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Fungi of the genus Ceratocystis are aggressive tree pathogens that cause serious diseases in several crops around the world. Ceratocystis wilt disease caused by C. cacaofunesta has been shown to be responsible for severe reductions in cacao production. In this study, headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used in combination with chemometric analysis for monitoring volatile organic compounds (VOCs) released from C. cacaofunesta. Low-molecular-weight esters, alcohols, ketones, and sulphur compounds were identified in the liquid broth. Monitoring the volatile profile over five days of fungal growth revealed that the concentrations of alcohol and esters were inversely proportional. Acetate esters were responsible for the intense fruity aroma of the C. cacaofunesta culture produced within the first hours after fungal inoculation, which decreased over time, and are likely associated with the attraction of insect vectors to maintain the life cycle of the pathogen. PCA revealed that 3-methylbutyl acetate was the metabolite with the highest factor loading for the separation of the VOC samples after 4 h of fungal growth, whereas ethanol and 3-methylbutan-1-ol had the highest factor loadings after 96 and 120 h. 3-Methylbutan-1-ol is a phytotoxic compound that is likely associated with host cell death since C. cacaofunesta is a necrotrophic fungus. Fungal VOCs play important roles in natural habitats, regulating developmental processes and intra- and interkingdom interactions. This is the first report on the volatiles released by C. cacaofunesta.
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Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry.
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Thermoanaerobacterium , Xilosa , Thermoanaerobacterium/genética , Genómica , Firmicutes , BiocombustiblesRESUMEN
Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.
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Saccharomyces cerevisiae , Xilosa , Saccharomyces cerevisiae/genética , Xilosa/genética , Ingeniería Metabólica/métodos , Fermentación , Etanol/metabolismoRESUMEN
Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , Pez Cebra , Macrófagos , PéptidosRESUMEN
Aspergillus welwitschiae causes bole rot disease in sisal (Agave sisalana and related species) which affects the production of natural fibers in Brazil, the main worldwide producer of sisal fibers. This fungus is a saprotroph with a broad host range. Previous research established A. welwitschiae as the only causative agent of bole rot in the field, but little is known about the evolution of this species and its strains. In this work, we performed a comparative genomics analysis of 40 Aspergillus strains. We show the conflicting molecular identity of this species, with one sisal-infecting strain sharing its last common ancestor with Aspergillus niger, having diverged only 833 thousand years ago. Furthermore, our analysis of positive selection reveals sites under selection in genes coding for siderophore transporters, Sodiumcalcium exchangers, and Phosphatidylethanolamine-binding proteins (PEBPs). Herein, we discuss the possible impacts of these gene functions on the pathogenicity in sisal.
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Agave , Agave/genética , Brasil , Aspergillus/genéticaRESUMEN
Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae's metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4FMY097 enhances robustness towards HMF, but the industrial fitness could not be fully recovered. The genetic network arising from the causative genes in the QTL window suggests that intracellular signaling TOR (Target of Rapamycin) and CWI (Cell Wall Integrity) pathways are regulators of this phenotype in FMY097. Because the QTL mapping did not result in one major allelic contribution to the evaluated trait, a background effect in FMY097's HMF resistance is expected. Quantification of NADPH - cofactor implied in endogenous aldehyde detoxification reactions - supports the former hypothesis, given its high availability in FMY097. Regarding thermotolerance, SEA4FMY097 grants BY4742 ability to grow in temperatures as high as 38 ºC in liquid, while allele PKC1FMY097 allows growth up to 40 ºC in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other > 1013S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness.
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Proteínas de Saccharomyces cerevisiae , Termotolerancia , Furaldehído/análogos & derivados , Redes Reguladoras de Genes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Termotolerancia/genéticaRESUMEN
BACKGROUND: The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. RESULTS: By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. CONCLUSIONS: Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the model's predictive capabilities.
