \u003cem\u003eAdPKS8\u003c/em\u003e; gel 7, NC region; gel 8, upstream region of AdPKS7ΔAT and downstream region of \u003cem\u003eHph\u003c/em\u003e; gel 9, downstream region of AdPKS7ΔAT and upstream region of \u003cem\u003eHph\u003c/em\u003e; gel 10, upstream region of AdPKS7ΔKS and downstream region of \u003cem\u003eHph\u003c/em\u003e; gel 11, downstream region of AdPKS7ΔKS and upstream region of \u003cem\u003eHph\u003c/em\u003e; gel 12, upstream region of AdPKS8ΔAT and downstream region of \u003cem\u003eHph\u003c/em\u003e; gel 13, downstream region of AdPKS8ΔAT and upstream region of \u003cem\u003eHph\u003c/em\u003e; gel 14, upstream region of AdPKS8ΔKS and downstream region of \u003cem\u003eHph\u003c/em\u003e; gel 15, downstream region of AdPKS8ΔKS and upstream region of \u003cem\u003eHph\u003c/em\u003e; gel 16, upstream region of ΔNC and downstream region of \u003cem\u003eHph\u003c/em\u003e; gel 17, downstream region of ΔNC and upstream region of \u003cem\u003eHph\u003c/em\u003e. Lane 1, wild-type \u003cem\u003eA. dauci \u003c/em\u003eFRA001 strain; Lane 2, AdPKS7ΔAT; lane 3, AdPKS7ΔKS; lane 4, AdPKS8ΔAT; lane 5, AdPKS8ΔKS; lane 6, ΔNC; lane 7, negative control. While the gels in this figure are cropped, the individual, whole gel photos are provided in \u003cstrong\u003eSupplementary document 1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/f6447c5f2512f752d614bbe2.png"},{"id":78257882,"identity":"78675c3c-0fa9-4661-a158-fc057fa80a7f","added_by":"auto","created_at":"2025-03-11 11:08:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1106040,"visible":true,"origin":"","legend":"\u003cp\u003eMycelial growth phenotyping analysis. \u003cstrong\u003e(a)\u003c/strong\u003eStatistical comparison of average area under the growth curve (AUC) values calculated from the radial mycelial growth of \u003cem\u003eA. dauci\u003c/em\u003e strains on V8 agar medium. Nine replicates per fungal strain were used and then analyzed using Tukey’s test. Different letters indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). \u003cstrong\u003e(b)\u003c/strong\u003e Obverse view of mycelial growth of \u003cem\u003eA. dauci\u003c/em\u003e strains on Petri dishes containing V8 agar media. Photographs were taken seven days post-transplantation of the mycelial plug into the center of the medium.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/78b632bef855eb7e24cc341f.png"},{"id":78257884,"identity":"eaa2881b-c3bd-4f4d-a516-e08f9393ae47","added_by":"auto","created_at":"2025-03-11 11:08:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1400062,"visible":true,"origin":"","legend":"\u003cp\u003eConidial phenotyping analysis. \u003cstrong\u003e(a)\u003c/strong\u003e Statistical comparison of mean conidial lengths (in mm) derived from 35 conidia per \u003cem\u003eA. dauci\u003c/em\u003e strain using Tukey’s post-hoc test. Different letters indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Mean conidial lengths were measured following an image-based measurement of conidia captured under a light microscope. \u003cstrong\u003e(b)\u003c/strong\u003e Micrographs of representative \u003cem\u003eA. dauci\u003c/em\u003e conidia for each strain, illustrating morphological differences. Scale bar = 100 mm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/199f1ef1c7e06c5c61d68294.png"},{"id":78257887,"identity":"12e520c5-0a0d-489e-b99b-b3d60fa1c159","added_by":"auto","created_at":"2025-03-11 11:08:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":240472,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC-UV absorption profiles of the organic exudates from various \u003cem\u003eA. dauci\u003c/em\u003e strains analyzed at 305 nm, in comparison with the aldaulactone standard. The line positioned at the retention time of 18.5 min indicates the aldaulactone peak. For all fungal strains, peak intensities were adjusted according to the ratio of mAU to the actual volume of raw organic extract injected into the HPLC column. Before launching, all organic exudates were adjusted to a final concentration of 5 mg/ml. \u003cstrong\u003eSupplementary document 2\u003c/strong\u003e presents the corresponding UV profiles showing the absorbance of compounds isolated at 18.5 min for all \u003cem\u003eA. dauci\u003c/em\u003estrains.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/1322f7c5964cd7a055d874c5.png"},{"id":78257886,"identity":"ca48c7db-8d0f-47ec-a43d-5eda965564c4","added_by":"auto","created_at":"2025-03-11 11:08:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":681690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn planta\u003c/em\u003e pathogenicity test results. \u003cstrong\u003e(a)\u003c/strong\u003e Statistical comparison of LogAUDPC values among various \u003cem\u003eA. dauci\u003c/em\u003e strains, as well as between the H1-susceptible and I2-partially resistant carrot genotypes. The values at the top indicate the \u003cem\u003ep-\u003c/em\u003evalues resulting from comparisons of each \u003cem\u003eA. dauci\u003c/em\u003e strain against the ΔNC strain within the H1-susceptible carrot genetic background. The values at the bottom represent \u003cem\u003ep-\u003c/em\u003evalues obtained from comparisons between the two carrot genotypes infected with the specified \u003cem\u003eA. dauci\u003c/em\u003e strain. Data were analyzed using the Waller-Duncan post-hoc test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), with values below 0.05 indicating significant differences. \u003cstrong\u003e(b)\u003c/strong\u003e Carrot leaves with varying LogAUDPC values, showing different levels of disease severity. Before photographing, the inoculated third-stage leaves of six-week-old carrots were detached from the whole plant, placed in a Petri dish lined with moist filter paper, and stored at 4°C for 2 days.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eSupplementary document 3\u003c/strong\u003e shows the conidia of \u003cem\u003eA. dauci\u003c/em\u003e FRA001 and mutant strains attached to the infected areas of carrot leaves, attesting that it was indeed the \u003cem\u003eA. dauci\u003c/em\u003e strain that produced the observed necrotic lesions.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/75bb1ad8d5bdab8e82012757.png"},{"id":92883750,"identity":"6c760ce4-b1ce-417f-9d5e-db707bf7f476","added_by":"auto","created_at":"2025-10-06 16:08:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5605774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/7177d696-e396-4ac5-b144-dfbea67e5ee1.pdf"},{"id":78257911,"identity":"4c101668-9ffc-4681-a454-03a7714a7bd2","added_by":"auto","created_at":"2025-03-11 11:08:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":57066285,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarydocumentsBernardinoetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6130137/v1/6f28b6213ac3305ea307f4e2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transformation of Alternaria dauci demonstrates the involvement of two polyketide synthase genes in aldaulactone production and fungal pathogenicity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe durable nature of partial or Quantitative Disease Resistance (QDR) in plants makes it a promising alternative to reduce the use of pesticides in agriculture. This is particularly true concerning pathogens with high evolutionary potential and necrotrophic pathogens\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Several genes control QDR and are associated with quantitative resistance loci (QRL) that each variably contribute to the phenotype\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Meanwhile, in qualitative disease resistance, R-genes are associated with total resistance to a disease. Their mechanism is generally based on the early detection of phytopathogen effectors. Specifically, R-genes facilitate a hypersensitivity response that causes the neighboring cells on the pathogen infection site to undergo programmed cell death\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The same response to necrotrophic pathogens would increase the plant\u0026rsquo;s susceptibility to the disease, as necrotrophic pathogens exploit cell death for their benefit. Hence, QDR appears to be a better means to mitigate the diseases caused by necrotrophic and hemi-biotrophic pathogens. However, in general, the levels of QDR obtained are insufficient to eliminate further phytosanitary measures such as the use of pesticides. Furthermore, breeding by exploiting QRLs is challenging as some of them can only be detected under certain environmental conditions or in specific genetic backgrounds\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As a result, observing QRL phenotypes and unveiling their underlying mechanisms have proven to be difficult.