Investigating the molecular participants of programmed cell death in plamodium falciparum

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy University of the Witwatersrand, Johannesburg, 2014Malaria, a disease resulting from infection by members of the Plasmodium genus, accounted for an estimated 627 000 deaths globally in 2012. The majority of these mortalities were due to P. falciparum infections and thus the species of focus in this study. Due to the rapid emergence of drug-resistant strains, novel avenues for research evaluating parasite survival and population regulation within the human host are now needed. Programmed cell death (PCD) is a well characterised means of self-regulation in metazoans, where a plethora of proteins and signals result in the destruction and/or removal of unnecessary, damaged or dangerous cells. A key protein participant is MDM2 which, via its SWIB/MDM2 domain, binds to the nuclear transcription factor p53 to promote p53 degradation and prevent apoptosis. SWIB/MDM2 domains additionally play key roles in transcription-dependent stress survival. No proven PCD molecular participants for P. falciparum exist but two SWIB/MDM2 homologues (PF3D7_0611400 (PfSWIB) and PF3D7_0518200 (PfMDM2)) and a putative p53 homologue (PF3D7_0522400 (Pfp53)) have been identified by bioinformatics. These were assessed experimentally in this study. Structural features of the SWIB/MDM2 domains of PfMDM2 and PfSWIB, suggested that they are chromatin remodelling factors. The domains were amplified from 3D7 P. falciparum genomic DNA, directionally cloned into the pGEX-4T-2 vector, and used for recombinant GST-fusion protein expression in E. coli. The soluble, tagged, domains were isolated and purified by affinity chromatography (PfMDM2, ~33kDa and PfSWIB, ~42kDa) and used, in conjunction with P. falciparum phage display library technology, for the identification of several novel binding partners. Two of these interactions were verified with in vitro binding assays, proving concentration dependent interactions between PfMDM2 and a conserved protein of unknown function; and PfSWIB and a putative Investigating the Molecular Participants of Programmed Cell Death in Plasmodium falciparum serine-threonine protein kinase (PfARK3). Transgenic P. falciparum parasites were created by transfection with pARL2-GFP vector constructs containing the PfSWIB and PfMDM2 genes. PfMDM2-GFP localized to the mitochondria under the control of an N-terminal signal sequence, under normal and heat stress conditions, the latter triggering PCD in the asexual intraerythrocytic parasite. PfSWIB-GFP localized to the cytoplasm under normal and heat stress conditions, but in a subpopulation of trophozoites it moved to the nucleus after exposure to elevated temperatures. PfMDM2 is hypothesized to play a role within the parasite mitochondrion, although its involvement in PCD is uncertain and may be unconventional, while PfSWIB is suggested to be involved in a stage-specific heat stress response. Pfp53 was found to have a putative DNA binding and tetramerization domain, based primarily on sequence alignments. A recombinant GST-tagged form (~87kDa) of these two domains was expressed in E. coli and purified by affinity chromatography. The ability of the recombinant protein to tetramerize was inconclusive, while in an electromobility shift assay it did not bind to a canonical p53 DNA binding consensus sequence identified in the parasite’s genome. The precise cellular function(s) for this protein requires further evaluation. This study represents the first characterisation of these three P. falciparum proteins. Several novel activities were identified for each and their role in PCD was evaluated by exposing parasites to febrile temperatures, which provided new information regarding heat stress regulation in P. falciparum

Expression Changes of Genes Related to Chromosome Remodeling Caused by Implantation with Low-energy N + Beam in Rice