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Agave plants present drought resistance mechanisms, commercial applications, and potential for bioenergy production. Currently, Agave species are used to produce alcoholic beverages and sisal fibers in semi-arid regions, mainly in Mexico and Brazil. Because of their high productivities, low lignin content, and high shoot-to-root ratio, agaves show potential as biomass feedstock to bioenergy production in marginal areas. Plants host many microorganisms and understanding their metabolism can inform biotechnological purposes. Here, we identify and characterize fungal transcripts found in three fiber-producing agave cultivars (Agave fourcroydes, A. sisalana, and hybrid 11648). We used leaf, stem, and root samples collected from the agave germplasm bank located in the state of Paraiba, in the Brazilian semiarid region, which has faced irregular precipitation periods. We used data from a de novo assembled transcriptome assembly (all tissues together). Regardless of the cultivar, around 10% of the transcripts mapped to fungi. Surprisingly, most root-specific transcripts were fungal (58%); of these around 64% were identified as Ascomycota and 28% as Basidiomycota in the three communities. Transcripts that code for heat shock proteins (HSPs) and enzymes involved in transport across the membrane in Ascomycota and Basidiomycota, abounded in libraries generated from the three cultivars. Indeed, among the most expressed transcripts, many were annotated as HSPs, which appear involved in abiotic stress resistance. Most HSPs expressed by Ascomycota are small HSPs, highly related to dealing with temperature stresses. Also, some KEGG pathways suggest interaction with the roots, related to transport to outside the cell, such as exosome (present in the three Ascomycota communities) and membrane trafficking, which were further investigated. We also found chitinases among secreted CAZymes, that can be related to pathogen control. We anticipate that our results can provide a starting point to the study of the potential uses of agaves' fungi as biotechnological tools.
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Agave , Ascomicetos , Basidiomycota , Micobioma , Agave/genética , Micobioma/genética , Transcriptoma/genética , MéxicoRESUMEN
BACKGROUND: Sugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. RESULTS: Potential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5'-NGG 20 nucleotide sequences and guidelines for the fast and cost-effective CRISPR editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer influence of the genetic background. Near-theoretical yields were achieved for all strains; however, the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane straw hydrolysate with remaining solid particles represented a challenge for xylose metabolization, and 3.65 ± 0.16 g/L xylitol titer was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONS: Although widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains' performance. We also pioneer the use of sugarcane straw as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor production of reduced sugars.
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The conversion of lignocellulosic polymers into monomeric sugars demands a plethora of enzymatic activities generally not produced by a single microorganism and induced by the carbon source. In this vein, this work investigates the synergy between the enzymes secreted by the cellulolytic model fungi Trichoderma reesei RUT-C30 (TR) and Penicillium oxalicum (PO) to deconstruct sugarcane straw (SCS) and energy cane bagasse (ECB). TR and PO secrete a similar profile of cellulose-active enzymes resulting in a comparable conversion of SCS and ECB into glucose. The synergy between the enzymes produced by both fungi to break down the cellulose fraction becomes evident by the improvement of glucose titers from ~35-54% and from ~10-17% in SCS and ECB conditions, respectively, reached with the mixture of the secretomes of both fungi. The effect of a hemicellulase-enriched secretome produced by TR is particularly seen in SCS where the xylose yield reached ~15% compared to 5% by PO, remaining unaltered following the mixture of secretomes. However, the secretion of enzymes active in the decorations of the main chain polymers possibly aid PO to access the hemicellulose fraction of ECB reaching xylose yields similar to TR in this condition.
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Saccharum , Trichoderma , Biomasa , Bastones , Celulosa/metabolismo , Glucosa , Hypocreales , Penicillium , Saccharum/metabolismo , Secretoma , Trichoderma/metabolismo , XilosaRESUMEN
BACKGROUND: Saccharomyces cerevisiae is largely applied in many biotechnological processes, from traditional food and beverage industries to modern biofuel and biochemicals factories. During the fermentation process, yeast cells are usually challenged in different harsh conditions, which often impact productivity. Regarding bioethanol production, cell exposure to acidic environments is related to productivity loss on both first- and second-generation ethanol. In this scenario, indigenous strains traditionally used in fermentation stand out as a source of complex genetic architecture, mainly due to their highly robust background-including low pH tolerance. RESULTS: In this work, we pioneer the use of QTL mapping to uncover the genetic basis that confers to the industrial strain Pedra-2 (PE-2) acidic tolerance during growth at low pH. First, we developed a fluorescence-based high-throughput approach to collect a large number of haploid cells using flow cytometry. Then, we were able to apply a bulk segregant analysis to solve the genetic basis of low pH resistance in PE-2, which uncovered a region in chromosome X as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed the allele GAS1, encoding a ß-1,3-glucanosyltransferase, as the casual variant in this region. The GAS1 sequence alignment of distinct S. cerevisiae strains pointed out a non-synonymous mutation (A631G) prevalence in wild-type isolates, which is absent in laboratory strains. We further showcase that GAS1 allele swap between PE-2 and a low pH-susceptible strain can improve cell viability on the latter of up to 12% after a sulfuric acid wash process. CONCLUSION: This work revealed GAS1 as one of the main causative genes associated with tolerance to growth at low pH in PE-2. We also showcase how GAS1PE-2 can improve acid resistance of a susceptible strain, suggesting that these findings can be a powerful foundation for the development of more robust and acid-tolerant strains. Our results collectively show the importance of tailored industrial isolated strains in discovering the genetic architecture of relevant traits and its implications over productivity.
