The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L.

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.

The repertoire of effector candidates in Colletotrichum lindemuthianum reveals important information about Colletotrichum genus lifestyle.

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean (Phaseolus vulgaris), and anthracnose is one of the most devastating diseases of this plant species. However, little is known about the proteins that are essential for the fungus-plant interactions. Knowledge of the fungus' arsenal of effector proteins is of great importance for understanding this pathosystem. In this work, we analyzed for the first time the arsenal of Colletotrichum lindemuthianum effector candidates (ClECs) and compared them with effector proteins from other species of the genus Colletotrichum, providing a valuable resource for studying the infection mechanisms of these pathogens in their hosts. Isolates of two physiological races (83.501 and 89 A2 2-3) of C. lindemuthianum were used to predict 353 and 349 ClECs, respectively. Of these ClECs, 63% were found to be rich in cysteine, have repetitive sequences of amino acids, and/or possess nuclear localization sequences. Several conserved domains were found between the ClECs. We also applied the effector prediction to nine species in the genus Colletotrichum, and the results ranged from 247 predicted effectors in Colletotrichum graminicola to 446 in Colletotrichum orbiculare. Twelve conserved domains were predicted in the effector candidates of all analyzed species of Colletotrichum. An expression analysis of the eight genes encoding the effector candidates in C. lindemuthianum revealed their induction during the biotrophic phase of the fungus on the bean.

Comparative analysis of the mitochondrial genome of the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose in common beans.

Fungi of the genus Colletotrichum are economically important and are used as models in plant-pathogen interaction studies. In this study, the complete mitochondrial genomes of two Colletotrichum lindemuthianum isolates were sequenced and compared with the mitochondrial genomes of seven species of Colletotrichum. The mitochondrial genome of C. lindemuthianum is a typical circular molecule 37,446 bp (isolate 89 A2 2-3) and 37,440 bp (isolate 83.501) in length. The difference of six nucleotides between the two genomes is the result of a deletion in the ribosomal protein S3 (rps3) gene in the 83.501 isolate. In addition, substitution of adenine for guanine within the rps3 gene in the mitochondrial genome of the 83.501 isolate was observed. Compared to the previously sequenced C. lindemuthianum mitochondrial genome, an exon no annotated in the cytochrome c oxidase I (cox1) gene and a non-conserved open reading frame (ncORF) were observed. The size of the mitochondrial genomes of the seven species of Colletotrichum was highly variable, being attributed mainly to the ncORF, ranging from one to 10 and also from introns ranging from one to 11 and which encode a total of up to nine homing endonucleases. This paper reports for the first time by means of transcriptome that then ncORFs are transcribed in Colletotrichum spp. Phylogeny data revealed that core mitochondrial genes could be used as an alternative in phylogenetic relationship studies in Colletotrichum spp. This work contributes to the genetic and biological knowledge of Colletotrichum spp., which is of great economic and scientific importance.

Draft Genome Sequences of Two Isolates of Colletotrichum lindemuthianum, the Causal Agent of Anthracnose in Common Beans.

Colletotrichum lindemuthianum is the causal agent of anthracnose in common beans, one of the main limiting factors of their culture. Here, we report for the first time, to our knowledge, a draft of the complete genome sequences of two isolates belonging to 83.501 and 89 A2 2-3 of C. lindemutuianum.

Use of the IRAP marker to study genetic variability in Pseudocercospora fijiensis populations.

Pseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.