Abstract: In order to investigate expression changes of genes related to chromosome remodeling induced by implantation with low-energy N + beam in rice, the differentially expressed genes related to chromosome remodeling in treated samples (6×10 17 N + /cm 2 ), were screened using Agilent gene chip. The results showed that, in the treated samples, 1 out of 30 genes related to histone deacetylase was up-regulated. Expressions of 48 genes related to histone acetyltransferases (HATs), two SNF2-related domain containing proteins and one SWIM Zn-finger domain containing protein were up-regulated, one SWIB/MDM2 domain containing protein and one TAFII55 protein conserved region domain containing protein were down-regulated, expression of 3 GCN5-related N-acetyltransferase domain containing proteins were significantly different, in which one was up-regulated and two down-regulated. There are several conserved protein domains related to chromosome remodeling on the Microarray, Expressions of 2 of 38 bromodomain related genes were changed of which one is up-regulated and the other down-regulated. Expressions of 2 of 7 chromodomain related genes and 8 of 36 SET related genes were up-regulated. Besides, one expression of 14 LSD1 type domain containing protein was significantly up-regulated. These findings suggest that the expression changes of genes related to chromosome remodeling may regulate the expression of certain genes responding to ion implantation, and induce the biological effect of the rice implanted by ion beam

Eukaryotic Components Remodeled Chloroplast Nucleoid Organization during the Green Plant Evolution.

Chloroplast (cp) DNA is thought to originate from the ancestral endosymbiont genome and is compacted to form nucleoprotein complexes, cp nucleoids. The structure of cp nucleoids is ubiquitously observed in diverse plants from unicellular algae to flowering plants and is believed to be a multifunctional platform for various processes, including cpDNA replication, repair/recombination, transcription, and inheritance. Despite its fundamental functions, the protein composition for cp nucleoids in flowering plants was suggested to be divergent from those of bacteria and algae, but the evolutionary process remains elusive. In this research, we aimed to reveal the evolutionary history of cp nucleoid organization by analyzing the key organisms representing the three evolutionary stages of eukaryotic phototrophs: the chlorophyte alga Chlamydomonas reinhardtii , the charophyte alga Klebsormidium flaccidum , and the most basal land plant Marchantia polymorpha . To clarify the core cp nucleoid proteins in C. reinhardtii , we performed an LC-MS/MS analysis using highly purified cp nucleoid fractions and identified a novel SAP domain-containing protein with a eukaryotic origin as a constitutive core component. Then, homologous genes for cp nucleoid proteins were searched for in C. reinhardtii , K. flaccidum , and M. polymorpha using the genome databases, and their intracellular localizations and DNA binding activities were investigated by cell biological/biochemical analyses. Based on these results, we propose a model that recurrent modification of cp nucleoid organization by eukaryotic factors originally related to chromatin organization might have been the driving force for the diversification of cp nucleoids since the early stage of green plant evolution

Exploring Chromatin-bound MDM2 Functions in Compromised Transcriptional Regulation of p53 Target Genes

MDM2 overexpression is a common occurrence in many types of cancer. A single nucleotide polymorphism (T to G) near the mdm2 promoter, termed mdm2 SNP309, leads to MDM2 overexpression. This polymorphism is associated with accelerated tumor formation, decreased sensitivity to DNA damage treatment and compromised p53 transcriptional activity. Two G/G SNP309 cancer cell lines MANCA and A875, a Burkitts\u27 lymphoma and melanoma respectively, express a stable wild-type p53 protein. We previously reported these cells have DNA damage resistant MDM2-p53 chromatin complexes and hypothesized that MDM2 is the contributing factor for the compromised p53 transcriptional activity. We created constitutive mdm2 shRNA cell lines to address MDM2 function. MDM2 knockdown in MANCA and A875 cells moderately increased expression of subsets of p53 target genes in a cell-type specific manner; although no additive effect was seen with DNA damage. Additionally, MDM2 knockdown did not affect p53 protein stability. We explored the mechanism for compromised p53 transcriptional activity in G/G SNP309 MANCA and A875 cell lines using chromatin immunoprecipitation analysis. When compared to fully activated T/T SNP309 ML-1 cells (myeloid leukemia with functional wild-type p53) treated with etoposide, MANCA and A875 cell lines displayed comparable recruitment of total and initiated RNA polymerase II at transcription start sites (TSS) for p21 and puma genes. This indicated that G/G SNP309 cells had functional transcription initiation at p53 target genes after DNA damage treatment. Next, we assessed transcriptional elongation using H3K36 trimethylation (H3K36me3) as a mark for active elongation. In ML-1 cells with etoposide treatment, we observed higher H3K36me3 at p21 and puma TSS than in either MANCA or A875 cells at these same regions. This suggested transcriptional elongation is compromised and also suggests that in G/G SNP309 cancer cells reactivation of p53 transcriptional activity is difficult. We sought to explain this phenomenon by examining the other well-known p53 negative regulator, MDMX, which is a homolog of MDM2. Interestingly, in MANCA cells the knockdown of MDM2 caused a substantial increase in MDMX protein levels. However, in A875 cells this was not observed. This suggests that for certain cell types, MDMX may function in some redundant roles for MDM2. Finally, we tested if inducing p53-independent cell death would be more effective than reactivating the wild-type p53 pathway. We treated the cells with 8-amino-adenosine, an inducer of p53-independent cell death. Indeed, treatment of MANCA and A875 cells with 8-amino-adenosine reduced cell viability more effectively than other chemotherapeutics