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Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.
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Viral infections pose intense burdens to healthcare systems and global economies. The correct diagnosis of viral diseases represents a crucial step towards effective treatments and control. Biosensors have been successfully implemented as accessible and accurate detection tests for some of the most important viruses. While most biosensors are based on physical or chemical interactions of cell-free components, the complexity of living microorganisms holds a poorly explored potential for viral detection in the face of the advances of synthetic biology. Indeed, cell-based biosensors have been praised for their versatility and economic attractiveness, however, yeast platforms for viral disease diagnostics are still limited to indirect antibody recognition. Here we propose a novel strategy for viral detection in Saccharomyces cerevisiae, which combines the transductive properties of G Protein-Coupled Receptors (GPCRs) with the Yeast Surface Display (YSD) of specific enzymes enrolled in the viral recognition process. The GPCR/YSD complex might allow for active virus detection through a modulated signal activated by a GPCR agonist, whose concentration correlates to the viral titer. Additionally, we explore this methodology in a case study for the detection of highly pathogenic coronaviruses that share the same cell receptor upon infection (i.e. the Angiotensin-Converting Enzyme 2, ACE2), as a conceptual example of the potential of the GPCR/YSD strategy for the diagnosis of COVID-19.
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COVID-19/diagnóstico , COVID-19/metabolismo , COVID-19/virología , Técnicas de Visualización de Superficie Celular , Interacciones Huésped-Patógeno , Receptores Acoplados a Proteínas G/metabolismo , SARS-CoV-2/fisiología , Técnicas del Sistema de Dos Híbridos , Animales , Técnicas Biosensibles , Línea Celular , Humanos , Técnicas de Diagnóstico Molecular , Saccharomyces cerevisiaeRESUMEN
Ethanol production has key differences between the two largest producing countries of this biofuel, Brazil and the USA, such as feedstock source, sugar concentration and ethanol titers in industrial fermentation. Therefore, it is highly probable that these specificities have led to genome adaptation of the Saccharomyces cerevisiae strains employed in each process to tolerate different environments. In order to identify particular adaptations, in this work, we have compared the genomes of industrial yeast strains widely used to produce ethanol from sugarcane, corn and sweet sorghum, and also two laboratory strains as reference. The genes were predicted and then 4524 single-copy orthologous were selected to build the phylogenetic tree. We found that the geographic location and industrial process were shown as the main evolutionary drivers: for sugarcane fermentation, positive selection was identified for metal homeostasis and stress response genes, whereas genes involved in membrane modeling have been connected with corn fermentation. In addition, the corn specialized strain Ethanol Red showed an increased number of copies of MAL31, a gene encoding a maltose transporter. In summary, our work can help to guide new strain chassis selection for engineering strategies, to produce more robust strains for biofuel production and other industrial applications.