\u003c/p\u003e \u003cp\u003eNo comprehensive model of QDR has been realized so far. Moreover, very different mechanisms of QDR have been uncovered, suggesting that such a unifying model does not exist\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Alternatively, it can be envisioned that several mechanisms concur to yield a general QDR level. These mechanisms include (i) morphological and developmental variations, (ii) basal defense involvement, (iii) chemical warfare, (iv) defense signal transduction pathways, and (v) QRLs as a weak version of R-genes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Recently, other mechanisms underlying QDR have been described, including vesicle trafficking, molecular chaperoning, and detoxification\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePhytotoxins are important determinants of plant diseases. Depending on their specificities, they can be classified as either non-host-specific toxins (NHSTs) or host-specific toxins (HSTs)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Both NHSTs and HSTs fall under either of the two categories based on their chemical nature: ribosome synthesis-dependent peptides or secondary metabolites (SMs), which house most of the phytotoxins\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Several mechanisms of how phytotoxins are deployed have been described, including hindering lipid metabolism and disrupting plant membrane function. For instance, AAL toxins produced by the fungus \u003cem\u003eAlternaria alternata\u003c/em\u003e are analogs of the plant\u0026rsquo;s ceramide synthase substrate. When exposed to this toxin, the plant cell undergoes rapid-fire production of sphingolipid precursors, leading to the loss of integrity of the plant\u0026rsquo;s plasma membrane\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. An R-gene, \u003cem\u003eAsc-1\u003c/em\u003e, from tomato plants of the \u003cem\u003easc/asc\u003c/em\u003e genotype, confers total resistance to AAL-toxin-induced programmed cell death\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Meanwhile, fusicoccin, a toxin produced by the fungus \u003cem\u003eFusicoccum amygdali\u003c/em\u003e, renders the stomata unable to close and ultimately causes plant wilting. It does so by irreversibly activating the plant plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase\u003csup\u003e10\u003c/sup\u003e. While these examples pertain to the involvement of toxins in total resistance, some phytotoxins are also involved in QDR.\u003c/p\u003e \u003cp\u003eFungal tricothecenes play a role in QDR in wheat infected with Fusarium head blight. In this pathosystem, \u003cem\u003eFhb7\u003c/em\u003e confers resistance to the disease by catalyzing the addition of glutathione (GSH) to a terminal epoxide of fungal tricothecenes\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A horizontally transferred glutathione S-transferase gene underlies this resistance mechanism conferred by \u003cem\u003eFhb7\u003c/em\u003e. Meanwhile, the HC toxin produced by the fungus \u003cem\u003eCochliobolus carbonum\u003c/em\u003e race 1 takes part in QDR in maize. The maize \u003cem\u003eHm1\u003c/em\u003e gene encodes an HC toxin reductase that inactivates the HC toxin\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Although \u003cem\u003eHm1\u003c/em\u003e confers major resistance, the partial loss-of-function of some \u003cem\u003eHm1\u003c/em\u003e alleles confers QDR\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTwo other phytotoxins have been involved in QDR: the SS toxin in the \u003cem\u003eStemphylium solani\u003c/em\u003e-\u003cem\u003eAllium sativum\u003c/em\u003e pathosystem and, in our laboratory, aldaulactone from \u003cem\u003eAlternaria dauci\u003c/em\u003e, the causal agent of Alternaria Leaf Blight (ALB) on carrots (\u003cem\u003eDaucus carota\u003c/em\u003e)\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The SS toxin causes plant cell death by inhibiting the H\u003csup\u003e+\u003c/sup\u003e-ATPase activity, NADH oxidation rate, and Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e reduction rate in a dose-dependent manner\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Thus, the SS toxin targets the standard redox system and the plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase, the latter mechanism being similar to that of fusicoccin. These findings bolster the long-standing notion that the plasma membrane is one of the primary sites of action of phytotoxins\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Meanwhile, aldaulactone is produced by the necrotrophic fungus \u003cem\u003eAlternaria dauci\u003c/em\u003e, causing ALB, the most prevalent and damaging foliar disease in carrots\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This disease is characterized by necrotic lesions surrounded by a chlorotic halo on carrot leaves. Out of the over seventy phytotoxins known to be produced by the phytopathogenic species of \u003cem\u003eAlternaria\u003c/em\u003e, six are known to be produced by \u003cem\u003eA. dauci\u003c/em\u003e: zinniol, alternariol, alternariol monomethyl ether, α-acetylorcinol, \u003cem\u003ep\u003c/em\u003e-hydroxybenzoic acid, and aldaulactone\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Moreover, the existence in its genome of 19 secondary metabolite gene clusters indicates that this list is most likely not exhaustive\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This quite diverse toxin weaponry perhaps helps \u003cem\u003eA. dauci\u003c/em\u003e infect a wide range of dicotyledonous plants, mainly inside but also outside of the \u003cem\u003eApiaceae\u003c/em\u003e family, with the carrot being its main host\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. There are various strains of \u003cem\u003eA. dauci\u003c/em\u003e, each characterized by different levels of aggressiveness. Similarly, various genotypes of carrots exhibit different levels of resistance to \u003cem\u003eA. dauci\u003c/em\u003e, broadening the disease resistance levels to a continuum\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, we took advantage of the diversity of both carrot resistance level and \u003cem\u003eA. dauci\u003c/em\u003e aggressiveness level to decipher the molecular basis of carrot QDR to the fungus. First, a correlation between plant partial resistance to \u003cem\u003eA. dauci\u003c/em\u003e with \u003cem\u003ein vitro\u003c/em\u003e cultured carrot cell resistance to fungal exudates was observed, indicating toxin resistance as a QDR mechanism\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Then, aldaulactone was isolated and characterized, dubbing it as an original \u003cem\u003eA. dauci\u003c/em\u003e-produced phytotoxin. It likely plays an important role in the carrot-\u003cem\u003eA. dauci\u003c/em\u003e interaction, as the production of aldaulactone was found to correlate with fungal pathogenicity levels\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In addition, \u003cem\u003ein vitro\u003c/em\u003e assays showed that I2- partially resistant carrot cells were less susceptible to aldaulactone when compared to H1-susceptible carrot cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These results prompted us to better understand the structure of aldaulactone and its biosynthetic pathway.