Functional genetics and genomics of the banana black Sigatoka pathogen Pseudocercospora fijiensis

The hemibiotroph Pseudocercospora fijiensis is the causal agent of the black Sigatoka disease on bananas; present worldwide being the reason of large economical losses and subject to chemical control as the best control method nowadays. Nonetheless, this fungus has shown being able to become resistant to different chemical fungicides and thus a successful global threat to bananas. The biology of this fungus is largely unknown due to the missing –omics tools applied to this pathosystem. The only manner to counteract this fungus is to understand its biology of survival and pathogenicity. Chapter 1. Is the introduction to the fungus, it constitutes an update to the knowledge of its biology and epidemiology. It demonstrates the importance of generating more data and tools to understand this powerful pathogen. Chapter 2. Presents the P. fijiensis genome sequence, the analysis of its main characteristics compared with other close fungi. A new genetic map is included counting putative core and dispensable scaffolds, whose high polymorphism was observed by electrophoretic karyotyping within isolates from the same field population. It is shown the massive genome expansion mainly due to repetitive DNA, particularly by LTR-retrotransposons, and how this can affect close sequences. The effect of RIP on the genome is analyzed and compared to that on other closely related fungi. The location close to repetitive sequence from the effector Pfavr4 was discussed under the scope of its epidemiology and the protein employed helped to elucidate the first putative resistant cognate gene in the resistant banana cultivar Calcutta 4. Further analysis of strains originated from populations with and without fungicide selection pressure provided estimates of dispersal of strains and genetic flow that will help to predict spatial patterns of fungicide evolution under different management strategies. Chapter 3. Describes the protocol for Agrobacterium-mediated transformation of P. fijiensis for both random and targeted mutagenesis. This method was successfully applied to the gene Pfavr4 and the Pfku70. The former is the first effector described in P. fijiensis, and the latter is the gene codifier of the KU70 protein, the main point of the non-homologous end joining (NHEJ) pathway that has been related to an increase on homologous recombination in several fungi and other eukaryotes, providing important tools for further use. This method was additionally employed to swap the Pfcyp51 gene promoter that helped to decipher biology of azole resistance in P. fijiensis, as described in chapter 4. Chapter 4. The appearance of resistant strains to azoles has been mostly correlated with non- synonymous point mutations in the coding sequence Pfcyp51 gene. In this chapter, we identified a 19 base pairs (bp) repeat element in the promoter region of this gene, by a simple PCR analysis, showed that copy number correlates positively with increased resistance to azoles, as well as the exposure to azole fungicides. We swapped the promoter of a resistant strain into a susceptible strain, and thus demonstrated that presence of the repeat element proportionally upregulates Pfcyp51 expression as well as tolerance to azoles. Besides the knowledge on genetic mechanism for azole resistance in P. fijiensis, the present study might offer another tool for optimizing the use of azoles in the control of black Sigatoka. Chapter 5. A general discussion of the results obtained in this thesis is offered with a broader point of view. Implications in ecology, pathology and further expectations on the control of this fungus, together with insights on trending topics of molecular tools for future research are included.