Functional genetics in ascomycetes pseudocercospora fijiensis (synonym mycosphaerella fijiensis) the pathogen of black sigatoka disease in banana
Pseudocercospora fijiensis (P. fijiensis), causal agent of the black Sigatoka disease (BSD) on banana, has spread globally since its discovery in Fiji 1963 in all the banana and plantain growing areas across the globe. It is an economically important disease in this crop. Several control measures including use of chemicals have been used against the disease. However, these control measures have not been effective. There is an indication that P. fijiensis is undergoing evolutionary changes by moving from low land warm climate to high land cool climate. This may furthermore complicate control of BSD. Mitogen Activated Protein Kinase (MAPK) signal transduction pathways are involved in wide dimension of cell regulation. Currently, there is lack of information for MAP kinase signalling pathways and their related genes which regulate fungal growth, pathogenicity or other phenotypes in ascomycete P. fijiensis. This is one of the major challenges affecting the development of management strategies for BSD. The research presented in this thesis provides novel insights into the identification and characterisation of MAPK genes in P. fijiensis, RNA interference mediated gene silencing in an ascomycetes P. fijiensis and Agrobacterium tumefaciens mediated transformation system. To identify genes regulating virulence in P. fijiensis, MAPK Hog1, Slt2 and Fus3 genes were isolated from P. fijiensis genomic DNA by PCR amplification and cloned in pGEM-T easy vector for Sanger sequencing. In this study, the in silico characterisation, the PfHog1, PfSlt2 and PfFus3 sequences were confirmed to be MAP kinase encoding genes belonging to Serine/Threonine catalytic domain. The functional analysis confirmed that the PfHog1 is important in hyper osmotic stress regulation, PfSlt2 involved in cell wall integrity, cell shape maintenance, and PfFus3 regulates cell growth. Protein structure model prediction confirmed these genes are >50% identical to MAPK structure of the Saccharomyces cerevisiae. Evolutionary analysis confirmed PfHog1, PfSlt2 and PfFus3 sequences are > 90% identical to their respective MAPK Hog1, Slt2 and Fus3 of strictly fungal pathogens. These findings are in line with the research question of virulence in P. fijiensis is regulated by MAP Kinase encoding genes. Genetic transformation procedure was developed for P. fijiensis Agrobacterium tumefaciens mediated transformation (ATMT) was done by co- cultivation of AGL1 strain and mycelia fragments. The ATMT of P. fijiensis using mycelia proved to be most suitable alternative method in functional genetics, for virulence assay and easily available for phenotype studies. This research demonstrated a novel RNAi-mediated gene silencing method in P. fijiensis through ATMT of mycelia fragments. The silencing vector pKOIISD1 revealed low expression of the target genes in silenced strains by >90% as compare to wild type P. fijiensis. This study confirmed that the research hypothesis of Agrobacterium tumefaciens mediated transformation is an alternative method of introducing transgenes in P. fijiensis mycelium fragments is true. To investigate the role of MAPK PfHog1 on osmotic stress adaptation, cultures of both wild type P. fijiensis and PfHog1 mutant strains were cultured on potato dextrose agar media supplemented with 1 M NaCl. PfHog1 mutant strains showed significant suppressed growth. Furthermore, the role of PfHog1, PfSlt2 and PfFus3 to virulence in P. fijiensis were determined by inoculating silenced strains on susceptible banana cultivar "Nakitembe". The silenced PfHog1, PfSlt2 and PfFus3/Kss1 strains showed significant reduced virulence characterised by low necrosis. Staining infected leaf tissues with lacto phenol cotton blue further confirmed the impaired penetration and mycelia growth of the silenced PfHog1 strains. In the PfSlt2 and PfFus3/Kss1 silenced strains, there was impaired infectious, invasive growth. Reduced virulence was further confirmed with low fungal biomass recovery from silenced PfHog1, PfSlt2 and PfFus3/Kss1 strains through quantification of the fungal biomass using absolute quantitative PCR. Collectively, these findings demonstrate that PfHog1 is critical for both osmotic stress regulation and virulence of P. fijiensis on its host banana. Meanwhile PfSlt2 and PfFus3 are important in plant infection and pathogenic growth of fungal pathogens. These results support the hypothesis of silencing MAP kinase encoding genes interfere with growth, development and virulence of P. fijiensis. Thus, PfHog1 PfSlt2 and PfFus3 could be interesting targets for the control of black Sigatoka disease in banana.