The Velvet transcription factor PnVeA regulates necrotrophic effectors and secondary metabolism in the wheat pathogen Parastagonospora nodorum.

The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation.

Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Development of an Efficient CRISPR/Cas9 System in Fusarium verticillioides and Its Application in Reducing Mycotoxin Contamination.

Fusarium verticillioides (F. verticillioides) is a globally recognized and highly impactful fungal pathogen of maize, causing yield losses and producing harmful mycotoxins that pose a threat to human and animal health. However, the genetic tools available for studying this crucial fungus are currently limited in comparison to other important fungal pathogens. To address this, an efficient CRISPR/Cas9 genome editing system based on an autonomously replicating plasmid with an AMA1 sequence was established in this study. First, gene disruption of pyrG and pyrE via nonhomologous end-joining (NHEJ) pathway was successfully achieved, with efficiency ranging from 66 to 100%. Second, precise gene deletions were achieved with remarkable efficiency using a dual sgRNA expression strategy. Third, the developed genome editing system can be applied to generate designer chromosomes in F. verticillioides, as evidenced by the deletion of a crucial 38 kb fragment required for fumonisin biosynthesis. Fourth, the pyrG recycling system has been established and successfully applied in F. verticillioides. Lastly, the developed ΔFUM1 and ΔFUM mutants can serve as biocontrol agents to reduce the fumonisin B1 (FB1) contamination produced by the toxigenic strain. Taken together, these significant advancements in genetic manipulation and biocontrol strategies provide valuable tools for studying and mitigating the impact of F. verticillioides on maize crops.

Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

A New ToxA Haplotype in the Wheat Fungal Pathogen Bipolaris sorokiniana.

The necrotrophic effector ToxA is a well-studied virulence factor produced by several fungal necrotrophs. Initially cloned from the wheat tan spot pathogen Pyrenophora tritici-repentis in 1996, ToxA was found almost a decade later in another fungal pathogen, Parastagonospora nodorum, and its sister species, Parastagonospora pseudonodorum. In 2018, ToxA was detected in a third wheat fungal pathogenic species, Bipolaris sorokiniana, which causes spot blotch disease. However, unlike the case with P. tritici-repentis and P. nodorum, the ToxA in B. sorokiniana has only been investigated in recent years. In this report, five Australian B. sorokiniana isolates were assessed for the presence of ToxA. Four isolates were found to contain ToxA. While one isolate harbored the previously reported ToxA haplotype sequence (ToxA19), three isolates contain a different haplotype, designated herein as ToxA25, which has a nonsynonymous mutation resulting in an amino acid change of glycine to arginine at position 168. Both B. sorokiniana ToxA isoforms, when heterologously expressed in Escherichia coli, exhibited the classic ToxA necrosis-inducing activity on ToxA-sensitive Tsn1 cultivars. Preliminary analysis of the B. sorokiniana isolates in Australian wheat cultivars showed that isolates with ToxA19, ToxA25, or ToxA-deficient displayed various degrees of virulence, with the most aggressive isolates observed for those producing ToxA. Differences in spot blotch disease severity between Tsn1 and tsn1 cultivars were observed; however, this was not limited to the ToxA-producing isolates. The overall results suggest that the virulence of the Australian B. sorokiniana isolates is diverse, with the significance of ToxA-Tsn1 interactions depending on individual isolates.

FvKex2 is required for development, virulence, and mycotoxin production in Fusarium verticillioides.

Fusarium verticillioides is one of the most important fungal pathogens causing maize ear and stalk rots, thereby undermining global food security. Infected seeds are usually unhealthy for consumption due to contamination with fumonisin B1 (FB1) mycotoxin produced by the fungus as a virulence factor. Unveiling the molecular factors that determine fungal development and pathogenesis will help in the control and management of the diseases. Kex2 is a kexin-like Golgi-resident proprotein convertase that is involved in the activation of some important proproteins. Herein, we identified and functionally characterized FvKex2 in relation to F. verticillioides development and virulence by bioinformatics and functional genomics approaches. We found that FvKex2 is required for the fungal normal vegetative growth, because the growth of the ∆Fvkex2 mutant was significantly reduced on culture media compared to the wild-type and complemented strains. The mutant also produced very few conidia with morphologically abnormal shapes when compared with those from the wild type. However, the kexin-like protein was dispensable for the male role in sexual reproduction in F. verticillioides. In contrast, pathogenicity was nearly abolished on wounded maize stalks and sugarcane leaves in the absence of FvKEX2 gene, suggesting an essential role of Fvkex2 in the virulence of F. verticillioides. Furthermore, high-performance liquid chromatography analysis revealed that the ∆Fvkex2 mutant produced a significantly lower level of FB1 mycotoxin compared to the wild-type and complemented strains, consistent with the loss of virulence observed in the mutant. Taken together, our results indicate that FvKex2 is critical for vegetative growth, FB1 biosynthesis, and virulence, but dispensable for sexual reproduction in F. verticillioides. The study presents the kexin-like protein as a potential drug target for the management of the devastating maize ear and stalk rot diseases. Further studies should aim at uncovering the link between FvKex2 activity and FB1 biosynthesis genes. KEY POINTS: •The kexin-like protein FvKex2 contributes significantly to the vegetative growth of Fusarium verticillioides. •The conserved protein is required for fungal conidiation and conidial morphology, but dispensable for sexual reproduction. •Deletion of FvKEX2 greatly attenuates the virulence and mycotoxin production potential of F. verticillioides.