\u003c/p\u003e \u003cp\u003eAldaulactone is a benzenediol lactone of a polyketide (PK) nature\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Generally, PK benzenediol lactones are comprised of two molecule families bearing a 1,3-benzenediol moiety connected to a macrolactone: either a dihydroxyphenylacetic acid lactone (DAL) or a resorcyclic acid lactone (RAL). Through bidimensional NMR analyses, aldaulactone was identified as a PK containing a DAL\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The biosynthesis of PKs entails polyketide synthase (PKS) genes that are usually organized in the same cluster. Tailoring enzymes that catalyze functional group transfer or redox reactions are also encoded by other genes found within the same cluster\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In fungi, two types of PKSs are known: type I and type III PKSs. While Type III fungal PKSs are less characterized, Type I fungal PKSs are widely studied since most fungal PKSs fall under this category\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Type I PKSs iteratively catalyze the head-to-tail Claisen condensation of acetyl-CoA, leading to the eventual formation of a polyketide\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These multifunctional enzymes can be grouped into three types, according to the resulting PK\u0026rsquo;s degree of reduction: non-reducing (NR)-, partially reducing (PR)-, and highly reducing (HR)-PKSs\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Moreover, PKSs are multidomain enzymes that harbor a minimal core module of three domains: ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe biosynthesis of some RALs such as zearalenone and 10,11-dehydrocurvularin have been described, owing to their genetic and molecular underpinnings\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In these pathways, the molecular backbones of the toxins are synthesized by the joint effort of Type I HR-PKS and NR-PKS. The same collaborative effort between the two Type I PKSs is also observed in the synthesis of DALs\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The regioselectivity of the cyclization spells out the difference between the synthesis of an RAL from that of a DAL: a C2-C7 aldol condensation produces an RAL, while a C3-C8 aldol condensation yields a DAL\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe construction of the biosynthetic pathway of aldaulactone requires probing into the genetic and molecular foundations of the PKSs involved. Bioinformatic analyses on the \u003cem\u003eA. dauci\u003c/em\u003e genome revealed a single cluster (cluster 8) harboring two PKSs\u0026mdash;\u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e\u0026mdash;thought to be involved in aldaulactone biosynthesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eAdPKS7\u003c/em\u003e encodes an HR-PKS, while the \u003cem\u003eAdPKS8\u003c/em\u003e encodes an NR-PKS. Based on the organization of both PKS genes in the cluster and the structure of aldaulactone, a biosynthetic pathway of this toxin was proposed\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The expression patterns of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e correlated with aldaulactone production under different experimental conditions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These findings support the hypothesis that both \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e are involved in aldaulactone biosynthesis.\u003c/p\u003e \u003cp\u003eHere, we present experiments designed to prove the implication of the two PKSs in aldaulactone biosynthesis and the involvement of aldaulactone in \u003cem\u003eA. dauci\u003c/em\u003e pathogenicity. To our knowledge, no \u003cem\u003eA. dauci\u003c/em\u003e transformation experiment has been published until now. The first aim of this study was thus to generate knock-out mutants of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e in the FRA001 strain of \u003cem\u003eA. dauci.\u003c/em\u003e Protoplast production, double-joint PCR-produced cassette uptake, and homologous recombination permitted the transformation of \u003cem\u003eA. dauci.\u003c/em\u003e Two domains were targeted in each of the two \u003cem\u003eAdPKS\u003c/em\u003e genes: the AT and the KS domains, which are minimally required to synthesize the aldaulactone backbone. In the mutants, the target domains were replaced by the Hygromycin Phosphotransferase gene (\u003cem\u003eHph\u003c/em\u003e), which confers resistance to hygromycin B. A non-coding mutant (\u003cem\u003ei.e\u003c/em\u003e., the target domain was outside of the coding region of both \u003cem\u003eAdPKS\u003c/em\u003e genes) was also constructed. In total, five mutants were generated: AdPKS7∆AT, AdPKS7∆KS, AdPKS8∆AT, AdPKS8∆KS, and ∆NC.\u003c/p\u003e \u003cp\u003eThe second aim of our study was to characterize the \u003cem\u003eA. dauci\u003c/em\u003e mutants based on their ability to produce aldaulactone and their pathogenicity on carrot leaves. We analyzed the organic exudates from the five fungal mutants and the wild-type FRA001 strain through HPLC-UV. In a controlled environment, we also infected carrot leaves of both the H1-susceptible genotype and the I2-partially resistant genotype using conidial suspensions from all mutants and the wild-type. Our results indicate that the transformation method employed on \u003cem\u003eA. dauci\u003c/em\u003e is efficient in carrying out functional validation experiments. Subsequently, through analyzing the mutants, we provided definitive proof of the function of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e in aldaulactone production and the important role of this toxin in \u003cem\u003eA. dauci\u003c/em\u003e pathogenicity on carrots.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTransformation of \u003cem\u003eA. dauci\u003c/em\u003e and molecular characterization of the transformed strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transformation of \u003cem\u003eA. dauci\u003c/em\u003e was realized in several steps, which include the generation of the hygromycin resistance cassette, protoplast production through enzymatic digestion, cassette DNA uptake in protoplasts, and selection of transformed fungal cells. The enzymatic cocktail Driselase/Kitalase allowed the \u003cem\u003eA. dauci\u003c/em\u003e FRA001 strain to produce a high yield of protoplasts after four to five hours of digestion. The protoplast transformation protocol originally developed for \u003cem\u003eA. brassicicola\u003c/em\u003e was adaptable to \u003cem\u003eA. dauci\u003c/em\u003e, although differences in hygromycin sensitivity necessitated some modifications in selection concentration. The initial transformation to create \u003cem\u003eA. dauci\u003c/em\u003e KO mutants was done using an agar layer containing 12 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e of hygromycin B, following the protocol for \u003cem\u003eA. brassicicola\u003c/em\u003e\u003csup\u003e43,44\u003c/sup\u003e. However, no \u003cem\u003eA. dauci\u003c/em\u003e transformants were recovered under this condition.\u003c/p\u003e\n\u003cp\u003eSubsequent hygromycin sensitivity assays with conidia from the \u003cem\u003eA. dauci\u0026nbsp;\u003c/em\u003eFRA001 and \u003cem\u003eA.\u003c/em\u003e \u003cem\u003ebrassicicola\u003c/em\u003e Abra43 strains revealed that \u003cem\u003eA. dauci\u003c/em\u003e is more sensitive to hygromycin B than \u003cem\u003eA. brassicicola\u0026nbsp;\u003c/em\u003eis, with complete inhibition of conidial germination at concentrations above 12 \u0026mu;g/ml. A second transformation attempt was thus conducted with a reduced hygromycin concentration of 2 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e to select KO mutants. This second transformation revealed that 2 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e hygromycin B was insufficient for effective selection, as both transformants and non-transformants traversed the hygromycin B-supplemented agar overlay. The transformants were then transplanted anew to PDA with either 5 or 8 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e hygromycin B. This process determined that an agar overlay supplemented with 8 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003eof hygromycin B was effective for initial selection, with subsequent transfer to PDA supplemented with 5 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e of hygromycin B to support sustained fungal colony growth without excessive inhibition.\u003c/p\u003e\n\u003cp\u003eThe molecular characterization of the wild-type \u003cem\u003eA. dauci\u003c/em\u003e FRA001 strain, along with all the mutants, was performed by PCR verification. Specifically, sequences corresponding to the internal transcribed spacer (\u003cem\u003eITS\u003c/em\u003e), hygromycin B phosphotransferase (\u003cem\u003eHph\u003c/em\u003e) gene, the acyltransferase (AT) and ketosynthase (KS) domains of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8,\u0026nbsp;\u003c/em\u003eand the non-coding region targeted in the \u0026Delta;NC strain were amplified via standard PCR and analyzed using gel electrophoresis. All \u003cem\u003eA. dauci\u003c/em\u003e strains exhibited amplification of the \u003cem\u003eITS\u003c/em\u003e, whereas only the mutant strains displayed amplification of the \u003cem\u003eHph\u003c/em\u003e (Figure 1). In the wild-type strain, the AT, KS, and non-coding regions were amplified. The targeted domains showed no amplification in the respective mutants.