Identification and characterization of novel type III secreted proteins in Chlamydia infection

Chlamydiae sind obligat intrazelluläre Parasiten, welche ein breites Spektrum von Organismen wie Tiere, Insekten oder Amöben infizieren. Diese Gram-negativen Bakterien stellen eine weltweit vermeidbare Hauptursache für Blindheit und Unfruchtbarkeit in Menschen dar. Infizieren Chlamydiae eine eukaryotische Wirtszelle, so bilden sie ein spezielles Kompartiment aus, die Inklusion, in der sie sich vermehren und von der infektiösen, metabolisch inaktiven Form in die nicht-infektiöse, aber metabolisch aktive Form konvertieren. Beide Formen besitzen ein Typ III Sekretionssystem, mit dem sie potentiell toxische Effektorproteine in die Wirtszelle translozieren. Aufgrund des intrazellulären Lebensstils von Chlamydiae konnten bis heute noch keine Strategien entwickelt werden, sie genetisch zu manipulieren. Dies wirkt sich erschwerend auf die Identifikation neuer Effektorproteine aus. Eine zusätzliche Komplikation wird dadurch hervorgerufen, dass die molekulare Erkennung von Effektoren durch die Typ III Sekretionsmaschinerie noch ungeklärt ist. Dennoch wird die Hypothese, dass sich dieses Erkennungssignal in den ersten 20 Aminosäuren des N-Terminus befinde, von einigen Versuchen gestützt. Um neue Typ III sezernierte Proteine zu identifizieren, bedienten wir uns des schon veröffentlichten Versuchsansatzes, bei dem der Sekretionstest von Chlamydiae-Effektoren in einem heterologen Typ III Sekretionssystem von Shigella flexneri durchgeführt wurde. Hierfür erstellten wir Chimären, welche aus den ersten 20 Aminosäuren des N-Terminus des potentiellen Effektors und einem Reporter-Protein, der Calmodulin-abhängigen Adenylatcyclase (Cya) von Bordetella pertussis, bestanden. Diese Chimären exprimierten wir in unterschiedlichen Shigella flexneri-Stämmen. Durch den Gebrauch eines Antikörpers gegen Cya konnten wir die Chimären lokalisieren und ihre Sekretionseigenschaften bestimmen. Um eine Liste von zu testenden Kandidaten zu erstellen machten wir uns das Wissen zunutze, dass viele Effektoren Signaturen von Proteindomänen, die typischerweise in Eukaryoten gefunden werden, besitzen. Durch die Software „Effective“ erhielten wir vorausberechnete Listen von Proteinen aus den Proteomen von Chlamydia trachomatis, Chlamydophila pneumoniae und Chlamydophila caviae, welche eine Anreicherung dieser eukaryotisch-ähnlichen Domänen besitzen. Anhand des oben erwähnten heterologen Sekretionstests konnten wir zeigen, dass 17/25 getesteten Kandidaten tatsächlich durch ein Typ III Sekretionssystem sezerniert wurden. Zusätzlich dazu unterzogen wir eine Auswahl an Proteinen der Umweltchlamydien, welche eine Rolle im Glykogenmetabolismus spielen, ebenfalls dem Sekretionstest. Diese Auswahl wurde hinsichtlich der Funktionen dieser Enzyme getroffen, welche diese bei einer Stabilisierung der Endosymbiosebildung zwischen einem Cyanobiont und einem heterotrophen Organismus erfüllt haben könnten. Wir zeigten, dass fast alle diese Enzyme sezerniert wurden, und dass einige von ihnen am engsten mit Pflanzenhomologen verwandt zu sein scheinen. Dies unterstützt die Hypothese, dass Chlamydiae eine unabdingbare Rolle in der frühen Entstehungsgeschichte der heutigen Pflanze einnahm. Weiters untersuchten wir Homologe dieser Enzyme in C. trachomatis und C. pneumoniae auf unterschiedliche Sekretionseigenschaften, da es bekannt ist, dass nur C. trachomatis Glykogen akkumuliert. Wir zeigten Unterschiede auf und gaben Anregungen für weitere Überlegungen.Chlamydiae are obligate intracellular parasites infecting a broad spectrum of organisms such as animals, insects and amoeba. These gram-negative bacteria are worldwide a major cause of preventable blindness and infertility in humans. Upon infection of a eukaryotic host cell Chlamydiae multiply within a parasitophorous compartment termed inclusion, where they also undergo conversion from an infectious, metabolically rather inert form to