Transcription factors that regulate development and light responses in neurospora crassa and aspergillus nidulans

Resumen

In this thesis we studied different transcription factors that regulate light and/or developmental responses in the ascomycetes Neurospora crassa and Aspergillus nidulans. The Chapter 1 described a possible mechanism to explain the defect in photoadaptation observed by mutations in RCO-1 or RCM-1 proteins. The WCC binding kinetics to light-regulated promoters is affected in a ¿rco-1 mutant. This fact, together a reduced expression of VVD is responsible for the altered light response of RCO-1/RCM-1-deficient strains. Light regulates the balance between asexual or sexual development in A. nidulans (Rodriguez-Romero et al., 2010), and it has been shown that light increases the accumulation of brlA mRNA (Mooney and Yager, 1990). In Chapter 2 we characterized the transient light-activation of brlA, including the two transcript brlA¿ and brlAß. Moreover, the transcription of other genes upstream of brlA were demonstrated to be also activated by light This light-dependent transcription requieres the photorreceptor complex FphA/LreA/LreB/VeA. Strains with deletions in either flbA, flbB or flbC do not present light-dependent brlA mRNA accumulation suggesting that the light-dependent activation of brlA occurs through the induction of these genes. Several environmental cues, including blue light, promote a developmental transition in the fungus Neurospora crassa that leads to the development of spores (conidia). Conidiation is controlled by FLUFFY (FL), a transcription factor with a binuclear zinc cluster domain. Blue light activates the transcription of fl through the transient binding of the WC complex to the fl promoter. The WC complex is a light-dependent transcription factor complex that is required for all the responses of Neurospora to light. Light activates the transcription of several conidiation genes in the fungus Aspergillus nidulans, and their Neurospora homologs have been identified in the Neurospora genome. We have assayed the activation by light of the Neurospora homologs of A. nidulans conidiation genes (flbA, flbC, flbD, stuA and medA), and the Neurospora conidiation gene con-10 as a control. Unlike con-10, none of the Neurospora homologs of the A. nidulans conidiation genes were induced by light in vegetative mycelia. However, we found that deletion of the regulatory gene fl resulted in light-dependent mRNA accumulation for all the conidiation genes. This result indicated that the absence of FL allows the binding of the WC complex to the promoter of these genes to activate transcription in a lightdependent manner. We have assayed the amount of WC proteins in the ¿fl and wild type strains but we did not find any difference between the two strains suggesting that FL and the WCC may compete for binding sites in close proximity in the promoter of these genes. We expect to identify additional genes deregulated by the absence of FL after massive sequencing of total RNA experiments (RNAseq) using a ¿fl mutant and wild-type strain in dark and light conditions. We have investigated the role of FL during conidiation in Neurospora using a tagged version of FL (FL::3XFLAG). We have found that FL is present in vegetative mycelia but the amount increses after blue-light exposure. In addition we have found similar amounts of FL during the vegetative and conidiation stages. In experiments with protein synthesis inhibitors we have found that FL is a very stable protein. Our results show that the regulatory role of FL cannot be exherted through the synthesis of the protein. We have observed several forms of FL due to phosphorylation, and and we have determined by mass spectrometry that FL::3XFLAG is phosphorylated in several residues. We have immunoprecipitated FL::3XFLAG in order to identify by mass spectrometry posible proteins interacting with FL. We have found that an hypothetical protein related with the ability to grow in the presence of trehalose in other organisms interacts with FL. The interaction has been tested in several conditions and seems to be very stable. Since FL is a transcription factor, we have use FL::3XFLAG strain to do ChIPseq (Chromatin Immunoprecipitation Sequencing) in order to identify the putative binding sites of FL to the DNA. We expect that the results from these experiments will help us to understand in more detail the role of FL in the activation of gene transcription during asexual development in Neurospora crassa. Conidiophore development in Neurospora crassa requires several genes and their genetic interactions have been established. Strains with mutations in aconidiate-2 (acon-2) or fluffyoid (fld) are blocked in the transition from filamentous to budding growth. Mutations in aconidiate-3 (acon-3) or fluffy (fl) allow the production of minor, but not the major constriction chains that are produced prior to the formation of conidia. Mutations in two conidial separation genes (csp-1 and csp-2) prevent the separation of cross walls to release free conidia. A strain with a mutation in gene fld is blocked at the formation of minor constrictions like acon-2 mutants but can produce some flecks of conidia under certain conditions of temperature or carbon starvation. The fld mutation was isolated spontaneously and was mapped by classical methods on chromosome IV between arg-14 and his-5. We looked for genes in this genomic region that could be responsible for the fld phenotype. One of them, locus NCU09739, had a point mutation in the fld strain: a deletion of a G nucleotide in position 1686 that lead to a change in the reading frame and a premature STOP codon. The resultant protein lacks 112 residues at C-terminus in fld mutant strain. In addition, we have found that the mutation in NCU09739 is inherited by fld strains after genetic crosses further supporting the proposal that fld is NCU09739. To further confirm that fld is NCU09739 we created a strain with a deletion of G1686 that showed the fld phenotype. The FLD protein is a putative zinc finger protein of 676 amino acids that contains a binuclear zinc finger cluster (Zn2Cys6) at N-terminus. The gene fld is not induced by light, but it is missregulated in the absence of fl suggesting an interaction between FLD and FL. FLD localizes in the nucleus and in the cytoplasm during vegetative growth in the wild type strain, and accumulates in aerial hyphae during conidiation. The localization of FLD during vegetative growth and during conidiation in mutants altered in conidiation will help us to understand the role of this transcription factor during condial development.