Species concepts in the economically important genus Colletotrichum have been undergoing revolutionary change in recent years. The old morphology-based species are rapidly being superseded by taxa that are largely defined by molecular sequence. Cryptic species are increasingly being recognized that cannot be reliably separated using morphological or cultural methods. In a number of cases, however, these demonstrably monophyletic units do not show marked host preference or shared pathogenic characteristics. This apparent paradox reduces the utility of the new classifications to the applied communities, especially to pathologists. We also know too little about the evolutionary significance of the varied nutritional/pathological strategies shown by Colletotrichum, especially the mechanisms by which colonies change status from endophyte/hemibiotroph to aggressive pathogen on the one hand, or to saprotroph on the other.

There will also be opportunities to exchange information relating to the rapidly increasing number of whole-genome sequencing projects that are planned or already in operation.

The meeting will bring together key workers from the academic and applied sectors to share knowledge, review progress and set priorities for further studies.

Organizers:

Michael Thon, University of Salamanca

Serenella Sukno, University of Salamanca

Location:

The meeting will take place as part of the 10th European Conference on Fungal Genetics  (ECFG10)  at NH Conference Centre Leeuwenhorst, Netherlands.   For more information about the conference, please see this page: at http://www.ecfg10.info/index.php

Data and Time: Monday 29 of March, 1:00 – 5:00 PM

Program:

1 PM opening words Dr. Michael Thon

1.10 PM Dr. Yasuyuki Kubo

Novel peroxin, Fam1p of Colletotrichum orbiculare is essential for pathogenesis and associates with Woronin bodies

1. 35 PM  Jochen Kleemann

Identification and characterization of candidate effector proteins secreted by the crucifer anthracnose fungus Colletotrichum higginsianum

2 PM Dr. Elaine Souza

Genetics analysis in Colletotrichum lindemuthianum

2.25 PM Riccardo Baroncelli

Evolutionary relationships in Colletotrichum acutatum populations

2.45.PM Break 30 minutes.

3.15 PM Emiel Ver Loren van Themaat

Towards a high quality genome and transcriptome of Colletotrichum higginsianum

3.40 PM Dr. Li Ju Ma/ Dr. L. Vaillancourt

Status of the C. graminicola genome sequence

4.05 PM Dr. Michael R. Thon

Genome wide computational analysis of C. graminicola for secreted proteins

4.30 PM discussion of genome annotation

A Genome Sequence for the Model Hemibiotroph Colletotrichum graminicola

Lisa Vaillancourt, Marty Dickman, Lijun Ma, Mike Thon, Jeff Osborn

Kentucky, USA

The goal of the on-going Colletotrichum genome project is to produce and release sequence (8X coverage) of the C. graminicola genome, together with a high-quality assembly and annotation. These resources will be made available to the public through the Broad Institute’s Fungal Genome Initiative web interface. A summary of the progress for the genome project so far will be presented. A major goal of the project is to provide tools that will contribute to understanding the functional and evolutionary relationships among different fungal lifestyles. Intracellular hemibiotrophy is a distinctive aspect of Colletotrichum pathology.  Following penetration of the host epidermis, most Colletotrichum species initially colonize living plant cells, breaching the cell wall but without penetrating the host cell plasma membrane or causing widespread cell death.  After a variable period of time the growth habit switches to necrotrophy, and widespread cell death and tissue destruction ensues. This switch in lifestyle is associated with morphological, genetic, and physiological changes in the hyphae. Our current understanding of the hemibiotrophic lifestyle in C. graminicola will be summarized.  Ideas for use of the genome data for further dissection of this lifestyle will be discussed.

FPC-DB: The Fungal Protein Cluster Database and its use for comparative fungal genomics

Mike Thon, Salamanca, Spain

Our group is developing the Fungal Protein Cluster Database (FPC-DB) to enable users to quickly locate information about the evolutionary history and the functions of fungal genes and gene families.  The proteins from fungal whole-genome sequencing projects are annotated with InterPro terms, SwissProt keywords, and Gene Ontology terms using an automated functional classification server previously developed in our group.  The proteins are then clustered into putative gene families using the MCL application. Each protein cluster includes multiple sequence alignments, phylogenetic trees and a summary of functional categories found in the cluster.  Users can perform queries using proteins IDs, protein functional categories, and species names as search terms. I will present an overview of FPC-DB and demonstrate how it can be used to compare gene family content in fungi.