Genome Analyses Reveal the Secondary Metabolites that Potentially Influence the Geographical Distribution of Fusarium pseudograminearum Populations.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, significantly impacts wheat yield and quality in China's Huanghuai region. The rapid F. pseudograminearum epidemic and FCR outbreak within a decade remain unexplained. In this study, two high-quality, chromosome-level genomes of F. pseudograminearum strains producing 3-acetyl-deoxynivalenol (3AcDON) and 15-acetyl-deoxynivalenol (15AcDON) toxins were assembled. Additionally, 38 related strains were resequenced. Genomic differences such as single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variations (SVs) among F. pseudograminearum strains were analyzed. The whole-genome SNP locus-based population classification mirrored the toxin chemotype (3AcDON and 15AcDON)-based classification, indicating the presence of genes associated with the trichothecene toxin gene cluster. Further analysis of differential SNP, indel, and SV loci between the 3AcDON and 15AcDON populations revealed a predominant connection to secondary metabolite synthesis genes. Notably, the majority of the secondary metabolite biosynthesis gene cluster loci were located in SNP-dense genomic regions, suggesting high mutability and a possible contribution to F. pseudograminearum population structure and environmental adaptability. This study provides insightful perspectives on the distribution and evolution of F. pseudograminearum and for forecasting the spread of wheat FCR, thereby aiding in the development of preventive measures and control strategies.

Type A Trichothecene Metabolic Profile Differentiation, Mechanisms, Biosynthetic Pathways, and Evolution in Fusarium Species-A Mini Review.

Trichothecenes are the most common Fusarium toxins detected in grains and related products. Type A trichothecenes are among the mycotoxins of greatest concern to food and feed safety due to their high toxicity. Recently, two different trichothecene genotypes within Fusarium species were reported. The available information showed that Tri1 and Tri16 genes are the key determinants of the trichothecene profiles of T-2 and DAS genotypes. In this review, polymorphisms in the Tri1 and Tri16 genes in the two genotypes were investigated. Meanwhile, the functions of genes involved in DAS and NEO biosynthesis are discussed. The possible biosynthetic pathways of DAS and NEO are proposed in this review, which will facilitate the understanding of the synthesis process of trichothecenes in Fusarium strains and may also inspire researchers to design and conduct further research. Together, the review provides insight into trichothecene profile differentiation and Tri gene evolutionary processes responsible for the structural diversification of trichothecene produced by Fusarium.

A Revised Nomenclature for ToxA Haplotypes Across Multiple Fungal Species.

ToxA is one of the most studied proteinaceous necrotrophic effectors produced by plant pathogens. It has been identified in four pathogens (Pyrenophora tritici-repentis, Parastagonospora nodorum, Parastagonospora pseudonodorum [formerly Parastagonospora avenaria f. sp. tritici], and Bipolaris sorokiniana) causing leaf spot diseases on cereals worldwide. To date, 24 different ToxA haplotypes have been identified. Some P. tritici-repentis and related species also express ToxB, another small protein necrotrophic effector. We present here a revised and standardized nomenclature for these effectors, which could be extended to other poly-haplotypic genes found across multiple species.

A global pangenome for the wheat fungal pathogen Pyrenophora tritici-repentis and prediction of effector protein structural homology.

