New insights into the evolution and structure of Colletotrichum plant-like subtilisins (CPLSs)

The Colletotrichum plant-like subtilisins (CPLSs) are a family of proteins found only in species of the phytopathogenic fungus Colletotrichum. CPLSs have high similarity to plant subtilisins and our previous work has shown that they were acquired by an ancient horizontal gene transfer event from plants. The rapid growth of sequence data in public databases enabled us to reexamine the structure and evolution of the CPLSs. A new plant subtilisin structural model aided us in refining the tertiary structure of CPLSs. Also, new information about protein interactions of plant subtilisin has provided new insights into the putative function of CPLSs. The availability of new genome sequences of members of the genus Colletotrichum gave us the opportunity to further validate our hypothesis that the CPLSs are unique to the Colletotrichum lineage. Together, this information furthers our knowledge of the potential role of the CPLSs in pathogenicity and the role of HGT in the genome evolution of plant pathogenic fungi.Fil: Armijos Jaramillo, Vinicio. Universidad de Salamanca; EspañaFil: Vargas, Walter Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad de Salamanca; EspañaFil: Sukno, Serenella A.. Universidad de Salamanca; EspañaFil: Thon, Michael R.. Universidad de Salamanca; Españ

New insights into the evolution and structure of Colletotrichum plant-like subtilisins (CPLSs)

The Colletotrichum plant-like subtilisins (CPLSs) are a family of proteins found only in species of the phytopathogenic fungus Colletotrichum. CPLSs have high similarity to plant subtilisins and our previous work has shown that they were acquired by an ancient horizontal gene transfer event from plants. The rapid growth of sequence data in public databases enabled us to reexamine the structure and evolution of the CPLSs. A new plant subtilisin structural model aided us in refining the tertiary structure of CPLSs. Also, new information about protein interactions of plant subtilisin has provided new insights into the putative function of CPLSs. The availability of new genome sequences of members of the genus Colletotrichum gave us the opportunity to further validate our hypothesis that the CPLSs are unique to the Colletotrichum lineage. Together, this information furthers our knowledge of the potential role of the CPLSs in pathogenicity and the role of HGT in the genome evolution of plant pathogenic fungi.Fil: Armijos Jaramillo, Vinicio. Universidad de Salamanca; EspañaFil: Vargas, Walter Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad de Salamanca; EspañaFil: Sukno, Serenella A.. Universidad de Salamanca; EspañaFil: Thon, Michael R.. Universidad de Salamanca; Españ

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host’s nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host’s nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.Fil: Vargas, Walter Alberto. Universidad de Salamanca; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sanz Martín, José M.. Universidad de Salamanca; EspañaFil: Rech, Gabriel E.. Universidad de Salamanca; EspañaFil: Armijos Jaramillo, Vinicio D.. Universidad de Salamanca; EspañaFil: Rivera Rodriguez, Lina Patricia. Universidad de Salamanca; EspañaFil: Echeverria, María de Las Mercedes. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Díaz Mínguez, José M.. Universidad de Salamanca; EspañaFil: Thon, Michael R.. Universidad de Salamanca; EspañaFil: Sukno, Serenella A.. Universidad de Salamanca; Españ

A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola

[EN] Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling

Population Genomics Provide Insights into the Global Genetic Structure of \u3ci\u3eColletotrichum graminicola\u3c/i\u3e, the Causal Agent of Maize Anthracnose

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and wholegenome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure

Molecular diversity of anthracnose pathogen populations associated with UK strawberry production suggests multiple introductions of three different Colletotrichum species.

Fragaria × ananassa (common name: strawberry) is a globally cultivated hybrid species belonging to Rosaceae family. Colletotrichum acutatum sensu lato (s.l.) is considered to be the second most economically important pathogen worldwide affecting strawberries. A collection of 148 Colletotrichum spp. isolates including 67 C. acutatum s.l. isolates associated with the phytosanitary history of UK strawberry production were used to characterize multi-locus genetic variation of this pathogen in the UK, relative to additional reference isolates that represent a worldwide sampling of the diversity of the fungus. The evidence indicates that three different species C. nymphaeae, C. godetiae and C. fioriniae are associated with strawberry production in the UK, which correspond to previously designated genetic groups A2, A4 and A3, respectively. Among these species, 12 distinct haplotypes were identified suggesting multiple introductions into the country. A subset of isolates was also used to compare aggressiveness in causing disease on strawberry plants and fruits. Isolates belonging to C. nymphaeae, C. godetiae and C. fioriniae representative of the UK anthracnose pathogen populations showed variation in their aggressiveness. Among the three species, C. nymphaeae and C. fioriniae appeared to be more aggressive compared to C. godetiae. This study highlights the genetic and pathogenic heterogeneity of the C. acutatum s.l. populations introduced into the UK linked to strawberry production