The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola

Stefan Wirsel, Halle, Germany

Report on the secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola, with information on new genes, and macroarray and Real-time RT-PCR expression data.

Role of STE12-like transcription factors in the production of microbial effectors inducing plant immunity in Colletotrichum-Legumes interactions

Valérie Jaulneau, Joanne Wong Sak Hoi, Marc Cazaux, Christophe Jacquet, Bernard Dumas

UMR 5546 CNRS-Université Paul Sabatier, Pôle de Biotechnologie Végétale, 24, Chemin de Borde Rouge,BP42617  Auzeville, 31326 Castanet-Tolosan France

In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. Recently, we showed that a STE12-like factor, named CLSTE12, regulates production of extracellular proteins such as pectinases and cell-wall associated proteins in the bean pathogen Colletotrichum lindemuthianum (Wong Sak Hoi et al., 2007). In the present study, we have investigated the role of CLSTE12 in the production of fungal effectors which induce plant defence responses in the case of host and non-host interactions. This study was done by using the model legume Medicago truncatula, a non-host plant for C. lindemuthianum, but a host for the related Colletotrichum specie, C. trifolii. Two M. truncatula lines showing contrasted phenotypes towards C. trifolii infection were used: Jemalong is resistant towards C. trifolii, whereas F83005.5 is susceptible (Ameline-Torregrosa et al., 2008). A STE12-like gene was isolated from C. trifolii, named CTSTE12 (Colletotrichum trifolii STE12) and a disrupted mutant was obtained by homologous recombination. C. lindemuthianum and C. trifolii strains (wild-type strains and STE12-like mutants) were used to inoculate the two Medicago truncatula lines. Inoculation of M. truncatula lines with a wild-type C. lindemuthianum strain induced the development of a hypersensitive response (HR) characterized by a necrotic spot at the site of inoculation and the induction defence gene coding PR proteins and enzymes involved in the synthesis of phytoalexins. Inoculation with CLSTE12 mutants or with the wild type strain induced similar responses in both lines. C. trifolii strains disrupted for CTSTE12 were non-pathogenic on the susceptible lines confirming the role of STE12-like proteins in pathogenesis. Interestingly, these strains were still able to produce a HR on the resistant lines, and also on the susceptible line. Together, these results demonstrate that perception of adapted and non-adapted Colletotrichum species occurs before the appressorium-mediated penetration through a STE12-like independent mechanism, and that suppression of this response on the susceptible plant required a functional STE12-like gene.

Ameline-Torregrosa, C. et al. (2008) Mol Plant-Microbe Interact 21 (1), 61-69

Wong Sak Hoi, J.W. et al. (2007) Mol Microbiol 64 (1), 68-82

Identification and functional analysis of ClaKEL1 gene encoding kelch repeat protein in Colletotrichum lagenarium.

Ayumu Sakaguchi, Kyoto, Japan

C. lagenarium is the causal agent of cucumber anthracnose disease. The infection process involves a series of change in fungal morphology, including a specialized infection structure appressorium formation. We have revealed that Schizosaccharomyces pombe tea1 homolog ClaKEL2 gene encoding for a kelch motif protein was involved in appressorium morphogenesis in C. lagenarium. A database reference of the genome sequences of filamentous fungi revealed that these have a number of predicted kelch family proteins. However, the roles of these proteins are not well known.

We isolated novel kelch repeat encoding gene ClaKEL1 from C. lagenarium. ClaKEL1 was isolated as the tagged gene in a pathogenicity deficient mutant P24 generated by restriction enzyme-mediated integration. ClaKEL1 encodes a putative protein of 555 amino acids containing kelch repeat at the C teminal region. The deduced amino acid sequence of ClaKEL1 had high homology with the amino acid sequence of other filamentous fungal hypothetical proteins. To elucidate the cellular role of Clakel1p, GFP-ClaKEL1 fusion gene was constructed. The GFP-Clakel1p signal corresponded to mRFP1-aTUB1 signal localizing at cytosolic microtubule or spindle microtubule. This result suggested that Clakel1p is a microtubule-associated protein.

To define the function of ClaKEL1, we isolated clakel1 knock-out mutants by homologous recombination. The clakel1 mutant did not differ from the wild type in growth rate on PDA medium, but had reduced conidiation. Conidia from clakel1 mutants could germinate same as wild type on glass slide, but the approximately 50% of the appressoria produced by clakel1 mutants were irregular in form, showing elliptic shape, thick neck and lateral germination. On the host cucumber leaves, appressoria of clakel1 mutant formed very few infection hyphae. In the wild type, 27% of the appressoria formed infection hyphae into host surface, while only 3% of the clakel1 mutant appressoria formed infection hyphae. Accordingly, compared with lesions produced by the wild type, clakel1 mutants produced small lesions on intact host leaves. These results indicate that ClaKEL1 is involved in appressorium morphogenesis and pathogenicity.

Mining candidate effectors from the secretome of Colletotrichum higginsianum appressoria

Jochen Kleemann, MPIZ, Köln, Germany

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify developmentally-regulated proteins secreted by appressoria that may function as virulence effectors required for the establishment of the biotrophic interaction, a cDNA library was prepared from mature appressoria formed in vitro. ESTs derived from sequencing 980 clones were assembled into 518 unique sequences and searched for putative open reading frames ab initio. Biocomputational predictors, including SignalP, TMHMM and Phobius, were used to screen the predicted amino acid translations for N-terminal signal peptides and to differentiate between soluble secreted proteins and membrane proteins. Fifty-three unique sequences (10 %) were predicted to encode proteins entering the secretory pathway, of which 26 were likely soluble extracellular proteins, including hydrolases and oxidoreductases known to be secreted from other fungi. Seven genes encoding secreted proteins of unknown function, including two Colletotrichum-specific sequences, were expressed early during plant infection and strongly upregulated or specifically expressed in mature appressoria and as such represent candidate effectors. Work is in progress to determine whether these proteins are higginsianum-specific and to evaluate their role in pathogenicity by means of targeted gene replacement and transient over-expression of the proteins in host epidermal cells.

Identification of biotrophy-related genes in Colletotrichum higginsianum 

Hiroyuki Takahara, MPIZ, Köln, Germany

The hemibiotrophic ascomycete Colletotrichum higginsianum initially invades Arabidopsis plants by developing specialised intracellular hyphae (IH) within living epidermal cells. In order to identify pathogenicity genes related to biotrophy, we established a novel method to isolate the IH from infected Arabidopsis leaves based on flow cytometry. A stage-specific cDNA library generated from isolated IH included a large number of genes encoding putative pathogenicity factors and secreted proteins. We also found many genes which are related to amino acid and vitamin metabolism and amino acid transporters. These results suggest that Colletotrichum IH are functionally equivalent to the haustoria of obligate biotrophs.

Development of a high throughput system for large-scale gene discovery in Colletotrichum higginsianum ~Efficient gene targeting by Colletotrichum higginsianum strains deficient for Neurospora crassa mus-51 gene homolog

Hiroshi Terada, Kie Tsuboi, Ai Mori, Gento Tsuji, and Yasuyuki Kubo

Kyoto, Japan

Colletotrichum higginsianum is the causal agent of anthracnose disease of Brassicaceae plants. This fungus has become an excellent candidate for studying fungal-plant interactions because it is able to infect a model plant, Arabidopsis thaliana.  The aim of our study is to develop a high throughput system for large-scale gene discovery in C. higginsianum.  We previously developed Agrobacterium tumefaciens-mediated transformation (AtMT) as a method for random insertional mutagenesis and targeted gene disruption in C. lagenarium.  This method allowed us to systemically carry out the isolation of mutants, gene rescue, and subsequent confirmation of the tagged genes.  In C. higginsianum, we confirmed that AtMT is an effective method for random insertional mutagenesis.  This method successfully allowed us to obtain a large population of transformants in a short period of time.  However on the other hand, we faced a difficulty of targeted gene disruption by AtMT.  We estimated the frequency of homologous integration for two pathogenicity-related genes, ChPKS1 coding for polyketide synthase and ChSSD1 coding for a putative regulator for cell wall integrity.  Although AtMT provided high efficiency of transformation in C. higginsianum, homologous integration occurred only 2.4% for ChPKS1 and 0.6% for ChSSD1, respectively.  This result suggested that it would be impractical to isolate homologous recombinants efficiently in C. higginsianum.  Thus, we focused on the Neurospora crassa mus-51 gene encoding a protein that plays a role for non-homologous end-joining of DNA breaks.  It was reported that the disruption strains of the gene showed a dramatic increase of homologous integration efficiency.  We isolated an orthologue of the mus-51 gene from C. higginsianum, and named it as ChMUS51.  Disruption strains of ChMUS51 were successfully isolated and used as recipients for targeted gene disruption.  Frequency of homologous integration was dramatically increased up to 97.5% for ChPKS1 and 98.3% for ChSSD1, respectively.  Next, we investigated the relationship between the length of the homologous sequence and the frequency of homologous integration.  Longer than 1000 bp homology was sufficient for effective isolation of recombinants and the frequency was over 95%.  However, shorter than 250 bp homology was insufficient for practical isolation of recombinants and the frequency was less than 3%. The chmus51 mutants also showed no defect in hyphal growth, infectious morphogenesis, and pathogenicity.  Thus, we concluded that the chmus51 disruption strain would be an efficient recipient for targeted gene disruption.

