Investigation of strategies of adaptation to the pathogenic life style of fungi from the Moniliophthora genus at various levels of biological organizations: species, biotypes, and geographic lineages

Abstract

The invasion of the main cacao-producing region of Brazil by the basidiomycete Moniliophthora perniciosa (causal agent of the witches' broom disease) caused the collapse of cacao production, a fundamental and unique raw material for the chocolate industry. Moniliophthora perniciosa and M. roreri are the most limiting pathogens for cacao (Theobroma cacao) cultivation in the Americas. Both species belong to the Agaricales, which are mostly saprophytic; pathogenicity has evolved in a few species of the order. Likewise, the lack of other phytopathogens closely related to M. perniciosa and M. roreri suggests that the evolution of phytopathogenicy in this genus has evolved recent changes in genes associated with the control of life styles and stages (biotrophic and necrotrophic), among others directly associated with pathogenicity. While M. roreri only attacks cacao pods, M. perniciosa is capable of infecting shoots, pods and flower buds; M. perniciosa is also classified into biotypes, which are host-specific. This large variation in life habits associated with the large genetic variability makes these fungal taxa an excelent model to investigate genomic changes associated with the evolution of phytopathogenicity. Therefore, we intend to perform comparative genomic analysis involving Agaricales species to verify species- and biotype-specific genetic differences; to evaluate evolutionary and selective rates between orthologs from species/biotypes; pathogenic lineages ancestrality; patterns of expansion and retraction of gene families; and the function divergence among gene families for each lineage. The comparison of M. perniciosa isolates and biotypes will allow the search for associations between genome structure and life styles. The genetic system that controls sexual reproduction, defined by two non-linked loci A and B, and genes associated with vegetative compatibility, genetically controlled by the vic loci (vegetative incompatibility), will be investigated as they may define gene flow in M. perniciosa. Transcriptomic analysis will be conducted to investigate factors that can lead to the success of M. perniciosa and M. roreri as pathogens. The comparison between compatible and incompatible interactions of M. perniciosa with its hosts (e.g. S-biotype x tomato and S-biotype x cacao, respectively) may indicate which are the key-factors related with infection successs or failure. Comparing shoot and pod infection by M. perniciosa with pod infection by M. roreri may help to elucidate how pathogens adopt specific infection strategies, and which are the general virulence factors related with Moniliophthora virulence. Recent reports in other pathosystems sugest that, besides effector proteins, pathogens may use small RNAs (sRNA) to directly manipulate the plant defense system, therefore expression of sRNA during infection with M. perniciosa will be evaluated. Mutants and transgenic lines in the 'Micro-Tom' (MT) background with changes in synthesis or perception of hormones will enable investigating the role of this class of compounds in the pathogenesis and defense against infection by M. perniciosa isolates from the S- and C-biotype. Additionally, we intend to develop new tools of genetic manipulation of the pathogen and hosts to assist future functional studies of this pathosystem, and develop transgenic lines of cacao and MT. The genomic information generated, together with the development of populational studies and genetic manipulations, will be applied to direct functional analysis of the pathogen essential genes. (AU)