a non-infectious, metabolically active form. Both forms possess a type III secretion system in order to translocate potentially toxic effector proteins to targets within the host cell. No genetic tools to manipulate Chlamydiae are available so far due to their obligate intracellular lifestyle, hampering the examination of putative secreted effectors. The identification of type III secreted proteins is additionally complicated by the fact that the molecular recognition of effectors by a type III machinery is still elusive. However, various experiments suggest a signal in the 20 amino terminal amino acids of the effectors. We made use of the already published approach of testing putative chlamydial effectors in a heterologous type III secretion system of Shigella flexneri in order to identify novel type III secreted proteins. For this purpose we designed chimeras consisting out of the 20 amino terminal amino acids of a candidate protein fused to a reporter, the calmodulin-dependent adenylate cyclase (Cya) of Bordetella pertussis. These chimeras were expressed in different strains of Shigella flexneri. By applying an antibody against Cya we could localize the chimeras and determine their characteristics of secretion. In order to create a list of candidates that we subsequently subjected to this secretion test we made use of the finding that many effectors have been shown to contain protein domain signatures that are typically found in eukaryotes. The software “Effective” gave us precalculated lists for proteins enriched in these “eukaryotic-like domains” in the proteomes of Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila caviae. With the previously mentioned heterologous secretion test we demonstrated that 17/25 chosen candidates were positive for type III secretion. The software “Effective” is thus a very useful approach, but it can only be used additionally, since other proteins we tested as positive were not listed by this software. Additionally, we tested an assortment of proteins of environmental Chlamydiae engaged in glycogen metabolism for secretion. This assortment was chosen regarding the function these enzymes could have fulfilled to stabilize the formation of endosymbiosis between a cyanobiont and a heterotrophic organism. We showed that almost all of these enzymes are indeed secreted and that some of them seem to be closest related to plant homologs, strengthening the hypothesis of an indispensable role of Chlamydiae in the early history of development of today’s plants. We further examined if these enzymes have different secretion characteristics in C. trachomatis and C. pneumoniae, knowing that only C. trachomatis accumulates glycogen. We could state differences here and gave suggestions for further thoughts

Analyses of domains and domain fusions in human proto-oncogenes

Background: Understanding the constituent domains of oncogenes, their origins and their fusions may shed new light about the initiation and the development of cancers. Results: We have developed a computational pipeline for identification of functional domains of human genes, prediction of the origins of these domains and their major fusion events during evolution through integration of existing and new tools of our own. An application of the pipeline to 124 well-characterized human oncogenes has led to the identification of a collection of domains and domain pairs that occur substantially more frequently in oncogenes than in human genes on average. Most of these enriched domains and domain pairs are related to tyrosine kinase activities. In addition, our analyses indicate that a substantial portion of the domain-fusion events of oncogenes took place in metazoans during evolution. Conclusion: We expect that the computational pipeline for domain identification, domain origin and domain fusion prediction will prove to be useful for studying other groups of genes. Originally published BMC Bioinformatics, Vol. 10, No. 88, Mar 200