\u003c/p\u003e\n\u003cp\u003eTo verify the correct insertion of the \u003cem\u003eHph\u0026nbsp;\u003c/em\u003egene in place of the target domains, PCR amplification was performed across the region spanning the upstream sequence of the respective \u003cem\u003eAdPKS\u0026nbsp;\u003c/em\u003egene (\u003cem\u003eAdPKS7\u003c/em\u003e or \u003cem\u003eAdPKS8\u003c/em\u003e) and the downstream sequence of \u003cem\u003eHph\u003c/em\u003e. Similarly, amplification was carried out for the downstream region of the \u003cem\u003eAdPKS\u0026nbsp;\u003c/em\u003egene and the upstream region of \u003cem\u003eHph\u003c/em\u003e. The presence of PCR products corresponding to both regions in the gel confirmed the correct insertion of \u003cem\u003eHph\u0026nbsp;\u003c/em\u003einto the genomes of the \u003cem\u003eA. dauci\u003c/em\u003e mutants. These results corroborate the successful knockout of the PKS genes, replaced by \u003cem\u003eHph\u003c/em\u003e in the PKS mutants, as well as the substitution of the non-coding region with \u003cem\u003eHph\u003c/em\u003e in the \u0026Delta;NC mutant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological profiling of \u003cem\u003eA. dauci\u003c/em\u003e mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo our knowledge, this study presents the first reported case of genetic transformation in \u003cem\u003eA. dauci\u003c/em\u003e. Consequently, we documented the morphological characteristics of the resulting mutants, focusing specifically on their radial mycelial growth on V8 agar medium and their conidial morphology and length.\u003c/p\u003e\n\u003cp\u003eThe mycelial growth rates of all five \u003cem\u003eA. dauci\u003c/em\u003e strains were evaluated by computing the Area Under the growth Curve (AUC) values. Figure 2a presents a statistical comparison of the mean AUC values across nine replicates per strain, analyzed using Tukey\u0026rsquo;s post-hoc test. The mycelial growth rates of all five mutant strains were slightly yet significantly faster than that of the wild-type. Moreover, the \u0026Delta;NC mutant displayed a growth rate that was statistically similar to those of the PKS mutants. A visual comparison of mycelial growths on V8 agar medium at seven days post-transplant is shown in Figure 2b. While differences in growth rate between the wild-type and mutant strains were subtle, a marked contrast in mycelial pigmentation was evident: the wild-type strain displayed a lighter hue compared to the black pigmentation observed in all mutant strains.\u003c/p\u003e\n\u003cp\u003eConidial phenotyping of the mutants was also conducted to evaluate further pleiotropic effects of the transformation. Micrographs of conidia per strain were captured using a light microscope, and conidial length was manually measured. Figure 3a shows a statistical comparison of mean conidial lengths for all \u003cem\u003eA. dauci\u003c/em\u003e strains, analyzed using Tukey\u0026rsquo;s post-hoc test. All mutants exhibited significantly longer average conidial lengths than the wild-type. Figure 3b visually compares representative conidia from each \u003cem\u003eA. dauci\u003c/em\u003e strain, highlighting the noticeably shorter conidium of the wild-type in contrast to the mutants. Under typical conditions, \u003cem\u003eA. dauci\u003c/em\u003e conidia display an ellipsoidal body with multiple transverse septa and one or more longitudinal septa, tapering into a long, slender filiform beak (Figure 3b, FRA001). While most conidia from the transformed strains retained this characteristic morphology, some displayed abnormal forms, such as bent bodies often with a clump of cells, as seen in the micrograph of the AdPKS7\u0026Delta;KS conidium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC-UV analysis for aldaulactone detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe organic exudates (OEs) of the various \u003cem\u003eA. dauci\u003c/em\u003e strains were analyzed using HPLC-UV to assess their ability to produce aldaulactone. For each fungal strain, three biological replicates were included. Since aldaulactone has a maximum absorbance at 305 nm, all chromatograms were examined at this wavelength\u003csup\u003e16\u003c/sup\u003e. Aldaulactone exhibits a retention time of 18.5 minutes, as seen in the chromatogram of the aldaulactone standard (\u003cem\u003ei.e.,\u0026nbsp;\u003c/em\u003epure aldaulactone; Figure 4). A peak corresponding to aldaulactone was detected in the wild-type and \u0026Delta;NC strains. However, this peak was absent in all PKS mutants.\u003c/p\u003e\n\u003cp\u003eTo confirm the identity of the purported aldaulactone detected in the wild-type and \u0026Delta;NC strains, the UV profiles of the compounds from the 18.5-min peak in each chromatogram were compared with that of aldaulactone. The profiles were consistent across three biological replicates, showing characteristic absorbance peaks at 196 nm and 302 nm (Supplementary Figure 1). These results confirm that the peak with a retention time of 18.5 min observed in both the wild-type and \u0026Delta;NC strains corresponds to aldaulactone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCarrot \u003cem\u003ein planta\u003c/em\u003e pathogenicity test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the potential pathogenicity of the mutant strains in inducing symptoms characteristic of Alternaria Leaf Blight (ALB), we inoculated carrot leaves with conidial suspensions from both the wild-type and mutant \u003cem\u003eA. dauci\u003c/em\u003e strains. The degree of pathogenicity of the \u003cem\u003eA. dauci\u003c/em\u003e strains on carrot leaves was then compared by analyzing the log-transformed Area Under the Disease Progression Curve (LogAUDPC) values. An illustration of symptom severity on carrot leaves showing different LogAUDPC values is provided in Figure 5a.\u003c/p\u003e\n\u003cp\u003eA Waller-Duncan post-hoc analysis was conducted to statistically compare between symptom severity of each \u003cem\u003eA. dauci\u003c/em\u003e strain. Given that the \u0026Delta;NC strain shares closer genetic similarity to the PKS mutants due to the insertion of the \u003cem\u003eHph\u003c/em\u003e gene, and exhibits similar morphological characteristics, we deemed it more appropriate to compare the pathogenicity of the PKS mutants to the \u0026Delta;NC strain rather than the wild-type FRA001 strain. In the H1-susceptible carrot genotype, the \u0026Delta;NC strain exhibited significantly higher pathogenicity compared to the PKS mutants, though no significant difference was observed relative to the wild-type strain (p \u0026lt; 0.05; Figure 5b). For the wild-type FRA001 strain, a significant difference in disease severity was noted between the H1-susceptible and I2-partially resistant carrot genotypes, with the former showing a markedly higher level of symptom severity. A similar pattern was observed for the \u0026Delta;NC strain, where the H1-susceptible genotype displayed significantly higher disease severity than the I2-partially resistant genotype. In contrast, no significant difference was seen between the H1-susceptible and I2-partially resistant leaves infected with any of the PKS mutants. The identity of the \u003cem\u003eA. dauci\u003c/em\u003e used for inoculation was confirmed by microscopic viewing of the conidia still attached to the carrot leaves 48h following leaf detachment (supplementary figure 2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a necrotrophic pathogen, \u003cem\u003eA. dauci\u003c/em\u003e is expected to rely on phytotoxins to establish pathogenicity in its carrot host. To date, six secondary metabolites (SMs) with a phytotoxic activity have been identified as being produced by \u003cem\u003eA. dauci\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. While the role of zinniol as a phytotoxin has been questioned, aldaulactone accounts for most\u0026mdash;but not all\u0026mdash;of the \u003cem\u003ein vitro\u003c/em\u003e toxicity of \u003cem\u003eA. dauci\u003c/em\u003e exudates to carrot cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In addition, aldaulactone production is strongly correlated with \u003cem\u003eA. dauci\u003c/em\u003e pathogenicity. Finally, a