Research on Colletotrichum at the University of Saskatchewan

S. Banniza, C. Armstrong-Cho, A. Cohen-Skali, G. Liu, J. Menat, B. Tar’an, A. Tullu, A. Vandenberg, S. Vail, J. Wang, Y. Wei

Dpts. of Plant Sciences and Biology, University of Saskatchewan, Saskatoon, Canada

A collaborative research group on Colletotrichum has been established at the University of Saskatchewan with the objective to gain a better understanding of host-pathogen interactions in this genus, and to exploit that knowledge in plant breeding and disease management.

Anthracnose of lentil (Lens culinaris) caused by C. truncatum is a major disease in lentil production in the Canadian Prairies. Two pathogenic races, Ct0 and Ct1, were identified in the population of Canadian C. truncatum (Buchwaldt et al., 2004: Phytopathology 94,236-243.), of which race Ct0 is highly aggressive and severely infects all germplasm of L. culinaris tested to date. Resistance to race Ct1 has been found in cultivated lentil and several potential resistance genes have been identified. Resistance to C. truncatum is not complete, but allows the disease to be managed without major economic loss in the field. A molecular marker for a dominant gene was identified and mapped to linkage group 6 in close proximity to a QTL for ascochyta blight resistance (Ascochyta lentis). Resistance to the more aggressive race was found in L. ervoides. Interspecific hybrids have been used to develop recombinant inbred lines that are currently used to study the genetics of resistance to these races. The ultimate objective is to identify the genes in the host plant conferring resistance to both races of C. truncatum.

Beyond pathogenicity, the two races of C. truncatum have not been well defined in terms of their interaction with the lentil host. We found that the infection process of both races appears to be very similar, clearly indicating that C. truncatum follows an intracellular hemibiotrophic infection strategy. Recent success in developing the teleomorph under controlled conditions allowed for a F₁ population of a cross between the two races to be developed. Ongoing pathogenicity testing of the progeny, marker identification and mapping of virulence are expected to shed some light on the genetic control of race identity. We are currently also performing 20,000 expression sequence tag (EST) analysis from cDNA libraries constructed from infected lentil leaves to further elucidate sequence variation and differences in gene expression between the two races.

In parallel, a Colletotrichum higginsianum-Arabidopsis pathosystem has been recently established in our research group. We are aiming at collecting a large set of Colletotrichum ESTs at the biotrophic stages of infection. Through extensive EST analysis and targeted-gene disruption, our research will focus on identification of Colletotrichum secretomes, effectors, and other potential virulence/pathogenicity factors. The unique hemibiotrophic parasitism of Colletotrichum on Arabidopsis, the best known host plant in defense and signaling, will facilitate to uncover additional resistance and defense signaling genes through pathogenicity screening on Arabidopsis T-DNA insertion populations from the TAIR.