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Etanol/metabolismo , Genoma Fúngico , Microbiología Industrial , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biocombustibles , Etanol/análisis , Fermentación , Genómica , Filogenia , Saccharomyces cerevisiae/clasificaciónRESUMEN
Xylose assimilation and fermentation are important traits for second generation ethanol production. However, some genomic features associated with this pentose sugar's metabolism remain unknown in yeasts. Comparative genomics studies have led to important insights in this field, but we are still far from completely understanding endogenous yeasts' xylose metabolism. In this work, we carried out a deep evolutionary analysis suited for comparative genomics of xylose-consuming yeasts, searching for of positive selection on genes associated with glucose and xylose metabolism in the xylose-fermenters' clade. Our investigation detected positive selection fingerprints at this clade not only among sequences of important genes for xylose metabolism, such as xylose reductase and xylitol dehydrogenase, but also in genes expected to undergo neutral evolution, such as the glycolytic gene phosphoglycerate mutase. In addition, we present expansion, positive selection marks, and convergence as evidence supporting the hypothesis that natural selection is shaping the evolution of the little studied methylglyoxal reductases. We propose a metabolic model suggesting that selected codons among these proteins caused a putative change in cofactor preference from NADPH to NADH that alleviates cellular redox imbalance. These findings provide a wider look into pentose metabolism of yeasts and add this previously overlooked piece into the intricate puzzle of oxidative imbalance. Although being extensively discussed in evolutionary works the awareness of selection patterns is recent in biotechnology researches, rendering insights to surpass the reached status quo in many of its subareas.
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Xilitol/metabolismo , Xilosa/metabolismo , Fermentación/genética , Fermentación/fisiología , Genómica/métodos , Filogenia , Selección Genética/genética , Selección Genética/fisiologíaRESUMEN
Here, we report the genome assembly of a Saccharomyces cerevisiae SA1-derived haploid (FMY097) indigenous strain isolated from a Brazilian ethanol distillery. FMY097 was recently reported to be a highly aldehyde-resistant strain capable of producing bioethanol in the presence of up to 40 mM furfural and 80 mM 5-hydroxymethylfurfural.
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BACKGROUND: The macaúba palm is a novel feedstock for oil production suitable for multiple uses, including as biodiesel and in the food and cosmetic industries. As an efficient alternative, the macaúba palm has limited genomic resources, particularly expressed sequence tag (EST) markers. We report a comprehensive set of validated EST-simple sequence repeat (SSR) markers by using transcriptome sequencing, its application in genetic diversity analysis and cross transferability in other palm trees with environmental and economic importance. RESULTS: In this study, a total of 418 EST-SSRs were identified to be unique for one transcript and region; 232 EST-SSRs were selected, with trinucleotide repeats being the most frequent motif, representing 380 (90.9%), followed by composited (4.5%), di- (3.6%), and hexanucleotides (3.6%). A total of 145 EST-SSRs (62.5%) were validated for consistent amplification in seventeen macaúba palm samples, and 100 were determined to be polymorphic with PIC values ranging from 0.25 to 0.77. Genetic diversity analysis was performed with the 20 most informative EST-SSR markers showing a distinct separation of the different groups of macaúba palm. Additionally, these 145 markers were transferred in six other palm species resulting in transferability rates of 99% (144) in Acrocomia intumescens, 98% (143) in Acrocomia totai, 80.7% (117 EST-EST) in African oil palm (Elaeis guineensis) and peach palm (Bactris gasipaes) samples, 70% (102) in the juçara palm (Euterpe edulis) and 71.7% (104) in the hat palm (Sabal causiarum). Analysis of genetic distance showed a high separation in accordance with geographic location, establishing distinct groups by genera. CONCLUSIONS: The EST markers identified in our study are a valuable resource and provide a genomic tool for genetic mapping and further genetic studies, as well as evaluation of co-location between QTLs and functionally associated markers.
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Arecaceae/genética , Variación Genética , Genoma de Planta/genética , Transcriptoma , Mapeo Cromosómico , Etiquetas de Secuencia Expresada , Marcadores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Repeticiones de Microsatélite/genética , Análisis de Secuencia de ARNRESUMEN
Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens.
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Agaricales/fisiología , Cacao/inmunología , Quitinasas/genética , Proteínas Fúngicas/genética , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Agaricales/genética , Secuencia de Aminoácidos , Cacao/microbiología , Quitinasas/química , Quitinasas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Enfermedades de las Plantas/microbiología , Alineación de SecuenciaRESUMEN
Draft genomes of three Salmonella enterica 4,[5],12:i:- (STi) strains isolated from human infections were obtained using Illumina sequencing. They were negative for the fljBA operon but positive for hin, and k-mer analyses revealed their identity as S. enterica 4,[5],12:i:- 08-1736 and S Typhimurium. A draft S Typhimurium sequence is described for comparison.