notable correlation was observed between carrot cell resistance to \u003cem\u003eA. dauci\u003c/em\u003e organic extracts and carrot plant resistance to the fungus, which led us to think that toxin resistance is a major resistance mechanism in the carrot-\u003cem\u003eA. dauci\u003c/em\u003e interaction\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo obtain a snapshot of the genetic determinants of secondary metabolism in \u003cem\u003eA. dauci\u003c/em\u003e, our lab has provided the first transcriptome of this species, along with the first appraisal of SM core gene diversity within Alternaria genomes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These investigations pinpointed \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e, both located in gene cluster 8, as candidate genes responsible for aldaulactone biosynthesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To confirm this, we conducted a functional validation of these genes that prompted us to produce and analyze KO mutants. For this to be realized, we adapted a transformation method originally developed for \u003cem\u003eA. brassicicola\u003c/em\u003e Abra43. The method was successfully applied to the \u003cem\u003eA. dauci\u003c/em\u003e FRA001 strain with a couple of modifications. First was the use of 8 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e hygromycin in the agar overlay, compared to 12 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e used for the transformation of \u003cem\u003eA. brassicicola\u003c/em\u003e Abra43. This adjustment of the hygromycin concentration suggests that the \u003cem\u003eA. dauci\u003c/em\u003e FRA001 cells exhibit lower resistance to hygromycin B than the \u003cem\u003eA. brassicicola\u003c/em\u003e Abra43 cells. Another modification involved extending the enzymatic digestion time for \u003cem\u003eA. dauci\u003c/em\u003e FRA001. While \u003cem\u003eA. brassicicola\u003c/em\u003e Abra43 cells underwent digestion for four hours, those of \u003cem\u003eA. dauci\u003c/em\u003e FRA001 were treated for six hours. To our knowledge, this is the first reported case of targeted genetic manipulation in \u003cem\u003eA. dauci\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMorphological profiling of the transformed \u003cem\u003eA. dauci\u003c/em\u003e strains revealed phenotypic effects associated with its genetic transformation. The significantly faster mycelial growth and longer conidia observed in all mutant strains including the ΔNC strain, compared to the wild-type, suggests that the transformation process itself impacted their physiology. Form variations in \u003cem\u003eA. dauci\u003c/em\u003e FRA001 mutant conidia were also observed alongside changes in conidial length: the conidia of these transformed strains occasionally exhibited clumps of cells in the conidial body, a feature absent in the wild-type FRA001 strain. These effects could be due to the insertion of foreign DNA, such as the \u003cem\u003eHph\u003c/em\u003e gene, which may have activated a general stress response pathway, potentially leading to physiological reprogramming. A study on the development of spontaneous hygromycin B resistance in the necrotrophic pathogen \u003cem\u003eMonilinia fructicola\u003c/em\u003e demonstrated that hygromycin B-resistant mutant colonies exhibited slower growth on the PDA medium when compared to the wild-type\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Although this study did not employ site-specific mutagenesis, it suggests that the insertion of the \u003cem\u003eHph\u003c/em\u003e gene might lead to developmental changes in fungi. Interestingly, similar conidial form variations have also been observed in other \u003cem\u003eA. dauci\u003c/em\u003e strains, such as FRA017, a wild strain resistant to the fungicide, iprodione. Also, similar morphological variations were observed in \u003cem\u003eA. dauci\u003c/em\u003e conidia exposed to environmental stress, such as fungicide\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the observed morphological changes can correspond to the expression of stress resistance-associated mechanisms. The shared morphological features observed among the ΔPKS mutants and the ΔNC strain, coupled with the insertion of \u003cem\u003eHph\u003c/em\u003e into their genomes, led us to use the ΔNC strain rather than the wild-type as the basis of comparison in experiments assessing the effects of \u003cem\u003eAdPKS\u003c/em\u003e gene knockouts.\u003c/p\u003e \u003cp\u003eOrganic exudates from the \u003cem\u003eA. dauci\u003c/em\u003e strains were analyzed by HPLC equipped with a photodiode array detector. Chromatograms were extracted at 305 nm, the wavelength where aldaulactone shows maximum absorbance. The ΔPKS mutants did not show the characteristic aldaulactone peak at 18.5 min. In contrast, both the wild-type and the ΔNC strains exhibited the aldaulactone peak, which is consistent across all three biological replicates. Moreover, the UV profile of the compound detected at 18.5 min in these two strains closely matches that of aldaulactone (Supplementary document 2). Minor peaks were likewise observed beyond the 18.5 min retention time across all fungal strains, suggesting that other metabolic pathways involved in the production of the compounds in these peaks were not affected by the transformation process. However, minor peaks detected before 18.5 min were present in the wild-type FRA001 strain but not in the transformed strains. Other metabolic pathways involved in the production of the compounds detected in these peaks may have been affected by the transformation process and not the knock-out of either of the \u003cem\u003eAdPKS\u003c/em\u003e genes, as the ΔNC strain did not show similar peaks. All in all, these results provide strong evidence that \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e are responsible for aldaulactone biosynthesis. In addition, they provide us with a set of isogenic strains that only differs in their production of aldaulactone.\u003c/p\u003e \u003cp\u003eTo evaluate the role of aldaulactone production in fungal pathogenicity, we conducted an \u003cem\u003ein planta\u003c/em\u003e pathogenicity test on six-week-old carrot leaves. In the H1-susceptible carrot genotype, a significant difference was observed between the ΔNC strain and all ΔPKS mutants, but not with the wild-type. This suggests that aldaulactone plays an essential role in the pathogenicity of \u003cem\u003eA. dauci\u003c/em\u003e. In contrast, no significant difference was observed across all fungal strains when inoculated on the I2-partially resistant carrot genotype. Aldaulactone production is the only component of pathogenicity that differs between the ΔNC and the ΔPKS mutants. In a previous study, we saw that the I2 cultivar resists aldaulactone toxicity\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A possible explanation for the \u003cem\u003ein planta\u003c/em\u003e observation could be that since the I2 genotype resists aldaulactone, it is solely the other components of \u003cem\u003eA. dauci\u003c/em\u003e pathogenicity that are responsible for the symptoms observed in this genotype. As a result, the absence or presence of aldaulactone alone does not affect the outcome of the confrontation with \u003cem\u003eA. dauci\u003c/em\u003e strains unable to produce aldaulactone, suggesting that the other components of I2 resistance are not strong enough to be detected in our setup when compared with that of the H1 genotype.\u003c/p\u003e \u003cp\u003eOur study demonstrates that the knockout of \u003cem\u003eAdPKS7\u003c/em\u003e or \u003cem\u003eAdPKS8\u003c/em\u003e, resulting in the complete loss of aldaulactone production, significantly reduces disease symptom severity in the H1-susceptible carrot genotype. This finding strongly indicates that aldaulactone is the primary phytotoxin responsible for causing ALB symptoms in H1-susceptible carrots. These results are in contrast with a recent study on the functional redundancy of pathogenicity factors in \u003cem\u003eBotrytis cinerea\u003c/em\u003e. In that study, multiple knockout mutants, lacking up to 12 cell-death-inducing proteins (CDIPs) and metabolites, exhibited a progressive decrease in virulence\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Interestingly, the reduction in virulence correlated with the increasing number of gene knockouts, and was not significant when less than four genes were deleted, suggesting functional redundancy among the pathogenicity factors. Despite the substantial reduction in known phytotoxins, significant phytotoxic activity persisted in the mutants' secretomes, pointing to the presence of yet-unidentified CDIPs in \u003cem\u003eB. cinerea\u003c/em\u003e. In stark contrast, our findings show that the knockout of \u003cem\u003eAdPKS7\u003c/em\u003e or \u003cem\u003eAdPKS8\u003c/em\u003e leads to a clear reduction in ALB symptoms in carrots. Unlike \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eA. dauci\u003c/em\u003e does not exhibit compensatory mechanisms to recover its pathogenicity when the aldaulactone biosynthesis pathway is disrupted. This underscores the central role of aldaulactone in \u003cem\u003eA. dauci\u003c/em\u003e pathogenicity on carrots.