The adaptive potential of plant fungal pathogens is largely governed by the gene content of a species, consisting of core and accessory genes across the pathogen isolate repertoire. To approximate the complete gene repertoire of a globally significant crop fungal pathogen, a pan genomic analysis was undertaken for Pyrenophora tritici-repentis (Ptr), the causal agent of tan (or yellow) spot disease in wheat. In this study, 15 new Ptr genomes were sequenced, assembled and annotated, including isolates from three races not previously sequenced. Together with 11 previously published Ptr genomes, a pangenome for 26 Ptr isolates from Australia, Europe, North Africa and America, representing nearly all known races, revealed a conserved core-gene content of 57 % and presents a new Ptr resource for searching natural homologues (orthologues not acquired by horizontal transfer from another species) using remote protein structural homology. Here, we identify for the first time a non-synonymous mutation in the Ptr necrotrophic effector gene ToxB, multiple copies of the inactive toxb within an isolate, a distant natural Pyrenophora homologue of a known Parastagonopora nodorum necrotrophic effector (SnTox3), and clear genomic break points for the ToxA effector horizontal transfer region. This comprehensive genomic analysis of Ptr races includes nine isolates sequenced via long read technologies. Accordingly, these resources provide a more complete representation of the species, and serve as a resource to monitor variations potentially involved in pathogenicity.

The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB.

BACKGROUND: In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements. RESULTS: A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr's adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr's effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb 'Starship' transposon (dubbed 'Horizon') with a clearly defined target site and target site duplications. 'Horizon' was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative 'Starship' (dubbed 'Icarus') in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and 'Icarus' were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as 'one-compartment' based on calculated gene distances and evolutionary rates. CONCLUSIONS: These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus.

Aspergillus flavus infects various crops with aflatoxins, and leads to aspergillosis opportunistically. Though H3K36 methylation plays an important role in fungal toxin metabolism and virulence, no data about the biological function of H3K36 methylation in A. flavus virulence has been reported. Our study showed that the Set2 histone methyltransferase family, AshA and SetB, involves in morphogenesis and mycotoxin anabolism by regulating related transcriptional factors, and they are important for fungal virulence to crops and animals. Western-blotting and double deletion analysis revealed that AshA mainly regulates H3K36me2, whereas SetB is mainly responsible for H3K36me3 in the nucleus. By construction of domain deletion A. flavus strain and point mutation strains by homologous recombination, the study revealed that SET domain is indispensable in mycotoxin anabolism and virulence of A. flavus, and N455 and V457 in it are the key amino acid residues. ChIP analysis inferred that the methyltransferase family controls fungal reproduction and regulates the production of AFB1 by directly regulating the production of the transcriptional factor genes, including wetA, steA, aflR and amylase, through H3K36 trimethylation in their chromatin fragments, based on which this study proposed that, by H3K36 trimethylation, this methyltransferase family controls AFB1 anabolism through transcriptional level and substrate utilization level. This study illuminates the epigenetic mechanism of the Set2 family in regulating fungal virulence and mycotoxin production, and provides new targets for controlling the virulence of the fungus A. flavus.AUTHOR SUMMARYThe methylation of H3K36 plays an important role in the fungal secondary metabolism and virulence, but no data about the regulatory mechanism of H3K36 methylation in the virulence of A. flavus have been reported. Our study revealed that, in the histone methyltransferase Set2 family, AshA mainly catalyzes H3K36me2, and involves in the methylation of H3K36me1, and SetB mainly catalyzes H3K36me3 and H3K36me1. Through domain deletion and point mutation analysis, this study also revealed that the SET domain was critical for the normal biological function of the Set2 family and that N455 and V457 in the domain were critical for AshA. By ChIP-seq and ChIP-qPCR analysis, H3K36 was found to be trimethylation modified in the promotors and ORF positions of wetA, steA, aflR and the amylase gene (AFLA_084340), and further qRT-PCR results showed that these methylation modifications regulate the expression levels of these genes. According to the results of ChIP-seq analysis, we proposed that, by H3K36 trimethylation, this methyltransferase family controls the metabolism of mycotoxin through transcriptional level and substrate utilization level. All the results from this study showed that Set2 family is essential for fungal secondary metabolism and virulence, which lays a theoretical groundwork in the early prevention and treatment of A. flavus pollution, and also provides an effective strategy to fight against other pathogenic fungi.

Genome-Wide Characterization Reveals Variation Potentially Involved in Pathogenicity and Mycotoxins Biosynthesis of Fusarium proliferatum Causing Spikelet Rot Disease in Rice.

Fusarium proliferatum is the primary cause of spikelet rot disease in rice (Oryza sativa L.) in China. The pathogen not only infects a wide range of cereals, causing severe yield losses but also contaminates grains by producing various mycotoxins that are hazardous to humans and animals. Here, we firstly reported the whole-genome sequence of F. proliferatum strain Fp9 isolated from the rice spikelet. The genome was approximately 43.9 Mb with an average GC content of 48.28%, and it was assembled into 12 scaffolds with an N50 length of 4,402,342 bp. There is a close phylogenetic relationship between F. proliferatum and Fusarium fujikuroi, the causal agent of the bakanae disease of rice. The expansion of genes encoding cell wall-degrading enzymes and major facilitator superfamily (MFS) transporters was observed in F. proliferatum relative to other fungi with different nutritional lifestyles. Species-specific genes responsible for mycotoxins biosynthesis were identified among F. proliferatum and other Fusarium species. The expanded and unique genes were supposed to promote F. proliferatum adaptation and the rapid response to the host's infection. The high-quality genome of F. proliferatum strain Fp9 provides a valuable resource for deciphering the mechanisms of pathogenicity and secondary metabolism, and therefore shed light on development of the disease management strategies and detoxification of mycotoxins contamination for spikelet rot disease in rice.

Host Genotype and Weather Effects on Fusarium Head Blight Severity and Mycotoxin Load in Spring Barley.

Epidemiology of Fusarium Head Blight (FHB) of spring barley is relatively little understood. In a five-year study, we assessed quantitative resistance to FHB in an assortment of 17 spring barley genotypes in the field in southern Germany. To this end, we used soil and spray inoculation of plants with F. culmorum and F. avenaceum. This increased disease pressure and provoked genotypic differentiation. To normalize effects of variable weather conditions across consecutive seasons, we used a disease ranking of the genotypes based on quantification of fungal DNA contents and multiple Fusarium toxins in harvested grain. Together, this allowed for assessment of stable quantitative FHB resistance of barley in several genotypes. Fungal DNA contents were positively associated with species-specific Fusarium toxins in single years and over several years in plots with soil inoculation. In those plots, plant height limited FHB; however, this was not observed after spray inoculation. A multiple linear regression model of recorded weather parameter and fungal DNA contents over five years identified time periods during the reproductive phase of barley, in which weather strongly influenced fungal colonization measured in mature barley grain. Environmental conditions before heading and late after anthesis showed strongest associations with F. culmorum DNA in all genotypes, whereas for F. avenaceum, this was less consistent where we observed weather-dependent associations, depending on the genotype. Based on this study, we discuss aspects of practical resistance breeding in barley relevant to improve quantitative resistance to FHB and associated mycotoxin contaminations.

Phylogenetic Diversity and Mycotoxin Potential of Emergent Phytopathogens Within the Fusarium tricinctum Species Complex.

Recent studies on multiple continents indicate members of the Fusarium tricinctum species complex (FTSC) are emerging as prevalent pathogens of small-grain cereals, pulses, and other economically important crops. These understudied fusaria produce structurally diverse mycotoxins, among which enniatins (ENNs) and moniliformin (MON) are the most frequent and of greatest concern to food and feed safety. Herein a large survey of fusaria in the Fusarium Research Center and Agricultural Research Service culture collections was undertaken to assess species diversity and mycotoxin potential within the FTSC. A 151-strain collection originating from diverse hosts and substrates from different agroclimatic regions throughout the world was selected from 460 FTSC strains to represent the breadth of FTSC phylogenetic diversity. Evolutionary relationships inferred from a five-locus dataset, using maximum likelihood and parsimony, resolved the 151 strains as 24 phylogenetically distinct species, including nine that are new to science. Of the five genes analyzed, nearly full-length phosphate permease sequences contained the most phylogenetically informative characters, establishing its suitability for species-level phylogenetics within the FTSC. Fifteen of the species produced ENNs, MON, the sphingosine analog 2-amino-14,16-dimethyloctadecan-3-ol (AOD), and the toxic pigment aurofusarin (AUR) on a cracked corn kernel substrate. Interestingly, the five earliest diverging species in the FTSC phylogeny (i.e., F. iranicum, F. flocciferum, F. torulosum, and Fusarium spp. FTSC 8 and 24) failed to produce AOD and MON, but synthesized ENNs and/or AUR. Moreover, our reassessment of nine published phylogenetic studies on the FTSC identified 11 additional novel taxa, suggesting this complex comprises at least 36 species.

The necrotrophic effector ToxA from Parastagonospora nodorum interacts with wheat NHL proteins to facilitate Tsn1-mediated necrosis.

