Mating type and infection-related gene analysis, sexual reproduction, microconidia, and conidial anastomosis tube events in Pyricularia oryzae

Abstract

Blast disease caused by Pyricularia oryzae is one of the main plant diseases in the world, affecting several species of grasses such as rice, wheat, triticale, barley, and rye. The genes that regulate sexual reproduction are called mating type genes. The Pmk1 MAPK, MPS1, and Osm1 MAPK pathways regulate appressorium formation, penetration, conidiation, invasive hyphal growth, and hyperosmotic stress. In this study, sexual reproduction using isolates of P. oryzae pathotypes Triticum and Oryzae, the sequences of the mating type genes and the expression of these genes under pairing conditions were evaluated. Knockout of the RGF1 and RGF2 genes of the P. oryzae isolate Ku80 was performed using the Split-Marker method. The interaction of RGF1 and RGF2 genes with the Pmk1, Mps1, and Osm1 MAPK pathways was evaluated by TEY (Pmk1 and Mps1) and TGY (Oms1) phosphorylation assays. Different concentrations of β-glucanase and Lysing enzymes were evaluated to obtain protoplasts of P. oryzae pathotype Triticum and transmission electron microscopy (TEM) was used to assess possible cellular damage in the protoplasts. The production of microconidia and the formation of conidia anastomosis tubes (CATs) of this pathogen were also evaluated. A fertile cross was obtained from the tester Guy11 isolate and wheat isolate 12.1.053i. Analysis of the mating type genes showed the presence of different haplotypes, and it was observed that some Single Nucleotide Polymorphisms (SNPs) may have led to alterations in the amino acid sequence and consequently in the predicted protein of MAT1-1-3b. A repeat region of cytosine and thymine (CT) dinucleotides located at the 5'-UTR of MAT1-1-3 showed that Triticum isolates have a smaller CT region, which may have influenced the expression of the MAT1-1-3 gene. The lack of MAT1-1-3 expression during the sexual cycle in the infertile pair also showed that this gene could play an important role in the fertility of the isolate. There was no change in the phosphorylation levels of Oms1 in the Δrgf1 and Δrgf2 mutants. Phosphorylation levels of Pmk1 and Mps1 were reduced in the Δrgf1 mutant and were not reduced in the Δrgf2 mutant, indicating that only RGF1 is involved in the activation of Pmk1 and Mps1 MAPK signaling. The Lysing enzyme compound was better for obtaining protoplasts and concentrations above 30 mg/mL affected their viability. Storage of protoplasts is not ideal at - 20°C even with the use of glycerol as a cryoprotectant. Microconidia were observed in oat agar medium and submerged culture using a 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.