| Magnaporthe oryzae is a major cereal pathogen which spreads rice blast disease via conidia, causing severe yield losses on rice and other plants. The regulation factor X (RFX) family transcription factors are highly conserved proteins, which contain 76 amino acid highly-conserved site-specific winged-helix DNA binding domain, in eukaryotic organisms ranging from yeasts to humans, with diverse functions among different species. Here, we show that the deletion of MoRFX1 in M. oryzae resulted in pleiotropic mutant phenotypes including reduced colony growth and conidiation, reduced conidial germination, delayed appressorium formation, lower appressorium turgor, weakened penetration ability, impaired virulence, and increased sensitivity to DNA damage (UV) and defects in maintaining genome integrity.On the morphology of spores, ΔMorfx1 produced one and two-celled conidia, but did not generate normal three-celled conidia. ΔMorfx1 differentiated fewer conidiophores in the aerial mycelia and produced fewer conidia on a conidiophore. ΔMorfx1 have longer and wider conidia than the wide type. On the nucleus and septum formation in hyphae and conidia, the nucleus and septum in conidia and hyphae appeared similar in the ΔMorfx1 and the wild-type strains. Every conidial and hyphal cell had one nucleus with a normal shape, however, a few micronuclei were visible in some of the AMorfxl hyphal cells, no such micronuclei were observed in wild-type cells. The vegetative hyphal cell length and width in ΔMorfx1 is bigger than the wide type. The inhibition ratio of the germination rate of ΔMorfx1 conidia exposed to UV light, H2O2, HU, MMS and CDDP was significantly increased as compared with the wide type, but the situation was opposite when exposed to bleomycin. The inhibition rates of growth of ΔMorfx1 were increased when exposed to MMS and HU, but reduced under bleomycin. When exposed to H2O2, the inhibition rates of growth of ΔMorfx1 were reduced, but the inhibition rates of aerial hyphae differentiation were increased. MoRFXl also plays a role in chitin metabolism, ΔMorfx1 had increased chitin content in the vegetative mycelia, but had no obvious change in the chitin content of the sporogenous mycelia. When exposed to CFW, CR and SDS, the conidial germination ofΔMorfx1 was inhibited more severely than that of the wide type. When exposed to CFW and CR, the inhibition rate of the mycelial growth of ΔMorfx1 was bigger than that of the wide type, whereas the inhibition rate of the mycelial growth of AMorfxl was reduced under SDS. Moreover, CFW, CR and SDS can interfere with the formation and stress response of the cell wall. As in conidia of the wild-type strain, the entire cellular content of ΔMorfx1 conidia was transferred into appressoria, the autophagic bodies in vacuoles occurred in bothΔMorfx1 and the wild-type.RNA-seq analysis of the wild-type and ΔMorfx1 strains in vegetative mycelia upon starvation revealed that 867 or 868 genes were up-regulated or down-regulated respectively, suggesting MoRFX1 had bipartite functions in regulating gene expression, not only as a transcriptional repressor, but also as a transcriptional activator in M. oryzae. Real-Time PCR results confirmed that the deletion of MoRFX1 affected the expression patterns of factors required for maintenance of genome stability including kinesin, kinetochore and DNA repair genes. MoRFX1 also altered the expression of chitin metabolism-related genes. In particular, the expression of chitin deacetylase gene MoCDA2, which was expressed abundantly in the wild-type, was nearly blocked in ΔMorfx1. Taken together, our results indicate that MoRFX1 is involved in the regulation of mycelial growth, conidial differentiation, and pathogenicity by modulating genome integrity and chitin metabolism during cell division. |
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