Genome communication in plants mediated by organelle-n-ucleus-located proteins

An increasing number of eukaryotic proteins have been shown to have a dual localization in the DNA-containing organelles, mitochondria and plastids, and/or the nucleus. Regulation of dual targeting and relocation of proteins from organelles to the nucleus offer the most direct means for communication between organelles as well as organelles and nucleus. Most of the mitochondrial proteins of animals have functions in DNA repair and gene expression by modelling of nucleoid architecture and/or chromatin. In plants, such proteins can affect replication and early development. Most plastid proteins with a confirmed or predicted second location in the nucleus are associated with the prokaryotic core RNA polymerase and are required for chloroplast development and light responses. Few plastid-nucleus-located proteins are involved in pathogen defence and cell cycle control. For three proteins, it has been clearly shown that they are first targeted to the organelle and then relocated to the nucleus, i.e. the nucleoid-associated proteins HEMERA and Whirly1 and the stroma-located defence protein NRIP1. Relocation to the nucleus can be experimentally demonstrated by plastid transformation leading to the synthesis of proteins with a tag that enables their detection in the nucleus or by fusions with fluoroproteins in different experimental set-ups. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'

New insights into the proteome of the transcriptionally active chromosome from chloroplasts

Plastiden besitzen eigene DNA (ptDNA), die zusammen mit einer Vielzahl an Proteinen in Strukturen verpackt ist, die den bakteriellen Chromosomen ähneln. Diese Nukleoproteinkomplexe werden als plastidäre Nukleoide oder Plastidenkerne bezeichnet. Im Unterschied zum Chromatin im Zellkern gibt es nur wenige Informationen über die Organisation und Dynamik der Nukleoide in den Plastiden. Zur Identifizierung von Proteinen des Transkriptionsaktiven Chromosoms (TAC) aus Spinatchloroplasten wurden Proteomanalysen durchgeführt. In einer voran-gegangenen Proteomanalyse mit einer TAC-Fraktion aus Senfchloroplasten wurde das DNA-Bindeprotein Whirly1 identifiziert. Mit dem spezifischen Antikörper konnte hier gezeigt werden, dass das Protein in der konventionell präparierten TAC I Fraktion, aber nicht in der hochaufgereinigten TAC-II-Fraktion von Gersten-chloroplasten vorkommt. Durch eine Proteomanalyse der hochaufgereinigten TAC II Fraktion konnten darüber hinaus sechs neue DNA-Bindeproteine identifiziert werden. Sie wurden als plastidäre nukleoidassoziierte Proteine (ptNAP) benannt. Eines der neu identifizierten ptNAP ist das Protein AtSWIB-1 (Arabidopsis thaliana SWIB domain-containing protein-1). Das Protein ist in der TAC-II-Fraktion ange-reichert und bindet an plastidäre DNA. Dies deutet darauf hin, dass das SWIB-1-Protein im Unterschied zum Whirly1-Protein eine integrale Komponente des TAC ist. Durch Fusionen mit dem GFP-Protein konnte eine duale Lokalisation des AtSWIB-1-Proteins im Zellkern und in den Nukleoiden der Plastiden gezeigt werden. Daten-bankrecherchen erlaubten die Identifizierung von drei weiteren SWIB-Domänen-proteinen von Arabidopsis thaliana, deren vorhergesagte plastidäre Lokalisation durch GFP-Fusionen bestätigt werden konnte. Zusammen mit SWIB-1 gehören sie zu den ersten Chromatinmodellierungsfaktoren, die außerhalb des Kerns vorkom-men. Aufgrund der Ergebnisse dieser Arbeit wird die Hypothese aufgestellt, dass Plastiden Chromatinmodellierungsproteine besitzen, die über Veränderungen in der Struktur der Nukleoide die Genexpression beeinflussen können.Chloroplasts possess their own DNA (ptDNA), which is packaged with proteins into structures analogous to bacterial chromosomes, termed nucleoids or plastid nuclei. In contrast to nuclear chromatin, there is only limited information on the organization and dynamics of the plastid nucleoids. In order to investigate the protein composition of the transcriptionally active chromosome (TAC) fractions from chloroplasts and to identify new components of it, proteomic analyses were performed. One of the DNA-binding proteins identified in a previous study was the Whirly1 protein. Immunological experiments with an antibody specific for HvWhirly1 allowed the detection of the protein in a conventionally prepared TAC-I fraction from barley chloroplasts but not in a highly purified TAC-II fraction. By proteome analyses with the highly purified TAC-II fraction, six new DNA-binding proteins were identified. They were named plastid nucleoid-associated proteins (ptNAP). One of newly identified ptNAPs is the AtSWIB 1 (Arabidopsis thaliana SWIB domain-containing protein-1) protein. Immuno-logical analyses with an antibody specific for AtSWIB-1 allowed the detection of the SWIB-1 protein in both the conventionally prepared TAC-I and the highly enriched TAC-II fraction from spinach chloroplasts indicating that SWIB-1 in contrast to Whirly1 is a core component of TAC. An AtSWIB-1:GFP fusion protein was shown to be dually located in the nucleus and in plastid nucleoids. Binding of the SWIB-1 protein to plastid DNA was confirmed by Southwestern analysis. Database searches revealed that SWIB-1 together with further five SWIB-domain proteins belong to a subgroup of the SWIB family being predicted to be targeted to organelles. Fusions with GFP protein confirmed a plastid location for three of them. Together with SWIB-1, these proteins are the first identified chromatin remodeling factors being imported into plastids. Based on these findings, it is hypothesized that plastids possess chromatin remodeling proteins in order to regulate the expression of their genes by introducing structural changes of the nucleoid