\u003c/p\u003e \u003cp\u003eOur previous findings have established that aldaulactone is toxic to carrot cells and also to tobacco leaves when directly injected into them, suggesting that aldaulactone may be classified as a non-host specific toxin (NHST)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This finding contrasts aldaulactone with the HC toxin, a host-specific toxin (HST) produced by \u003cem\u003eCochliobolus carbonum\u003c/em\u003e race 1. While the HC toxin targets specific maize genotypes owing to the absence of the \u003cem\u003eHM1\u003c/em\u003e gene in their genomes, aldaulactone exhibits toxicity across distant plant species, such as \u003cem\u003eD. carota\u003c/em\u003e and \u003cem\u003eN. benthamiana\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This broader host range suggests that aldaulactone potentially targets conserved cellular processes, a hallmark of NHSTs. The resistance mechanisms against NHSTs, such as aldaulactone, are often associated with QDR rather than race-specific total resistance typical of HSTs. In this context, the partial resistance observed in the carrot I2 genotype likely demonstrates an array of possible defense responses, such as detoxification mechanisms and cellular defense mechanisms, among others. This is in contrast with the HC toxin resistance mechanism in maize, where the single dominant \u003cem\u003eHM1\u003c/em\u003e gene encodes a specific HM toxin reductase that inactivates the toxin. The NHST classification of aldaulactone thus corresponds to the general notion of this group of toxins in that they are not the sole determinant of pathogenicity\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In turn, the ALB symptoms observed on carrot leaves infected with the ΔPKS mutants are attributed to the other components of \u003cem\u003eA. dauci\u003c/em\u003e pathogenicity and not aldaulactone.\u003c/p\u003e \u003cp\u003eBoth the ingenuity and downfall of toxin resistance mechanisms in plants are reflected in the plants\u0026rsquo; evolutionary race against necrotrophic pathogens. For instance, the \u003cem\u003ePc2\u003c/em\u003e gene renders some plants susceptible to victorin, a toxin produced by the necrotrophic fungus, \u003cem\u003eCochliobolus victoriae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Various \u003cem\u003eTsn\u003c/em\u003e genes, on the other hand, recognize effectors encoded by \u003cem\u003eTox\u003c/em\u003e genes in \u003cem\u003eParastagonospora nodorum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Upon recognition of the effectors, the \u003cem\u003eTsn\u003c/em\u003e genes activate programmed cell death, a means for necrotrophic fungi such as \u003cem\u003eP. nodorum\u003c/em\u003e to undermine plant defense responses and promote infection. These are examples of the inverse gene-for-gene hypothesis, where a susceptible gene, instead of an R-gene, is compatible with the gene of a pathogen encoding an avirulence factor. Another example is the \u003cem\u003eAsc1\u003c/em\u003e gene in tomatoes that confers susceptibility to AAL-toxin produced by \u003cem\u003eAlternaria alternata\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The mutation in \u003cem\u003eAsc1\u003c/em\u003e in some tomato cultivars is responsible for the susceptibility to the toxin. These cases show that plants resist fungal-derived phytotoxins in various ways, whether by detoxifying the toxins or by lacking susceptibility genes. However, how carrots resist aldaulactone is yet to be determined. As mentioned, the NSHT nature of aldaulactone suggests that resistance against it may not depend on a single R-gene, but rather on general cellular defense mechanisms or perhaps, on the inverse gene-for-gene hypothesis.\u003c/p\u003e \u003cp\u003eAll in all, we provided an efficient site-directed transformation method for \u003cem\u003eA. dauci\u003c/em\u003e, offering a valuable tool for genetic manipulation in this species. Through the analysis of the transformed strains, definitive proof of the roles of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e in aldaulactone production was provided. Furthermore, fungal transformation gave us isogenic lines that helped us isolate the role of aldaulactone in both fungal pathogenicity and carrot plant resistance. This study represents the first demonstration of the role of both aldaulactone itself and aldaulactone resistance in the carrot-\u003cem\u003eA. dauci\u003c/em\u003e interaction. Further research is needed to elucidate the toxin mode of action and the resistance mechanisms involved in this pathosystem. That is, studies streamlined on identifying the molecular target of aldaulactone and the key defense pathways it activates could reveal whether the resistance against it fits into known mechanisms or follows an entirely different one. Beyond the recessive resistance to necrotrophic effectors such as those encoded by \u003cem\u003eP. nodorum Tox\u003c/em\u003e genes, there are only three examples where pathogen resistance genes were shown to encode toxin resistance factors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Lengthening this short list would invite insights into the diversity of plant toxin resistance mechanisms, a crucial part of plant resistance mechanisms against necrotrophic pathogens.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eFungal material and fungal growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlternaria dauci\u0026nbsp;\u003c/em\u003ewas grown on Petri dishes containing V8 agar and incubated at 24\u0026deg;C in darkness for 14 d. Only one strain of \u003cem\u003eA. dauci\u0026nbsp;\u003c/em\u003ewas used in this paper: the FRA001 strain, which is moderately aggressive on carrot and was also used in other studies\u003csup\u003e28\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e16,27\u003c/sup\u003e. It was collected as described in \u003csup\u003e28,54\u003c/sup\u003e and is freely available from the COMIC collection (COMIC-SFR Quasav, 42 rue Georges Morel, 49070 Beaucouz\u0026eacute; cedex, France).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor liquid cultures, all fungal strains were grown in Difco\u003csup\u003eTM\u003c/sup\u003e Potato Dextrose Broth (PDB) as in\u003csup\u003e16\u003c/sup\u003e. Ten colonized agar plugs (about 5 mm in diameter) were taken from solid V8 culture and ground for 1 min in 40 mL of PDB. Sixty mL of PDB was then used to rinse any adhering mycelia on the sterile metal grinder (DuPont Instruments, Sorvall\u003csup\u003e\u0026reg;\u003c/sup\u003e Omni-Mixer), then poured into the flask containing the earlier collected 40 mL suspension. The 100-mL suspension was then sealed and incubated in the dark at 24\u0026deg;C for 72 h with an agitation of 125 rpm (MINITRON\u003csup\u003e\u0026reg;\u003c/sup\u003e, Infors HT). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC-UV analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFRA001 PDB culture suspensions for HPLC-UV analysis were grown for 72 h. Then, the raw exudates were recovered by serial filtration using 200 \u0026micro;m, 50 \u0026micro;m, and 1 \u0026micro;m sterile nylon membranes from Sefar Nitex (Sefar AG, Heiden, Switzerland) as in \u003csup\u003e15\u003c/sup\u003e. The mycelia were weighed and stored at -80\u0026deg;C for future use, while the filtered raw exudates were neutralized to pH 7 and underwent liquid-liquid extraction through the addition of an equal volume of ethyl acetate. This was done three times to ensure that the organic exudate (OE) was maximally extracted. The OEs were then dried over sodium sulphate and evaporated under reduced pressure, and stored as described in \u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe OEs were resuspended in HPLC-grade methanol (5 mg\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e) and centrifuged at 11,000g for 10 min. The supernatants were analyzed using a Shimadzu Prominence-I LC-2030 3D coupled with PDA detector and assisted by LabSolutions software. The mobile phase (flow rate: 0.7 mL\u0026middot;min\u003csup\u003e-1\u003c/sup\u003e) was composed of water and HPLC-grade methanol with the following gradient: 90% water and 10% methanol at 0 min, then 100% methanol after 25 min, maintained for 5 min. All analyses were performed at 25\u0026deg;C on an UPTISPHERE C18-ODB column (150 x 4.6 mm; 3 \u0026micro;m). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic DNA extraction and standard PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genomic DNA of \u003cem\u003eA. dauci\u003c/em\u003e was extracted using the microwave miniprep method as described by \u003csup\u003e55\u003c/sup\u003e. The molecular characterization of fungal strains through standard PCR (Thermocycler T100, Bio-rad) was performed on various genomic regions, with the internal transcribed spacer (ITS) region as a positive control. The PCR mixtures contained 1x Green GoTaq\u0026reg; Flexi Buffer (Promega), 1.25u of GoTaq\u0026reg; Hot Start DNA polymerase (Promega), 0.1 \u0026micro;M of each primer, 1.5 mM of MgCl\u003csub\u003e2\u003c/sub\u003e solution, 0.2 mM of dNTP, 1 \u0026micro;L of the DNA suspension, and diluted to a total volume of 25 \u0026micro;L using nuclease-free water. The primers used in this study are listed and detailed in \u003cstrong\u003eSupplementary document 4\u003c/strong\u003e. The PCR programs used vary depending on the regions amplified. Detailed programs are specified in \u003cstrong\u003eSupplementary document 5\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene replacement cassette production and fungal transformation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene replacement cassettes were created by the double-joint PCR method as detailed by \u003csup\u003e56\u003c/sup\u003e. They bore the 5\u0026rsquo; and 3\u0026rsquo; flanking regions of each target gene fused with the\u003cem\u003e\u0026nbsp;Hph\u003c/em\u003e gene (LT726869) conferring resistance to hygromycin B. For each amplification, the Phusion Hot Start II High-Fidelity DNA polymerase pack (Thermo Fisher Scientific) and the appropriate primers were used. The final volume for each reaction was 50 \u0026micro;L, which was comprised of Phusion Hot Start DNA polymerase (0.02 u\u0026middot;\u0026micro;L\u003csup\u003e-1\u003c/sup\u003e), 3% v/v DMSO, 1x Buffer HF, dNTP (0.2 mM each), primers (20 \u0026micro;mol each), and 1 \u0026micro;L of the DNA. The first round of PCR amplified the 5\u0026rsquo; and 3\u0026rsquo; flanking regions of the target gene, as well as the \u003cem\u003eHph\u003c/em\u003e gene (1699 bp) from the plasmid pCB1636\u003csup\u003e57\u003c/sup\u003e. The second round of PCR ligated the products from the first PCR round to form the gene replacement cassette. The third round of PCR amplified the entire cassette from the second PCR round. Products of the first and third rounds of PCR were purified using the NucleoSpin\u003csup\u003e\u0026reg;\u003c/sup\u003e Gel and PCR Clean-up kit (Macherey Nagel) according to the manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtoplast production was initiated by culturing \u003cem\u003eA. dauci\u003c/em\u003e conidia (minimum of 80 cfu\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e) in 100 mL of PDB for 15 h at 24\u0026deg;C with an agitation of 175 rpm. The mycelia were then collected by centrifugation (10 min, 1700 g), washed in 0.7 M NaCl, and then centrifuged again using the same conditions. This washing step was performed twice before adding the mycelia to a 20-mL digestion solution containing Driselase (20 mg\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e; Sigma D9515-5G) and Kitalase (10 mg\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e; Wako 1W114-0037) prepared in 0.7 M NaCl. The resulting mixture was carefully mixed by inversion every 30 min for 6 h. The protoplasts were collected by centrifugation (7 min, 1700 g) and then delicately resuspended in 10 mL STC buffer (1.2 M Sorbitol, 10 mM Tris pH 7.5, 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e). Centrifugation was repeated and the protoplasts were resuspended in 1 mL STC at a concentration between 10\u003csup\u003e6\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e protoplasts\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e. For each mutant, 10-20 ng of the gene replacement cassette was added to 500 \u0026micro;L of protoplast suspension, which was then incubated on ice for 20 min. PEG [6 % w/v Polyethylene glycol MW 3350 (Sigma-Aldrich P3640), 10 mM Tris-HCl pH 7.5, 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e]\u0026nbsp;was added three times, with the suspension warmed in the hand in between each addition. The\u0026nbsp;suspension was gently mixed, incubated on ice for 5 min, and added with 1 mL of STC. Then, 250 \u0026micro;L of the protoplast suspension was added to 20 mL of the regeneration medium (1 M sucrose, 0.1% w/v yeast extract, 0.1% w/v casein hydrolase, and 1.6% w/v bacteriological agar), and the resulting mixture was poured into a Petri dish. After 24 h, a 10-mL agar overlay supplemented with hygromycin B (8 \u0026micro;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e) was poured. The plates were stored at 24\u0026deg;C in the dark.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe hygromycin B-resistant mutants were identified based on their ability to cross the agar overlay 5 to 7 days after. They were then sub-cultured on PDA media supplemented with hygromycin B (5 \u0026micro;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e). The purity of the mutants was checked by standard PCR as described above. In cases where the mutants were not pure (\u003cem\u003ei.e\u003c/em\u003e., when a band corresponding to the target gene appeared on the agarose gel), monosporing on PDA media supplemented with hygromycin B (5 \u0026micro;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e) was conducted. The mutant strains were conserved in cryotubes with 1 mL 30% glycerol and stored at -80\u0026deg;C. In total, five \u003cem\u003eA. dauci\u0026nbsp;\u003c/em\u003emutants were constructed from the \u003cem\u003eA. dauci\u003c/em\u003e FRA001 strain: AdPKS7∆AT, AdPKS7∆KS, AdPKS8∆AT, AdPKS8∆KS, and ∆NC. The ∆NC strain was generated by knocking out a non-coding region 946 bp downstream the \u003cem\u003eAdPKS7\u003c/em\u003e gene through its replacement with \u003cem\u003eHph\u003c/em\u003e. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFungal phenotyping\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mycelial radial growth assay was carried out according to \u003csup\u003e58\u003c/sup\u003e. Briefly, agar disks were sourced from the margin of a 7d-old colony grown on V8 agar media and were transferred to another V8 agar media. The radius of the mycelial colony was measured in cm at 3-, 4-, 5-, 6-, and 7 days post-transfer. The area under the growth curve was then calculated as in \u003csup\u003e59\u003c/sup\u003e and photos of the obverse view of the Petri dishes bearing the mycelia were captured on the final day of measurement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConidial phenotyping was performed by first scraping the surface of 14-day-old fungal colonies from V8 agar medium with 100 mL 0.05% Tween 20 solution. Three \u0026micro;L of the conidial suspension was mounted on a slide and then viewed under a light microscope at 40x magnification (ZEISS Axio Imager 2). The length from the tip of the conidial body to the end of the filiform beak was measured, with at least 35 measurements of different conidia for each strain. The ImageJ software was used to measure conidial lengths. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant material and \u003cem\u003ein planta\u0026nbsp;\u003c/em\u003epathogenicity test\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eDaucus carota\u003c/em\u003e genotypes used in this study were H1 (susceptible) and I2 (partially resistant). The H1 and I2 seeds were obtained as described in \u003csup\u003e15\u003c/sup\u003e. Plant cultivation was performed according to \u003csup\u003e21\u003c/sup\u003e. Briefly, plants were grown in pots containing peat moss/sand mixture in greenhouse conditions (16h of day, 22\u0026deg;C day/19\u0026deg;C night, 60% humidity) for six weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlant inoculation procedures have been described in detail in \u003csup\u003e19\u003c/sup\u003e. Sporulation of \u003cem\u003eA. dauci\u0026nbsp;\u003c/em\u003estrains was realized by individually growing the fungi on V8 agar and incubating them at 24\u0026deg;C in darkness for 14 d. The conidial suspensions were adjusted to 200 conidia mL\u003csup\u003e-1\u003c/sup\u003e in 0.05% Tween 20. The third leaf of six-week-old carrot plants was inoculated with the conidial suspension. For this, leaves still-attached to the plant were placed in incubation chambers made of a Petri dish anchored to the substrate. In an incubation chamber, a moist filter paper was placed at the bottom, allowing the leaf to rest on top of the paper and be held in place by paper clips and 6 mm nuts. Forty drops of 5 \u0026micro;L of the conidial suspension were applied on the adaxial side of the leaf using a micropipette. Symptom intensity was evaluated at 7-, 11-, and 13-days post-inoculation and is expressed as the number of symptoms per conidia. A value of 1 was assigned to a necrotic lesion smaller than the drop deposited, while a value of 10 was assigned to the lesion larger than the drop. The ratio of the number of lesions per viable conidia was calculated at each scoring date, and the area under the disease progression curve (AUDPC) was determined according to \u003csup\u003e19\u003c/sup\u003e. In each condition (\u003cem\u003ei.e\u003c/em\u003e., carrot genotype inoculated with the fungal strain), three technical replicates were made. This was done in four repetitions. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using the R v3.2.4 software in R Studio v1.0.136\u003csup\u003e60\u003c/sup\u003e. Normality and homoscedasticity of residues were checked using a Shapiro test and graphical assessments, respectively. To determine significant differences between fungal strains in the morphological characterization experiments, one-way analysis of variance (ANOVA) alongside Tukey\u0026rsquo;s post-hoc test was used. Meanwhile, the \u003cem\u003ein planta\u003c/em\u003e pathogenicity test, the AUDPC values were log-transformed (LogAUDPC), then underwent the same ANOVA, but coupled with the Waller-Duncan multiple comparisons test. In all three analyses, significant differences were noted at the \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAdditional information\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.B. and P.P. designed and managed the project. J.M.B., R.B., and P.P. wrote the article. M.G., E.N., N.B.S., and J.Colou performed fungal transformation. J.M.B., J.C., E.N., M.G., J.Colou, and F.B. contributed to the molecular analysis and characterization of fungal strains. J.M.B. and A.R. performed the morphological profiling of fungal strains. B.H. and F.B. managed the fungal collection. J.M.B., E.N., J.K., A.R., J.J.H., and D.B. produced, extracted, and analyzed the fungal extracts. J.M.B., E.N., J.K., A.R., and S.A. conducted the in planta pathogenicity test. J.M.B. and R.B. performed the statistical analyses. All authors reviewed the manuscript and approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would alike to thank J\u0026eacute;r\u0026ocirc;me Collemare of the Westerdijk Fungal Biodiversity Institute, Julie Chong of Universit\u0026eacute; de Haute-Alsace, and Sandrine Giraud of Universit\u0026eacute; d\u0026rsquo;Angers for their valuable insights in the fulfillment of this scientific work. They would also like to thank the following people from the Institut de Recherche en Horticulture et Semences (IRHS): Thomas Guillemette of the FungiSem team for his insights on fungal transformation, Mathilde Briard of the QuaRVeg team for the provision of carrot seeds, Aurelia Rolland and Fabienne Simonneau of the IMAC team for the realization of the conidial phenotyping, and Kaat Hellyn and Daniel Sochard of the PHENOTIC team for participating in the in planta experiments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are available upon request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePoland, J. A., Balint-Kurti, P. J., Wisser, R. J., Pratt, R. C. \u0026amp; Nelson, R. J. 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(2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alternaria dauci, transformation, toxin, biosynthesis, pathogenicity","lastPublishedDoi":"10.21203/rs.3.rs-6130137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6130137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemical warfare between the host and the pathogen plays a crucial role in plant-necrotrophic pathogen interactions, but examples of its involvement in quantitative disease resistance in plants are poorly documented. In the \u003cem\u003eDaucus carota-Alternaria dauci\u003c/em\u003e pathosystem, the novel toxin aldaulactone has been identified as a key factor in both fungal pathogenicity and the carrot\u0026rsquo;s partial resistance to the pathogen. Bioinformatic analyses have pinpointed a secondary metabolism gene cluster that harbors two polyketide synthase genes, \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e, that are likely responsible for the biosynthesis of aldaulactone. Here, we present the functional validation of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e as genes responsible for aldaulactone production in \u003cem\u003eA. dauci\u003c/em\u003e. We generated knock-out \u003cem\u003eA. dauci\u003c/em\u003e mutants for \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e by replacing essential domains with a hygromycin resistance gene, marking the first reported case of genetic manipulation in \u003cem\u003eA. dauci\u003c/em\u003e. Following transformation, the mutants were analyzed for toxin production via HPLC-UV and assessed for pathogenicity \u003cem\u003ein planta\u003c/em\u003e. Aldaulactone production was abolished in all PKS mutants, which also exhibited significantly reduced pathogenicity on H1-susceptible carrot leaves. These findings confirm the roles of \u003cem\u003eAdPKS7\u003c/em\u003e and \u003cem\u003eAdPKS8\u003c/em\u003e in aldaulactone biosynthesis and their contribution to fungal pathogenicity.\u003c/p\u003e","manuscriptTitle":"Transformation of Alternaria dauci demonstrates the involvement of two polyketide synthase genes in aldaulactone production and fungal pathogenicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-11 11:07:55","doi":"10.21203/rs.3.rs-6130137/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-06T04:15:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-28T05:10:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T12:34:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52590201726135917142773202593621514706","date":"2025-04-02T15:07:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119739938256623106805263683879783654414","date":"2025-03-28T09:41:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-17T14:52:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-17T14:46:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-11T12:34:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-07T09:26:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-28T16:06:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26a9f272-544a-4696-af2e-dfc5afb62ac7","owner":[],"postedDate":"March 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45350303,"name":"Biological sciences/Genetics/Microbial genetics/Fungal genetics"},{"id":45350304,"name":"Biological sciences/Microbiology/Fungi/Fungal pathogenesis"},{"id":45350305,"name":"Biological sciences/Genetics"},{"id":45350306,"name":"Biological sciences/Microbiology"},{"id":45350307,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-10-06T16:02:12+00:00","versionOfRecord":{"articleIdentity":"rs-6130137","link":"https://doi.org/10.1038/s41598-025-17441-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-02 15:57:35","publishedOnDateReadable":"October 2nd, 2025"},"versionCreatedAt":"2025-03-11 11:07:55","video":"","vorDoi":"10.1038/s41598-025-17441-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-17441-z","workflowStages":[]},"version":"v1","identity":"rs-6130137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6130137","identity":"rs-6130137","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}