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

PoGO: Prediction of Gene Ontology terms for fungal proteins

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

Identification and Comparison of Colletotrichum Secreted Effector Candidates Reveal Two Independent Lineages Pathogenic to Soybean

Colletotrichum is one of the most important plant pathogenic genus of fungi due to its scientific and economic impact. A wide range of hosts can be infected by Colletotrichum spp., which causes losses in crops of major importance worldwide, such as soybean. Soybean anthracnose is mainly caused by C. truncatum, but other species have been identified at an increasing rate during the last decade, becoming one of the most important limiting factors to soybean production in several regions. To gain a better understanding of the evolutionary origin of soybean anthracnose, we compared the repertoire of effector candidates of four Colletotrichum species pathogenic to soybean and eight species not pathogenic. Our results show that the four species infecting soybean belong to two lineages and do not share any effector candidates. These results strongly suggest that two Colletotrichum lineages have acquired the capability to infect soybean independently. This study also provides, for each lineage, a set of candidate effectors encoding genes that may have important roles in pathogenicity towards soybean offering a new resource useful for further research on soybean anthracnose management

Quantitative Detection of Double-Stranded RNA-Mediated Gene Silencing of Parasitism Genes in Heterodera glycines

The introduction of a double-stranded RNA (dsRNA) into an organism to induce sequence-specific RNA interference (RNAi) of a target transcript has become a powerful technique to investigate gene function in nematodes and many organisms. Data provided here indicate that the inclusion of 1–2 mM spermidine and 50 mM octopamine and a 24 hr incubation period of nematodes in double-stranded RNA (dsRNA) soaking solutions resulted in a considerable increase in the percentage of nematodes that ingested dsRNA as compared to previous reports. This modified dsRNA soaking method was coupled with quantitative real-time RT-PCR (qRT-PCR) analyses to assess the potential silencing of the Heterodera glycines parasitism gene transcripts Hg-pel-1 and Hg-4E02 that are expressed within the esophageal gland cells of preparasitic H. glycines J2. The Hg-pel-1 transcript was most efficiently silenced with one dsRNA construct (ds267) at the highest dsRNA soaking concentration of 5.0 mg/ml, while the Hg-4E02 transcript was more efficiently silenced at the 2.5 mg/ml dsRNA concentration as compared to 5.0 mg/ml. A dsRNA construct (ds285) complementary to a different sequence within the Hg-pel-1 transcript than construct ds267 induced only minimal silencing of the Hg-pel-1 transcript at 2.5 mg/ml. The results suggest that both dsRNA concentration and sequence relative to the transcript targeted are critical for maximizing potential RNAi effects in parasitic nematodes

Comparative Transcriptomic Provides Novel Insights into the Soybean Response to Colletotrichum truncatum infection

Soybean (Glycine max) is among the most important crops in the world and its production can be threatened by biotic diseases, such as anthracnose. Soybean anthracnose is a seed-borne disease mainly caused by the hemibiotrophic fungus Colletotrichum truncatum. Typical symptoms are pre- and post-emergence damping-off, necrotic lesions on cotyledons, petioles, leaves, and pods. Anthracnose symptoms can appear early in the field, causing major losses to soybean production. In preliminary experiments, we observed that the same soybean cultivar can have a range of susceptibility towards different strains of C. truncatum, while the same C. truncatum strain can cause varying levels of disease severity in different soybean cultivars. To gain a better understanding of the molecular mechanisms regulating the early response of different soybean cultivars to different C. truncatum strains, we performed pathogenicity assays to select two soybean cultivars with significantly different susceptibility to two different C. truncatum strains and analyzed their transcriptome profiles at different time-points of interaction (0, 12, 48, and 120 hours post-inoculation - hpi). The pathogenicity assays showed that the soybean cultivar Gm1 is more resistant to C. truncatum strain 1080, and it is highly susceptible to strain 1059, while cultivar Gm2 shows the opposite behavior. However, if only trivial anthracnose symptoms appeared in the More Resistant Phenotype (MRP; Gm1-1080; Gm2-1059) upon 120 hpi, in the More Susceptible Phenotype (MSP; Gm-1059; Gm2- 1080) plants show mild symptoms already at 72 hpi, after which disease evolved rapidly to severe necrosis and plant death. Interestingly, several genes related to different cellular responses of the plant immune system (pathogen recognition, signaling events, transcriptional reprogramming, and defense-related genes) were commonly modulated at the same time-points only in both MRP. The list of differentially expressed genes (DEGs) specific to the more resistant combinations and related to different cellular responses of the plant immune system may shed light on the important host defense pathways against soybean anthracnose

Nitric oxide synthase-like protein in pea (Pisum sativum L.)

Nitric oxide synthase activity was detected in pea (Pisum sativum L.) leaf extracts using a citrulline formation assay that is typically employed in mammalian systems. A total protein extraction method was modified from that used in mammalian systems based on biochemical activities such as the use of protease inhibitors, pH, and precipitation with salts and organic solvents. Physiological aspects in plants, such as effects of chemicals that induce systemic resistance to NOS activity and immunodetection of an NOS-like protein, were also studied. The NOS-like protein was partially isolated using liquid chromatography and characterized based on mammalian NOS inhibitor and cofactor requirements. Correlation of NOS activity and NOS-like gene expression during incompatible and compatible pea–bacteria interactions were investigated using interactions of Ralstonia solanacearum and Pseudomonas syringae pv. pisi, respectively, with pea. NOS activity was detected using citrulline formation assay. Gene expression was measured using real-time reverse transcription-polymerase chain reactions and a 348-bp probe designed from a cloned cDNA fragment of pea that was homologous to NOS of snail and AtNOS1/AtNOA1 of Arabidopsis. The possibility of NO production from various sources in cells of pea is also discussed

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

The FTF gene family regulates virulence and expression of SIX effectors in Fusarium oxysporum

[EN] The FTF (Fusarium transcription factor) gene family comprises a single copy gene, FTF2, which is present in all the filamentous ascomycetes analysed, and several copies of a close relative, FTF1, which is exclusive to Fusarium oxysporum. An RNAmediated gene silencing system was developed to target mRNA produced by all the FTF genes, and tested in two formae speciales: F. oxysporum f. sp. phaseoli (whose host is common bean) and F. oxysporum f. sp. lycopersici (whose host is tomato). Quantification of the mRNA levels showed knockdown of FTF1 and FTF2 in randomly isolated transformants of both formae speciales. The attenuation of FTF expression resulted in a marked reduction in virulence, a reduced expression of several SIX (Secreted In Xylem) genes, the best studied family of effectors in F. oxysporum, and lower levels of SGE1 (Six Gene Expression 1) mRNA, the presumptive regulator of SIX expression. Moreover, the knockdown mutants showed a pattern of colonization of the host plant similar to that displayed by strains devoid of FTF1 copies (weakly virulent strains). Gene knockout of FTF2 also resulted in a reduction in virulence, but to a lesser extent. These results demonstrate the role of the FTF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum and suggest that the control of effector expression is the mechanism involved

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