Colletotrichum spp. associated with coffee berry like disease in Vietnam

¹Phuong Nguyen, ²Olga Vinnere Pettersson and ¹Erland Liljeroth

¹ Department of Plant Protection Biology, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53 Alnarp, Sweden

² Department of Evolution, Genomics and Systematics, Molecular Evolution, Uppsala University, Norbyv. 18C, 752 36 Uppsala, Sweden

Isolates of Colletotrichum associated with disease on coffee berries in Vietnam were identified and characterized morphologically and molecularly. Species-specific primers for amplification of the rDNA ITS1 (internal transcribed spacer) and the mtSSU (mitochondrial small subunit rRNA gene) regions were used for identification of C. gloeosporioides and C. acutatum. The genetic variation among isolates of C. gloeosporiodes was investigated with random amplified polymorphic DNA (RAPD)(38 isolates) and microsatellite-primered PCR (MpPCR)(52 isolates) markers. Cluster analysis showed that the isolates mainly grouped in accordance with geographical distributions. We found a higher genetic variation among isolates from the north compared to the south of the country and a moderate gene differentiation between populations from the north and the south. However, there was no differentiation among sublocations within the northern and the southern populations. A four gamete test indicated a high level of recombination, particularly in the south. The geographic differences may be explained by different histories of coffee cultivation in different parts of Vietnam.

Three isolates of Colletotrichum having falcate conidia, two isolates of C. acutaum and 34 isolates of C. gloeosporioies from Vietnam were further investigated by sequencing both the ITS and the mtSSU regions. Three African reference isolates of C. kahawae, causing coffee berry diseases (CBD), were also investigated as comparison. The results confirmed the identification of C. gloeosporioides and C. acutatum. The isolates with falcate conidia can be either C. dematium or C. capsici. All Vietnamese isolates, except two, could use tartrate as a sole carbon source. Two Vietnamese isolates and the CBD reference isolates could not utilize tartrate. However, according to the ITS1 sequence analysis and combination of ITS and mtSSU, the three reference CBD isolates fell separately from the clade of all Vietnamese C. gloeosporioides isolates. We also observed that the symptoms caused by the Vietnamese isolates on both hypocotyls and green berries were less severe than symptoms caused by the reference CBD isolates. This is indicative of the absence of C. kahwae on coffee in Vietnam. No significant correlation between pathogenicity and genotype was found among the Vietnamese isolates of C. gloeosporioides.

Genotypic and phenotypic diversity in Colletotrichum pathosystems

S. Sreenivasaprasad¹, P. Talhinhas^1,2, S. Muthumeenakshi¹, J. Neves-Martins² and H. Oliveira²

¹Warwick HRI, University of Warwick, UK; ISA, Lisbon, Portugal 

We are using a range of approaches including the use of the Colletotrichum higginsianum – Arabidopsis model system to understand the molecular basis of population diversity and varied pathogenicity lifestyles in C. acutatum. Colletotrichum species populations from Olive within a single geographic location comprised five molecular groups A2 – A6 of C. acutatum, along with sporadic occurrence of C. gloeosporioides. A spatio-temporal survey over a six year period enabled an understanding of the dynamics of these populations with reference to the level and patterns of olive cultivation and the incidence of anthracnose. Diagnostic PCR and histopathological observations were useful in understanding the epidemiology of the main olive anthracnose pathogen. On a global scale, C. acutatum populations from various hosts, split into a number of molecular groups with varying bio-geographic association patterns. Moreover, C. acutatum exhibits different pathogenic strategies on various hosts, but the components regulating these processes are poorly understood. We envisage the use of recently developed and emerging molecular genomic technologies to gain further insights into these aspects.

Colletotrichum vs Neurospora: what are the roles of conidial anastomosis tube fusion? 

M. Gabriela Roca and Nick D. Read

Institute of Cell Biology, Rutherford Building, University of Edinburgh, Edinburgh, EH9 3JH

Conidial anastomosis tubes (CATs) are specialized hyphae that were first described in Colleotrichum lindemuthianum. CAT fusion results in conidial germlings becoming interconnected but its functional roles are little understood. Nevertheless, CAT fusion is very common in different ascomycete species, and may have a significant impact on the ecology of these fungi in nature. We have compared CATs and CAT fusion in Colletotrichum and the model organism Neurospora crassa. Recent experimental results suggest that CAT fusion may play multiple roles.  First, it may improve the chances of colony establishment by allowing heterogeneously distributed nutrients and/or water to be shared between different germlings. Second, it may provide a stimulus for fused germlings to grow more rapidly than unfused germlings, and thus shorten the period of colony establishment.  Third, it may provide a mechanism for gene transfer in the absence of sexual reproduction. Evidence for CAT fusion having these different roles will be presented and discussed.

Venue: European Fungal Genetics meeting (ECFG9), David Hume Tower Conference Room