Abstract
Carbon fixation pathway of plants (CFPP) in photosynthesis converts solar energy to biomass, bio-products and biofuel. Intriguingly, a large number of heterotrophic fungi also possess enzymes functionally associated with CFPP, raising the questions about their roles in fungal development and in evolution. Here, we report on the presence of 17 CFPP associated enzymes (ten in Calvin-Benson-Basham reductive pentose phosphate pathway and seven in C4-dicarboxylic acid cycle) in the genome of Sclerotinia sclerotiorum, a heterotrophic phytopathogenic fungus, and only two unique enzymes: ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) and phosphoribulokinase (PRK) were absent. This data suggested an incomplete CFPP-like pathway (CLP) in fungi. Functional profile analysis demonstrated that the activity of the incomplete CLP was dramatically regulated during different developmental stages of S. sclerotiorum. Subsequent experiments confirmed that many of them were essential to the virulence and/or sclerotial formation. Most of the CLP associated genes are conserved in fungi. Phylogenetic analysis showed that many of them have undergone gene duplication, gene acquisition or loss and functional diversification in evolutionary history. These findings showed an evolutionary links in the carbon fixation processes of autotrophs and heterotrophs and implicated the functions of related genes were in course of continuous change in different organisms in evolution.
Highlights
MethodsCDNA production and quantification of gene expression by quantitative reverse transcription PCR (qRT-PCR) amplification
The functional profile analysis and the gene functional enrichment analysis demonstrated many KEGG metabolic pathways were involved in multiple developmental processes of S. sclerotiorum (Table S4 and S6)
Our functional profile analysis showed an incomplete CFPP-like pathway (CLP) was dramatically activated during the infection, sclerotial myceliogenic germination and sclerotial carpogenic germination of S. sclerotiorum (Table S4), and this CLP was selected for further study
Summary
CDNA production and quantification of gene expression by qRT-PCR amplification. To evaluate the dynamic expression levels of the selected genes in wild-type strain Ep-1PNA367 which were cultured on PDA from the first day to the twelfth day respectively, the cultures were collected every day to extract the total RNA. To evaluate the expression levels of the selected genes in different transformants, the transformants and the wild-type strain were cultured on PDA until the day when the expression of corresponding genes reached the highest level that is validated by the dynamic expression detection in the previous step, and the extraction of the total RNA of these cultures, cDNA synthesis and the qRT-PCR were conducted according to Zhu et al.[34]. Data acquisition and analysis were performed using the Bio-Rad CFX Manager Software (version 2.0)
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