Population Genomics Provide Insights into the Global Genetic Structure of Colletotrichum graminicola, the Causal Agent of Maize Anthracnose

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and whole-genome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure. IMPORTANCE Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems. We employed a population genomics approach to investigate the genetic diversity and reproductive biology of the maize anthracnose pathogen (Colletotrichum graminicola) in 14 countries. We found that the populations are correlated with their geographical origin and that migration between countries is ongoing, possibly caused by the movement of infected plant material. This result has direct implications for disease management because migration can cause the movement of more virulent and/or fungicide-resistant genotypes. We conclude that genetic recombination is frequent (in contrast to the traditional view of C. graminicola being mainly asexual), which strongly impacts control measures and breeding programs aimed at controlling this disease.This research was supported by grants AGL2015-66362-R, RTI2018-093611-B-100, and PID2021-125349NB-100, funded by the Ministry of Science and Innovation (MCIN) of Spain AEI/10.13039/501100011033; and by grant SA165U13 funded by the Junta de Castilla y Léon. F.R. was supported by grant FJC2020-043351-I financed by MCIN/AEI /10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. R.B. was supported by the postdoctoral program of USAL (Program II). F.B.C.-F. was supported by grant BES-2016-078373, funded by MCIN/AEI/10.13039/501100011033. S.B. was supported by a fellowship program from the regional government of Castilla y León. W.B. was supported by a productivity fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 307855/2019-8). Genome sequencing was funded by the UNC Microbiome Core, which is funded in part by the Center for Gastrointestinal Biology and Disease (CGIBD P30 DK034987) and the UNC Nutrition Obesity Research Center (NORC P30 DK056350). P.D.E. was partially supported by the USDA National Institute of Food and Federal Appropriations under Project PEN04660 and accession no. 1016474.Peer reviewe

Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum

Background: Many species belonging to the genus Colletotrichum cause anthracnose disease on a wide range of plant species. In addition to their economic impact, the genus Colletotrichum is a useful model for the study of the evolution of host specificity, speciation and reproductive behaviors. Genome projects of Colletotrichum species have already opened a new era for studying the evolution of pathogenesis in fungi. Results: We sequenced and annotated the genomes of four strains in the Colletotrichum acutatum species complex (CAsc), a clade of broad host range pathogens within the genus. The four CAsc proteomes and secretomes along with those representing an additional 13 species (six Colletotrichum spp. and seven other Sordariomycetes) were classified into protein families using a variety of tools. Hierarchical clustering of gene family and functional domain assignments, and phylogenetic analyses revealed lineage specific losses of carbohydrate-active enzymes (CAZymes) and proteases encoding genes in Colletotrichum species that have narrow host range as well as duplications of these families in the CAsc. We also found a lineage specific expansion of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLPs) families within the CAsc. Conclusions: This study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content

First report of apple bitter rot caused by Colletotrichum godetiae in the UK

Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008

PoGO: Prediction of Gene Ontology terms for fungal proteins

Abstract Background Automated protein function prediction methods are the only practical approach for assigning functions to genes obtained from model organisms. Many of the previously reported function annotation methods are of limited utility for fungal protein annotation. They are often trained only to one species, are not available for high-volume data processing, or require the use of data derived by experiments such as microarray analysis. To meet the increasing need for high throughput, automated annotation of fungal genomes, we have developed a tool for annotating fungal protein sequences with terms from the Gene Ontology. Results We describe a classifier called PoGO (Prediction of Gene Ontology terms) that uses statistical pattern recognition methods to assign Gene Ontology (GO) terms to proteins from filamentous fungi. PoGO is organized as a meta-classifier in which each evidence source (sequence similarity, protein domains, protein structure and biochemical properties) is used to train independent base-level classifiers. The outputs of the base classifiers are used to train a meta-classifier, which provides the final assignment of GO terms. An independent classifier is trained for each GO term, making the system amenable to updating, without having to re-train the whole system. The resulting system is robust. It provides better accuracy and can assign GO terms to a higher percentage of unannotated protein sequences than other methods that we tested. Conclusions Our annotation system overcomes many of the shortcomings that we found in other methods. We also provide a web server where users can submit protein sequences to be annotated.</p