Abstract
AbstractIsw2 is known to bind DNA, using a Myb-type DNA binding domain and with another part of the protein interact with histones in the closest nucleosome to pull the two closest nucleosomes closer to the binding site using ATP. These bindings hinder these nucleosomes’ sliding movements and affect histone tail modification and DNA methylation but give more space for sliding and access for transcription factors further away from the Isw2 DNA binding sites. In a previous paper, we have knocked out the Magnaporthe oryzae Isw2 protein and complemented it with a GFP-labelled construct. We found that the MoISW2 is needed, especially in the later necrotrophic stages of infection when the plant starts defending itself.Using published transcriptomic data, we have now found that MoISW2 is mainly co-regulated with Histone 4 (MoHIS4), which likely indicates that MoIsw2 predominately interacts with MoHis4. We performed a ChIP-seq experiment using MoIsw2-GFP to map the binding sites and investigated if MoIsw2 regulates genes by the limited sliding type mechanism and if this regulation could be involved in pathogenicity regulation. Common motifs were found in the binding DNA sequences using MEME. Interestingly, these motifs were mainly intergenic, and a dominant palindrome motif was present in 200 sequences.We further performed an RNAseq for ΔMoisw2 compared with the Ku80 background strain and used genes sorted in physical order on the supercontigs to determine the effect of the mutation on the regulation of the genes physically close to the MoIsw2 binding sites. The genes close to the binding site were more absolutely regulated in the ΔMoisw2 mutant as expected due to the limited sliding mechaninsm. As nucleosomes are evenly distributed at roughly 200bp distance, there should be an interference pattern of regulation of genes centered around the MoIsw2 DNA binding site if the genes are roughly evenly distributed now given more space to slide due to this mechnism, which could be confirmed. We queried the NCBI database again and checked where the about 200 palindromic sequences were found in the genome to investigate if MoIsw2 binds to any known structures in the DNA. The palindromic sequences were found in conserved retrotransposon elements that generally contain palindromes. Since these retrotransposon elements can move place, they can create new regulatory variations with MoIsw2. Many avirulence genes recognized by different host plant cultivars and typically vary in expression between pathogen isolates were close to the MoIsw2 DNA binding site. That could mean that mobile elements associated with some MoIsw2 DNA binding sites are involved in creating variations in the pathogenicity of different M. oryzae strains. To test this idea, we searched through the transcriptomic data of our strain (70-15) and another strain (98-05) for variation in AVR expression during infection and if differences in MoIsw2 binding could be the cause for this variation and it seems to be that. Finally, we found that most of the genes upregulated in the ΔMoisw2 mutant regulate biomass growth (DNA-binding and quality control, and TFs overrepresented). In contrast, the ones downregulated have to do with mainly mitochondrial oxidative phosphorylation, oxidative stresses, and secondary metabolites.Similar results for enriched and depleted genes were found for all genes in our ChIP-seq data for genes closest to MoIsw2 binding. We conclude that MoIsw2 interaction with nucleosomes and binding to palindromic DNA in transposons located between genes creates a possibility for an evolutionary fast variation of offspring regulatory pattern without loss of genes through mutation and fast creation of M.oryzae strains with new cultivar host ranges in response to defenses by resistant cultivars and consequently a fast break of resistance. MoISW2 seems to lay a mechanistic ground for natural adaptation-directed fast evolution (NADFE) of M. oryzae, which is discussed as a new concept for Eukaryotes.
Publisher
Cold Spring Harbor Laboratory