The plant pathogen Parastagonospora nodorum secretes necrotrophic effectors to promote disease. These effectors induce cell death on wheat cultivars carrying dominant susceptibility genes in an inverse gene-for-gene manner. However, the molecular mechanisms underpinning these interactions and resulting cell death remain unclear. Here, we used a yeast two-hybrid library approach to identify wheat proteins that interact with the necrotrophic effector ToxA. Using this strategy, we identified an interaction between ToxA and a wheat transmembrane NDR/HIN1-like protein (TaNHL10) and confirmed the interaction using in planta co-immunoprecipitation and confocal microscopy co-localization analysis. We showed that the C-terminus of TaNHL10 is extracellular whilst the N-terminus is localized in the cytoplasm. Further analyses using yeast two-hybrid and confocal microscopy co-localization showed that ToxA interacts with the C-terminal LEA2 extracellular domain of TaNHL10. Random mutagenesis was then used to identify a ToxA mutant, ToxAN109D , which was unable to interact with TaNHL10 in yeast two-hybrid assays. Subsequent heterologous expression and purification of ToxAN109D in Nicotiania benthamiana revealed that the mutated protein was unable to induce necrosis on Tsn1-dominant wheat cultivars, confirming that the interaction of ToxA with TaNHL10 is required to induce cell death. Collectively, these data advance our understanding on how ToxA induces cell death during infection and further highlight the importance of host cell surface interactions in necrotrophic pathosystems.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

Dynamics of Fusarium Mycotoxins and Lytic Enzymes during Pea Plants' Infection.

Fusarium species are common plant pathogens that cause several important diseases. They produce a wide range of secondary metabolites, among which mycotoxins and extracellular cell wall-degrading enzymes (CWDEs) contribute to weakening and invading the host plant successfully. Two species of Fusarium isolated from peas were monitored for their expression profile of three cell wall-degrading enzyme coding genes upon culturing with extracts from resistant (Sokolik) and susceptible (Santana) pea cultivars. The extracts from Santana induced a sudden increase in the gene expression, whereas Sokolik elicited a reduced expression. The coherent observation was that the biochemical profile of the host plant plays a major role in regulating the fungal gene expression. In order to uncover the fungal characteristics in planta, both pea cultivars were infected with two strains each of F. proliferatum and F. oxysporum on the 30th day of growth. The enzyme activity assays from both roots and rhizosphere indicated that more enzymes were used for degrading the cell wall of the resistant host compared to the susceptible host. The most commonly produced enzymes were cellulase, ß-glucosidase, xylanase, pectinase and lipase, where the pathogen selectively degraded the components of both the primary and secondary cell walls. The levels of beauvericin accumulated in the infected roots of both cultivars were also monitored. There was a difference between the levels of beauvericin accumulated in both the cultivars, where the susceptible cultivar had more beauvericin than the resistant one, showing that the plants susceptible to the pathogen were also susceptible to the toxin accumulation.

Current Approaches for Advancement in Understanding the Molecular Mechanisms of Mycotoxin Biosynthesis.

Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.

Transcriptome profiling in susceptible and tolerant rubber tree clones in response to cassiicolin Cas1, a necrotrophic effector from Corynespora cassiicola.

Corynespora cassiicola, a fungal plant pathogen with a large host range, causes important damages in rubber tree (Hevea brasiliensis), in Asia and Africa. A small secreted protein named cassiicolin was previously identified as a necrotrophic effector required for the virulence of C. cassiicola in specific rubber tree clones. The objective of this study was to decipher the cassiicolin-mediated molecular mechanisms involved in this compatible interaction. We comparatively analyzed the RNA-Seq transcriptomic profiles of leaves treated or not with the purified cassiicolin Cas1, in two rubber clones: PB260 (susceptible) and RRIM600 (tolerant). The reads were mapped against a synthetic transcriptome composed of all available transcriptomic references from the two clones. Genes differentially expressed in response to cassiicolin Cas1 were identified, in each clone, at two different time-points. After de novo annotation of the synthetic transcriptome, we analyzed GO enrichment of the differentially expressed genes in order to elucidate the main functional pathways impacted by cassiicolin. Cassiicolin induced qualitatively similar transcriptional modifications in both the susceptible and the tolerant clones, with a strong negative impact on photosynthesis, and the activation of defense responses via redox signaling, production of pathogenesis-related protein, or activation of the secondary metabolism. In the tolerant clone, transcriptional reprogramming occurred earlier but remained moderate. By contrast, the susceptible clone displayed a late but huge transcriptional burst, characterized by massive induction of phosphorylation events and all the features of a hypersensitive response. These results confirm that cassiicolin Cas1 is a necrotrophic effector triggering a hypersensitive response in susceptible rubber clones, in agreement with the necrotrophic-effector-triggered susceptibility model.