Transcriptome Analysis and SNP Identification Reveal That Heterologous Overexpression of Two Uncharacterized Genes Enhances the Tolerance of Magnaporthe oryzae to Manganese Toxicity

Abstract

Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. Manganese is a crucial trace element that constitutes the cofactors of many enzymes. However, excessive Mn 2+ can be toxic for both prokaryotes and eukaryotes. The mechanism of fungal genetics and metabolism in response to Mn 2+ stress remains understudied, warranting further studies. Magnaporthe oryzae is well-established as the most destructive pathogen of rice. A field strain, YN2046, more sensitive to Mn 2+ toxicity than other strains, was obtained from a previous study. Herein, we explored the genetic mechanisms of Mn 2+ sensitivity in YN2046 through comparative transcriptomic analyses. We found that many genes previously reported to participate in Mn 2+ stress were not regulated in YN2046. These non-responsive genes might cause Mn 2+ sensitivity in YN2046. Weight gene correlation network analysis (WGCNA) was performed to characterize the expression profile in YN2046. Some overexpressed genes were only found in the Mn 2+ tolerant isolate YN125. Among these, many single nucleotide polymorphism (SNP) were identified between YN125 and YN2046, which might disrupt the expression levels of Mn responsive genes. We cloned two uncharacterized genes, MGG_13347 and MGG_16609 , from YN125 and transformed them to YN2046 with a strong promoter. Our results showed that the heterologous overexpression of two genes in YN2046 restored its sensitivity. Transcriptomic and biochemical analyses were performed to understand Mn tolerance mechanisms mediated by the two heterologous overexpressed genes. Our results showed that heterologous overexpression of these two genes activated downstream gene expression and metabolite production to restore M. oryzae sensitivity to Mn, implying that SNPs in responsive genes account for different phenotypes of the two strains under Mn stress. IMPORTANCE Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. The comparative transcriptome analysis showed that fewer genes were regulated in the Mn-sensitive strain. WGCNA and SNP analyses found that mutations in promoter and coding sequence regions might disrupt the expression of genes involved in Mn detoxification in the sensitive strain. We transferred two unannotated genes that were cloned from the Mn-tolerant strain into a sensitive strain with strong promoters, and the transformants exhibited an enhanced tolerance to Mn 2+ toxicity. Transcriptome and biochemistry results indicated that heterologous overexpression of the two genes enhanced the tolerance to Mn toxicity by reactivation of downstream genes in M. oryzae .