Investigating a BIR-containing plasmodium falciparum protein and identifying its binding partners

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2018.The majority of the worldwide malaria deaths are caused by the Plasmodium falciparum parasite and unfortunately parasite strains are emerging that are resistant to not only artemisinin, but also the partner drugs used in current antimalarial combination therapy. The intraerythrocytic P. falciparum life stage is characterised by exponential, asexual proliferation that could cause the premature death of the human host before the sexual gametocytes have had enough time to develop and be taken up by the mosquito vector to continue its lifecycle. It is hypothesised that P. falciparum maintains its population at a level low enough to allow for the transmission of these gametocytes by using a form of regulated cell death (RCD). The molecular members of this cell death pathway are currently unclear, but a putative P. falciparum inhibitor of apoptosis protein (PfIAP; PF3D7_0519600) has previously been identified. Metazoan IAP proteins play anti-apoptotic roles in cells by interacting and inhibiting pro-apoptotic caspases, but also perform other functions. Analysis of the PfIAP protein using bioinformatic tools revealed that it contains one conserved baculoviral IAP repeat (BIR) domain and that this P. falciparum BIR domain is structurally similar to the BIR domains of various human IAP proteins. mRNA extracted from asexual P. falciparum parasites was used to construct a biotin-tagged phage display library, which was used in biopanning experiments with two regions of the PfIAP protein, expressed as recombinant GST-tagged proteins. Four binding partners were identified for the N-terminal BIR domain of the protein, while two proteins were identified as interacting partners for the C-terminal region of PfIAP. Of these, a double C2-like domain-containing protein (PfDOC2) and the high molecular weight rhoptry protein 3 (PfRhopH3) were expressed as recombinant His-tagged proteins and verified as PfIAP binding partners by in vitro binding assays. Transgenic P. falciparum parasites were generated expressing a GFP-tagged PfIAP BIR domain, which localised to the cytoplasm under both normal and high temperature conditions which mimic febrile malaria, a physiological trigger of RCD. Knockout experiments of the pfiap gene using the CRISPR-Cas9 genome editing tool suggested that this gene could be essential for the survival of asexual P. falciparum parasites. This study offers the first details of a putative P. falciparum inhibitor of apoptosis protein and suggests that it could have non-apoptotic roles in the parasite, given the diverse functions of the binding partners that comprise the PfIAP protein-protein network.LG201

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