Banca de DEFESA: DÉRICA GONÇALVES TAVARES

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
DISCENTE: DÉRICA GONÇALVES TAVARES
DATA: 30/09/2021
HORA: 14:00
LOCAL: Google meet - https://meet.google.com/szt-kmtr-mev
TÍTULO:

Analysis of mating type genes in sexual reproduction, microconidia and conidial anastomosis tube (CAT) events, and study of infection-related genes in Pyricularia oryzae

PALAVRAS-CHAVES:

Wheat blast, Crossing, Gene expression, Gene knockout, Genetic transformation

PÁGINAS: 100
GRANDE ÁREA: Ciências Agrárias
ÁREA: Agronomia
SUBÁREA: Fitossanidade
ESPECIALIDADE: Microbiologia Agrícola
RESUMO:

Blast disease caused by Pyricularia oryzae affecs several species of grasses like rice, wheat, triticale, barley, and rye and is one of the main plant diseases in the world. Studies of genes related to sexual cycle and infection pathways can help to elucidate potential sources of genetic variability in this pathogen. In this work, we performed sexual reproduction using different P. oryzae isolates, structural analysis of MAT1-1 and MAT1-2 idiomorph sequences, and expression of mating type genes under crossing conditions. Low fertility was observed in wheat P. oryzae isolates. However, fertile cross was obtained from Guy11 tester and 12.1.053i wheat isolate. Analysis of mating type genes showed presence of different haplotypes separating isolates of wheat and rice into different groups. MAT1-1 idiomorph and MAT1-1-1 gene showed higher haplotypic diversity. Single nucleotide polymorphism (SNP) may have led to alterations in the amino acid sequence and consequently in the predicted protein of MAT1-1-3b gene. A region of cytosine and thymine (CT) dinucleotides located at the 5'-UTR of MAT1-1-3 gene showed that sequences of wheat isolates have a smaller CT region, which may have influenced the expression of MAT1-1-3 gene. Lack of MAT1-1-3 gene expression during sexual cycle in infertile isolates also showed that this gene can play an important role in the fertility of P. oryzae. Several signaling pathways are related to pathogenicity in fungi. Pmk1, Mps1, and Osm1 MAPK pathways regulate appressorium formation, penetration, conidiation, invasive hyphae growth, and hyperosmotic stress. RGF1 and RGF2 genes appear to function upstream to these pathways and may play roles in their regulation. The objective of this work was to perform the knockout of RGF1 and RGF2 genes in P. oryzae Ku80 isolate and to evaluate the interaction of these genes with the Pmk1, Mps1, and Osm1 MAPK pathways. Mutants were obtained by the Split-Marker method and TEY (Pmk1 and Mps1) and TGY (Oms1) phosphorylation assays were performed using proteins extracted from the mutants. There was no change in phosphorylation levels in Oms1 in ∆rgf1 and ∆rgf2 mutants. Phosphorylation levels of Pmk1 and Mps1 were reduced in ∆rgf1 and were not reduced in ∆rgf2 indicating that only RGF1 is involved in the activation of Pmk1 and Mps1 MAPK signaling. Protoplast-mediated genetic transformation is widely used in filamentous fungi and the enzymatic compound is an important factor in this process. We evaluated the use of different concentrations of the enzymatic compounds β-Glucanase from Trichoderma longibrachiatum (Sigma) and Lysing Enzymes from Trichoderma harzianum (Sigma) to obtain protoplasts of P. oryze pathotype Triticum. Enzyme compounds were tested at concentrations of 30 and 60 mg/mL. For the best enzyme compound the concentrations of 5, 10, 20, 30, 60, and 90 mg/mL were tested, and transmission electron microscopy (TEM) was used to assess cellular changes in protoplasts at concentrations of 60 and 90 mg/mL. Storage of protoplast at -20 °C using glycerol as cryoprotectant was also evaluated. The Lysing Enzyme compound was better for protoplast release and concentrations above 30 mg/mL affect its viability. Storage of protoplasts is not ideal at -20 ºC even with the use of cryoprotectant, which seems to have caused greater osmotic stress on the cells. In this work we also evaluated production of microconidia and formation of conidia anastomosis tubes (CATs) in P. oryzae pathotype Triticum. Microconidia were observed on oatmeal agar and can also be produced in submerged culture using complete medium. Few CAT events were observed due to the high germination rate of P. oryzae conidia. However, this is the first report of CAT events in Pyricularia and such events could be used in studies related to the parasexuality of this fungus.

MEMBROS DA BANCA:
Externo à Instituição - SILVINO INTRA MOREIRA - UFLA (Suplente)
Externo à Instituição - NATÁLIA CHAGAS FREITAS - EMBRAPA (Suplente)
Interno - LUDWIG HEINRICH PFENNING (Membro)
Externo à Instituição - FABIANO JOSÉ PERINA - UFLA (Membro)
Interno - EUSTAQUIO SOUZA DIAS (Membro)
Externo à Instituição - EDUARDO SEITI GOMIDE MIZUBUTI - UFV (Membro)
Presidente - EDUARDO ALVES (Membro)