Moniliophthora perniciosa development: key genes involved in stress-mediated cell wall organization and autophagy.

Moniliophthora perniciosa is a basidiomycete responsible for the witches' broom disease in cacao (Theobroma cacao L.). Chitin synthase (CHS), chitinase (CHIT) and autophagy (ATG) genes have been associated to stress response preceding the formation of basidiocarp. An analysis of literature mining, interactomics and gene expression was developed to identify the main proteins related to development, cell wall organization and autophagy in M. perniciosa. TORC2 complex elements were identified and were involved in the response to the nutrient starvation during the fungus development stages preceding the basidiocarp formation. This complex interacted with target proteins related to cell wall synthesis and to polarization and cell division (FKS1, CHS, CDC42, ROM2). Autolysis and autophagy processes were associated to CHIT2, ATG8 and to the TORC1 complex (TOR1 and KOG1), which is central in the upstream signalization of the stress response due to nutrient starvation and growth regulation. Other important elements that participate to steps preceding basidiocarp formation were also identified (KOG1, SSZ1, GDI1, FKS1, CCD10, CKS1, CDC42, RHO1, AVO1, BAG7). Similar gene expression patterns during fungus reproductive structure formation and when treated by rapamycin (a nutritional related-autophagy stress agent) were observed: cell division related-genes were repressed while those related to autolysis/autophagy were overexpressed.

A 16S rDNA PCR-based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information.

BACKGROUND: Subunits of ribosomal RNA genes (rDNAs) characterized by PCR-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology. However, relevant factors have shown to interfere in the experimental outputs in a variety of systems. In this work, a 'theoretical' to 'actual' delta approach was applied to data on culturable mock bacterial communities (MBCs) to study the levels of losses in operational taxonomic units (OTUs) detectability. Computational and lab-bench strategies based on 16S rDNA amplification by 799F and U1492R primers were employed, using a fingerprinting method with highly improved detectability of fragments as a case-study tool. MBCs were of two major types: in silico MBCs, assembled with database-retrieved sequences, and in vitro MBCs, with AluI digestions of PCR data generated from culturable endophytes isolated from cacao trees. RESULTS: Interfering factors for the 16 s rDNA amplifications, such as the type of template, direct and nested PCR, proportion of chloroplast DNA from a tropical plant source (Virola officinalis), and biased-amplification by the primers resulted in altered bacterial 16S rDNA amplification, both on MBCs and V. officinalis leaf-extracted DNA. For the theoretical data, the maximum number of fragments for in silico and in vitro cuts were not significantly different from each other. Primers' preferences for certain sequences were detected, depending on the MBCs' composition prior to PCR. The results indicated overall losses from 2.3 up to 8.2 times in the number of OTUs detected from actual AluI digestions of MBCs when compared to in silico and in vitro theoretical data. CONCLUSIONS: Due to all those effects, the final amplification profile of the bacterial community assembled was remarkably simplified when compared to the expected number of detectable fragments known to be present in the MBC. From these findings, the scope of hypotheses generation and conclusions from experiments based on PCR amplifications of bacterial communities was discussed.

Genome sequence and effectorome of Moniliophthora perniciosa and Moniliophthora roreri subpopulations.

BACKGROUND: The hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity. RESULTS: Candidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection. CONCLUSIONS: The present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity.